Note: Descriptions are shown in the official language in which they were submitted.
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METHODS AND APPARATUS FOR DECONTAMINATING FLUIDS
BACKGROUND OF THE INVENTION
FIELD OF THE INVENTION:
The present invention relates to methods for decontaminating fluids, including
protein-containing biological fluids, in particular blood products, other
natural biologicals,
t 0 and synthetic biotechnology products. The present invention also relates
to apparatus useful
for decontaminating fluids, including protein-containing biological fluids, in
particular blood
products, other natural biologicals, and synthetic biotechnology products. The
present
invention further relates to apparatus for contacting ozone with a liquid.
15 DISCUSSION OF THE BACKGROUND:
Protein-containing biological fluids are important for a number of reasons. In
particular, protein-containing fluids such as whole blood and blood products,
such as red
blood cells, platelets, and plasma, are important components of the health
care system.
Likewise, modern health care is also dependent on other important protein-
containing
20 biological fluids, including synthetic biotechnology products such as
recombinant clotting
factors, as well as natural biological products, such as antitoxins and
vaccines.
Unfortunately, the source of these fluids and the fact that these fluids
contain proteins make
them susceptible to contamination by a variety of infectious agents, such as
parasites,
bacteria, fungi, and viruses.
25 The common factor in all of these contaminants is that they contain DNA
and/or
RNA. Decontamination of the protein-containing fluid thus does not necessarily
require the
removal of the contaminating agents, but only the disruption of the
contaminating agents'
DNA and/or RNA so that these agents cannot propagate and thus spread disease.
The approach of attacking DNA and/or RNA is particularly useful in the blood
3o industry because red blood cells, platelets and plasma, which are the
useful components of
blood for transfusion and pharmaceutical manufacture, contain no DNA or RNA.
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Furthermore, the leukocytes, or white blood cells, do contain DNA and RNA, but
it is
desirable to destroy this material to eliminate graft versus host disease
(GVHD), as recently
recommended for general transfusion practice.
Because of these potential benefits, several techniques have been developed to
attack
DNA and/or RNA in blood and blood products. The main target of this work is
plasma,
which is the straw-colored material left after the cellular blood components
have been
removed. Rich in proteins and nutrients, plasma can harbor many contaminants,
but the
smallest of the above contaminants, and thus the most difficult to treat, are
the viruses.
Specifically, potentially lethal viruses, such as HIV and Hepatitis B, are of
great concern.
1o These contaminants pose a great hazard when contaminated units are
inadvertently included
in the large pools of plasma used for the manufacture of pharmaceuticals, thus
possibly
leading to large scale infection among the treated population.
The existing techniques to eliminate such pathogens from plasma were recently
summarized at the 1998 AABB annual meeting (Transfusion Transmitted Diseases
(Prions;
Bacteria and Parasites); Selected Topics in Transfusion-Transmitted
Infections: American
Association of Blood Banks Annual Meeting, The Compendium, 1998) and the 1999
CHI
annual blood safety and screening symposium (Safety Issues: New Inactivation
Technologies: Plasma; Cambridge Healthtech Institute's Fifth Annual Blood
Safety &
Screening Symposium, Feb 23-24, 1999). These techniques can be roughly divided
into two
2o groups: (1) those that can treat only enveloped viruses, and (2) those that
can treat both
enveloped and non-enveloped viruses.
Beginning with the techniques that can treat only enveloped viruses, the most
notable
example is the solvent/detergent combinations that are specifically directed
at the viral
envelope itself. In particular, the Red Cross and V. I. Technologies are now
actively
promoting one such product as Plas+SD. Intended for direct transfusion,
Plas+SD provides
some degree of safety and uniform product consistency. There are, however,
concerns over:
(1) cost; (2) residual solvent/detergent left in the product; (3) the use of a
donor pool, albeit a
relatively small one at about 2,000 units; (4) the inability to treat non-
enveloped viruses; (5)
the impact of new or emerging viruses (A. Pereira; "Cost-effectiveness of
transfusing virus-
3o inactivated plasma instead of standard plasma," Transfusion, vol. 39, pp.
479-487 (1999));
and (6) recent recalls (V. I. Tech Sees $3M, Or 24c/Shr 2Q Chg from Pdt
Recall: Wall Street
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Journal/Dow Jones Newswires, July 14, 1999).
Another technique for treating enveloped viruses is very high static pressure,
on the
order of 45,000 to 60,000 psi. However, the required pressure vessels are
quite expensive
and this technique, though used elsewhere (S. Denys et al, "Modeling
conductive heat
s transfer and process uniformity during batch high-pressure processing of
foods," Biotechnol.
Prog., vol. 16( 1 ), pp. 92-1 O l (2000)), is still under development in the
plasma industry.
Beyond the limitation to enveloped viruses, there are also the practical
problems of
contamination and/or leaking of the pump oil, as well as rupture of the plasma
bag. One
variation is to freeze the plasma (D. W. Bradley, et al, "Pressure cycling
technology: a novel
1o approach to virus inactivation in plasma," Transfusion, vol. 40(2), pp. 193-
200 (2000)), but
this process is relatively slow and raises the problem of freeze damage to the
plasma proteins.
Of the many techniques capable of treating both enveloped and non-enveloped
viruses, the most common example is intense light exposure. At high enough
frequencies, in
the UVC to gamma range, the energy in the light disrupts the basic structure
of the
15 contaminants. However, at these energies, there is also the problem of
oxygen radical
formation. To prevent these radicals from damaging the proteins, quenching
agents are
typically added to the plasma. Unfortunately, these agents are expensive, at
least partially
toxic, and must be removed before the plasma can be used. To avoid such
problems, a
limited exposure technique has recently been reported, but the results to date
show only
20 partial success, as well as some degree of protein damage (K. M. Remington,
"Identification
of Critical Parameters and Application to UVC Viral Inactivation in the
Absence of
Additives," Cambridge Healthtech Institute's Sixth Annual Blood Product Safety
Symposium, Feb. 13-15, 2000).
An extension of these direct light exposure techniques is the addition of a
light-
25 sensitive compound, such as methylene blue, to the plasma. When activated
by light of the
appropriate wavelength, this compound then attacks the contaminants. Like the
above
solvent/detergent processes, however, there are concerns over costs and the
effects of residual
material in the so-treated plasma.
Yet another approach commonly used for both enveloped and non-enveloped
viruses
3o is heat treatment, typically with steam. Obviously, however, this approach
is not suitable for
heat-sensitive proteins and is not used for single plasma units.
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Finally, there are also other techniques under development, such as various
ozone
processes, but these processes are typically expensive and difficult to
execute in the closed
environment required for plasma processing. In addition, ozone-based methods
suffer from
the disadvantage of requiring long treatment times. On the other hand, ozone
itself is cheap
and is quite effective given sufficient processing time, and leaves no toxic
residue (M. M.
Kekez, S. A. Sattar; "A new ozone-based method for virus inactivation:
preliminary study,"
Phys. Med. Biol., vol. 42, pp. 2027-2039 (1997); U.S. Patent No. 4,632,980;
and U.S. Patent
No. 5,882,591 ).
To achieve better results, some of the above decontamination techniques have
been
1 o combined. For example, the combination of the heat and solventldetergent
processes is quite
effective against pathogens such as HIV (B. Horowitz; "Virus Inactivation by
Solvent/Detergent Treatment and the Manufacture of SD-Plasma," Vox Sang, vol.
74, Suppl.
l, pp. 203-206 (1998)).
Unfortunately, all of the above decontamination techniques, as well as others,
have
serious problems. The underlying difficulty is that the contaminant viral DNA
and/or RNA
are both proteins, any thus any technique that disrupts these contaminants can
also cause
significant damage to the desired proteins in the treated fluid. This is of
great concern
because damaged proteins are less effective clinically. For example, excessive
decontamination damage of this protein will reduce the concentration in the
fibrin glues used
2o during surgery, and the resulting glue will thus not be capable of either
approximating a
wound or inducing hemostasis. Furthermore, damaged proteins also induce
antibody
formation, thereby making future treatment quite difficult (Barbara A. Konkle;
"New
Products for Patients with Hemophilia or von Willebrand Disease," American
Association of
Blood Banks Armual Meeting, The Compendium, Baltimore, MD, pp. 111-115, 1998).
In addition, the contaminants and the desired proteins are also so similar
that any
technique that completely destroys all of the contaminants would also destroy
all of the
desired proteins. For this reason, no practical decontamination technology can
be completely
effective, and thus some small degree of contamination will always remain in
the treated
fluid. This is a particular problem for lethal contaminants such as HIV and
Hepatitis B. In
3o such cases, the goal is thus to reduce the contaminant as much as possible.
In practice,
acceptable levels are generally considered to be a logarithmic reduction
factor (LRF) of 6,
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which means that 1 part in 1 million survives treatment.
Of course, because erythrocytes and platelets also have proteins similar to
those found
in contaminant, DNA and RNA, the problems of protein damage and incomplete
decontamination also extend to these blood components.
Furthermore, similar problems also arise in the treatment of biologics other
than blood
products. Specifically, these other biologics, whether of synthetic or natural
origin, should
contain no untreated genetic material of their own, and should also not be
contaminated with
foreign DNA and/or RNA. On the other hand, the proteins in these biologics are
similar to
the proteins in the contaminating DNA and/or RNA. The net result of any
treatment is thus
again at least some protein damage, along with limited decontamination.
Finally, blood products and other biologics are also subject to several other
problems.
For example, in the modern health care environment, costs must be carefully
controlled, both
for capital equipment and any disposables. Likewise, technician time and
training must be
kept to minimum levels. Beyond these cost factors, however, there are also
several process
concerns. Specifically, the overall processing time must be kept as short as
possible, as
demonstrated by the recent and ongoing shortages in various intravenous
immunoglobulins
(IVIGs). In addition, there is only a limited supply of starting material,
which must therefore
be treated as efficiently as possible. Of course, all of the above concerns
must be met, while
also satisfying stringent regulatory requirements for safety and efficacy,
along with full
2o documentation.
The most difficult problem in decontamination work, however, is the
possibility of
contamination by agents that do not follow the normal DNA or RNA infection
route.
Specifically, recent work indicates that infections may also proceed by
distortions in protein
shape. In this case, the underlying agent is referred to as a "prion" and the
resulting disease is
commonly called "mad cow disease" in the bovine form, "scrapie" in the sheep
form, and
Creutzfeldt-Jakob Disease in the human form. Although their resistance to
conventional
decontamination technologies in fact characterizes prions, recent work
indicates that these
infectious agents may be at least partially susceptible to gamma irradiation,
and possibly
subject to sonic or ozone effects as well. It is therefore understood that the
following
3o techniques that are designed to protect proteins during decontamination for
conventional
agents can also be applied to protect proteins during prion decontamination.
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A method for freeing blood and blood components of viable enveloped viruses by
contacting the blood or blood product with ozone is disclosed in U.S. Patent
No. 4,632,980.
U.S. Patent No. 5,882,591 discloses a method and apparatus for disinfecting
biological fluids,
such as plasma/serum, through the interaction with gases, such as ozone.
SUMMARY OF THE INVENTION
Thus, there remains a need for effective processes for decontaminating fluids,
including protein-containing biological fluids, such as plasma. In particular,
there remains a
to need for processes for decontaminating protein-containing biological
fluids, such as plasma,
which may be applied to individual units as well as pooled units, and which
afford improved
protection against infectious agents, including viruses. In addition, these
processes must be
fast, efficient, inexpensive, and cause minimum damage to the desired
proteins. There also
remains a need for apparatus which are useful for carrying out such processes.
15 Accordingly, it is one object of the present invention to provide novel
methods for
decontaminating fluids.
It is another object of the present invention to provide novel methods for
decontaminating protein-containing biological fluids.
It is another object of the present invention to provide novel methods for
2o decontaminating plasma.
It is another object of the present invention to provide novel methods for
decontaminating human plasma.
It is another object of the present invention to provide novel methods for
decontaminating protein-containing biological fluids which provide a high
level of protection
25 from infectious agents.
It is another object of the present invention to provide novel methods for
decontaminating plasma which provide a high level of protection from
infectious agents.
It is another object of the present invention to provide novel methods for
decontaminating human plasma which provide a high level of protection from
infectious
3o agents.
It is another object of the present invention to provide novel methods for
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decontaminating protein-containing biological fluids which provide a high
level of protection
from viruses.
It is another object of the present invention to provide novel methods for
decontaminating plasma which provide a high level of protection from viruses.
It is another object of the present invention to provide novel methods for
decontaminating human plasma which provide a high level of protection from
viruses.
It is another object of the present invention to provide novel methods for
decontaminating plasma which may be readily applied to individual plasma
units.
It is another object of the present invention to provide novel methods for
1 o decontaminating human plasma which may be readily applied to individual
plasma units.
It is another object of the present invention to provide novel methods for
decontaminating plasma which may be readily applied to batch processing of
pooled plasma
units.
It is another object of the present invention to provide novel methods for
15 decontaminating human plasma which may be readily applied to batch
processing of pooled
plasma units.
It is another object of the present invention to provide novel apparatus
useful for
decontaminating fluids.
It is another object of the present invention to provide novel apparatus
useful for
2o decontaminating protein-containing biological fluids.
It is another object of the present invention to provide novel apparatus
useful for
decontaminating plasma.
It is another object of the present invention to provide novel apparatus
useful for
decontaminating human plasma.
25 It is another object of the present invention to provide novel apparatus
useful for
decontaminating protein-containing biological fluids which provide a high
level of protection
from infectious agents.
It is another object of the present invention to provide novel apparatus
useful for
decontaminating plasma which provide a high level of protection from
infectious agents.
3o It is another object of the present invention to provide novel apparatus
useful for
decontaminating human plasma which provide a high level of protection from
infectious
7
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agents.
It is another object of the present invention to provide novel apparatus
useful for
decontaminating protein-containing biological fluids which provide a high
level of protection
from viruses.
It is another object of the present invention to provide novel apparatus
useful for
decontaminating plasma which provide a high level of protection from viruses.
It is another object of the present invention to provide novel apparatus
useful for
decontaminating human plasma which provide a high level of protection from
viruses.
It is another object of the present invention to provide novel apparatus
useful for
to decontaminating plasma which may be readily applied to individual plasma
units.
It is another object of the present invention to provide novel apparatus
useful for
decontaminating human plasma which may be readily applied to individual plasma
units.
It is another object of the present invention to provide novel apparatus
useful for
decontaminating plasma which may be readily applied to batch processing of
pooled plasma
15 units.
It is another object of the present invention to provide novel apparatus
useful for
decontaminating human plasma which may be readily applied to batch processing
of pooled
plasma units.
It is another object of the present invention to provide novel apparatus for
contacting
2o ozone with a liquid.
These and other objects, which will become apparent during the following
detailed
description, have been achieved by the inventor's discovery, in a first main
embodiment, of a
method for decontaminating plasma by:
(a) treating plasma with ultrasonic energy.
25 The inventor has also discovered, in a second main embodiment, that plasma
may be
effectively decontaminated by a method involving:
(a') a step for the treatment of plasma with ultrasonic energy.
The inventor has further discovered, in a third main embodiment, that a fluid,
such as
a protein-containing biological fluid, may be effectively decontaminated by a
method
30 involving:
(a) treating a fluid with ultrasonic energy, while degassing the fluid.
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The inventor has also discovered, in a fourth main embodiment, that such
fluids may
be effectively decontaminated by a method involving:
(a') a step for the treatment of a fluid with ultrasonic energy, while
degassing the
fluid.
The inventor has further discovered, in a fifth main embodiment, that a fluid,
such as
a protein-containing biological fluid may be effectively decontaminated by a
method
involving:
(a) simultaneously treating a fluid with at least two different frequencies of
ultrasonic energy.
l0 The inventor has also discovered, in a sixth main embodiment, that such
fluids may
be effectively decontaminated by a method involving:
(a') a step for the simultaneous treatment of a fluid with at least two
different
frequencies of ultrasonic energy.
The inventor has also discovered, in a seventh main embodiment, that such
fluids may
be effectively decontaminated by a method involving:
(a) treating a fluid with ultrasonic energy to obtain a de-oxygenated fluid;
and
(b) irradiating said de-oxygenated fluid.
The inventor has also discovered, in an eighth main embodiment, that such a
fluid
may be effectively decontaminated by a method involving:
(a') a step for the treatment of a fluid with ultrasonic energy to obtain a de-
oxygenated fluid; and
(b') a step for the irradiation of said de-oxygenated fluid.
The inventor has further discovered, in a ninth main embodiment, that such
fluids
may be effectively decontaminated by a method involving:
(a) treating a fluid with ultrasonic energy to obtain a de-oxygenated fluid;
and
(b) contacting said de-oxygenated fluid with a pulsed electric field.
The inventor has also discovered, in a tenth main embodiment, that such a
fluid may
be effectively decontaminated by a method involving:
(a') a step for the treatment of a fluid with ultrasonic energy to obtain a de-
oxygenated fluid; and
(b') a step for contacting said de-oxygenated fluid with a pulsed electric
field.
9
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The inventor has further discovered, in an eleventh main embodiment, that a
fluid,
such as a protein-containing biological fluid may be effectively
decontaminated by a method
involving:
(a) treating a fluid with ultrasonic energy to obtain a de-oxygenated fluid;
and
(b) contacting said de-oxygenated fluid with ozone.
The inventor has also discovered, in a twelfth main embodiment, that such a
fluid
may be effectively decontaminated by a method involving:
(a') a step for the treatment of a fluid with ultrasonic energy to obtain a de-
oxygenated fluid; and
(b') a step for the treatment of said de-oxygenated fluid with ozone.
The inventor has further discovered, in a thirteenth main embodiment, that a
fluid,
such as protein-containing biological fluid, may be effectively decontaminated
by a method
involving:
(a) mixing a fluid with ozone, to obtain an ozone-containing fluid; and
(b) treating said ozone-containing fluid with ultrasonic energy.
The inventor has also discovered, in a fourteenth main embodiment, that such a
fluid
may be effectively decontaminated by a method involving:
(a') a step for mixing a fluid with ozone, to obtain an ozone-containing
fluid; and
(b') a step for the treatment of said ozone-containing fluid with ultrasonic
energy.
2o The inventor has further discovered, in a fifteenth main embodiment, that a
fluid, such
as a protein-containing biological fluid, may be effectively decontaminated by
a method
involving:
(a) treating a fluid with ultrasonic energy to obtain a de-oxygenated fluid;
(b) contacting said de-oxygenated fluid with ozone, to obtain an ozone-
containing
fluid; and
(c) treating said ozone-containing fluid with ultrasonic energy.
The inventor has also discovered, in a sixteenth main embodiment, that such a
fluid
may be effectively decontaminated by a method involving:
(a' ) a step for the treatment of a fluid with ultrasonic energy to obtain a
de-
oxygenated fluid;
(b') a step for the treatment of said de-oxygenated fluid, to obtain an ozone-
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containing fluid; and
(c') a step for the treatment of said ozone-containing fluid with ultrasonic
energy.
The inventor has further discovered, in a seventeenth main embodiment, that a
fluid,
including a protein-containing biological fluid, may be effectively
decontaminated by a
method involving:
(a) treating a fluid with ultrasonic energy to obtain a de-oxygenated fluid;
(b) irradiating said de-oxygenated fluid, to obtain an irradiated fluid; and
(c) contacting said irradiated fluid with ozone, to obtain an ozone-containing
fluid.
1 o The inventor has also discovered, in an eighteenth main embodiment, that
such a fluid
may be effectively decontaminated by a method involving:
(a') a step for the treatment of a fluid with ultrasonic energy to obtain a de-
oxygenated fluid;
(b') a step for the irradiation of said de-oxygenated fluid, to obtain an
irradiated
fluid; and
(c') a step for the treatment of said irradiated fluid, to obtain an ozone-
containing
fluid.
The inventor has further discovered, in a nineteenth main embodiment, that a
fluid,
including a protein-containing biological fluid, may be effectively
decontaminated by a
method involving:
(a) treating a fluid with ultrasonic energy to obtain a de-oxygenated fluid;
(b) irradiating said de-oxygenated fluid, to obtain an irradiated fluid;
(c) contacting said irradiated fluid with ozone, to obtain an ozone-containing
fluid; and
(d) treating said ozone-containing fluid with ultrasonic energy.
The inventor has also discovered, in a twentieth main embodiment, that such a
fluid
may be effectively decontaminated by a method involving:
(a') a step for the treatment of a fluid with ultrasonic energy to obtain a de-
oxygenated fluid;
(b') a step for the irradiation of said de-oxygenated fluid, to obtain an
irradiated
fluid;
11
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(c') a step for the treatment of said irradiated fluid, to obtain an ozone-
containing
fluid; and
(d') a step for the treatment of said ozone-containing fluid with ultrasonic
energy.
The inventor has also discovered, in a twenty-first main embodiment, that a
fluid,
including a protein-containing biological fluid, may be effectively
decontaminated by means
of an apparatus which contains:
( 1 ) a chamber for containing a fluid;
(2) a vacuum source coupled to the chamber; and
(3) a source of ultrasonic energy coupled to the chamber,
o wherein said chamber comprises (i) a flat panel, (ii) an inlet, and (iii) an
outlet; and wherein
said flat panel of said chamber and said inlet are dimensioned such that a
fluid flowing
through said inlet and across said flat panel to said outlet will form a thin
film and travel in
plug flow at least during some portion of its flow across said flat panel.
The inventor has further discovered, in a twenty-second main embodiment, that
such
a fluid may be effectively decontaminated by means of an apparatus which
contains:
(1') a means for containing said fluid;
(2') means for contacting said fluid with a vacuum; and
(3') a means for introducing ultrasonic energy into said means for containing
said
fluid,
wherein said means for containing said fluid comprises (i) a means for the
introduction of
said fluid into said containing means, (ii) a means for said fluid to flow
through said
containing means, and (iii) a means for the removal of said fluid from said
containing means;
and wherein said containing means is dimensioned such that a protein-
containing fluid
flowing through said containing means will form a thin film and travel in plug
flow at least
during some portion of its flow through said containing means.
The inventor has also discovered, in a twenty-third main embodiment, that a
fluid,
including a protein-containing biological fluid, may be effectively
decontaminated by means
of an apparatus which contains:
(1) a chamber for containing a fluid;
3o (2) a vacuum source coupled to the chamber;
(3) a source of ultrasonic energy coupled to such chamber; and
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(4) a source of UV, gamma, or x-ray radiation.
The inventor has also discovered, in a twenty-fourth main embodiment, that a
fluid,
including a protein-containing biological fluid, may be effectively
decontaminated by means
of an apparatus which contains:
(1') a means for containing said fluid;
(2') means for contacting said fluid with a vacuum;
(3') a means for introducing ultrasonic energy into said means for containing
said
fluid; and
(4') a means for the treatment of said fluid with UV, gamma, or x-ray
radiation.
to The inventor has also discovered, in a twenty-fifth main embodiment, that a
fluid,
including a protein-containing biological fluid, may be effectively
decontaminated by means
of an apparatus which contains:
( 1 ) a chamber for containing a fluid;
(2) a vacuum source coupled to the chamber;
(3) a source of ultrasonic energy coupled to such chamber; and
(4) a source of ozone,
wherein said chamber comprises: (i) an inlet for introducing ozone from the
source of ozone;
(ii) an inlet for introducing plasma; and (iii) a device for mixing ozone from
the source of
ozone with a fluid.
2o The inventor has further discovered, in a twenty-sixth main embodiment,
that a fluid,
including a protein-containing biological fluid, may be effectively
decontaminated by means
of an apparatus which contains:
(1') a means for containing said fluid;
(2' ) a means for contacting said fluid with a vacuum;
(3') a means for introducing ultrasonic energy into said means for containing
said
fluid; and
(4') a means for generating ozone,
wherein said means for containing said fluid comprises: (i) a means for the
introduction of
ozone from said means for generating ozone into said containing means; (ii) a
means for the
3o introduction of said fluid into said containing means; and (iii) a means
for mixing said ozone
from said means for generating ozone with said fluid in said containing means.
13
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The inventor has also discovered, in a twenty-seventh main embodiment, that a
fluid,
including a protein-containing biological fluid, may be effectively
decontaminated by means
of an apparatus which contains:
( 1 ) a chamber for containing a fluid;
(2) a vacuum source coupled to the chamber;
(3) a source of UV, gamma, or x-ray radiation;
(4) a source of ultrasonic energy coupled to such chamber; and
(5) a source of ozone,
wherein said chamber comprises: (i) an inlet for introducing ozone from the
source of ozone;
(ii) an inlet for introducing plasma; and (iii) a device for mixing ozone from
the source of
ozone with a fluid.
The inventor has further discovered, in a twenty-eighth main embodiment, that
a
fluid, including a protein-containing biological fluid, may be effectively
decontaminated by
means of an apparatus which contains:
(1') a means for containing said fluid;
(2') a means for contacting said fluid with a vacuum;
(3') a means for the treatment of said fluid with UV, gamma, or x-ray
radiation.
(4') a means for introducing ultrasonic energy into said means for containing
said
fluid; and
(S') a means for generating ozone,
wherein said means for containing said fluid comprises: (i) a means for the
introduction of
ozone from said means for generating ozone into said containing means; (ii) a
means for the
introduction of said fluid into said containing means; and (iii) a means for
mixing said ozone
from said means for generating ozone with said fluid in said containing means.
The inventor has further discovered, in a twenty-ninth main embodiment, that
ozone
may be effectively contacted with a liquid with an apparatus which comprises:
( 1 ) a substrate which has a lower surface and an upper surface and which has
a
plurality of passage-ways connecting said lower surface with said upper
surface;
3o (2) a source of ultrasonic energy coupled to said substrate, such that said
ultrasonic energy is introduced into the liquid by the vibration of said
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substrate;
(3) a source of ozone connected to said lower surface of said substrate.
BRIEF DESCRIPTION OF THE DRAWINGS
A more complete appreciation of the invention and many of the attendant
advantages
thereof will be readily obtained as the same become better understood by
reference to the
following detailed description when considered in connection with the
accompanying
drawings, wherein:
Figure I is a flow chart which depicts one embodiment of the method according
to the
1 o present invention;
Figure 2 is a schematic representation of an ultrasonic degassing chamber
according
to the present invention;
Figure 3 is a schematic representation of an ultrasound treatment apparatus
according
to the present invention;
Figure 4 is a schematic representation of another embodiment of a combined
ultrasound treatment and UV treatment apparatus according to the present
invention;
Figure 5 is a schematic representation of a combined ozone and ultrasound
treatment
apparatus according to the present invention;
Figure 6 is a cross-sectional view of an UV treatment chamber and components
according to the present invention;
Figure 7 is a schematic representation of an ozone contactor according to the
present
invention;
Figure 8 is a schematic representation of an ozone contactor according to the
present
invention;
Figure 9 is a schematic representation of a preferred embodiment of an ozone
contactor;
Figure 10 is a schematic representation of a preferred embodiment of another
ozone
contactor;
Figure 11 is a schematic representation of another ozone contactor which is
useful for
3o platelets.
CA 02481144 2004-10-04
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DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Thus, the present invention provides novel methods and apparatus for
decontaminating a fluid. In certain preferred embodiments, the fluid is a
liquid such as a
protein-containing biological fluid. Suitable protein-containing biological
fluids include
body fluids, such as whole blood, saliva, semen, spinal fluid, etc. In
addition to whole blood,
the protein-containing biological fluid may be a blood product, such as
plasma, sera, and the
red blood cell (erythrocyte) or platelet fractions of whole blood. The protein-
containing
biological fluid may also be any natural or synthetic protein-containing fluid
derived from
various in vitro or in vivo processes, such as a fermentation broth.
1 o In a particularly preferred embodiment of the present invention, the
protein-
containing biological fluid is plasma. The present methods and apparatus are
discussed in
detail below primarily in the context of the decontamination of plasma.
However, it is to be
understood that the present methods and apparatus may also be used to
decontaminate the
protein-containing biological fluids discussed above, including foodstuffs
(including eggs)
and reaction mixtures containing fermentation products such as those obtained
by
recombinant DNA technology. The present invention is applicable not only to
protein-
containing biological fluids, but other heat-sensitive materials as well. In
particular, the
pulsed electric field (PEF) methods discussed below are effective for
decontaminating apple
juice. Thus, in the context of the present methods, the fluid may be any
liquid which is
2o desired in decontaminated form, and includes juices such as apple juice,
orange juice, tomato
juice, etc. In the context of the present invention, the term fluid also
includes liquid-like
materials which are not typically thought of as liquids. Thus, the present
methods and
apparatus may also be applied to the decontamination of eggs, etc. for in
vitro fertilization
(IVF). The present methods may also be applied to foodstuffs, which do not
necessarily have
to include proteins. Likewise, the present methods may be used anywhere there
is a problem
of contamination, particularly in regard to PEF.
The plasma to be treated may be that of any mammal, such as dog, cat, cow,
horse,
pig, chimpanzee, and human. In a preferred embodiment, the plasma to be
decontaminated is
human plasma.
3o The plasma to be decontaminated may be collected by any conventional
technique,
such as from whole blood donation or apheresis, in which cells are returned to
the donor.
16
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Whole blood donation involves taking less volume from the donor (about 200
ml), but
requires a fairly long time (on the order of months) between donations.
Apheresis involves
taking a greater volume from the donor (about 600 ml) but, since cells are
returned to the
donor, requires a shorter time (on the order of weeks) between donations. The
collection of
plasma is described in AABB (American Association of Blood Banks) Press
Technical
Manual, 13'h Edition, Baltimore MD, 1999, which is incorporated herein by
reference.
The plasma to be decontaminated may be an individual unit obtained from a
single
donor. Alternatively, the plasma to be decontaminated may be obtained by
pooling a large
number of individual units taken from a correspondingly large number of
donors.
1o As noted above, a method of decontaminating plasma need not remove or even
inactivate all infectious agents to be considered useful. In fact, many
methods of
decontaminating plasma are specifically designed to address only certain types
of infectious
agents, e.g., enveloped viruses, and none guarantees removal or inactivation
of 100 % of even
the infectious agent for which it is designed. Accordingly, in the context of
the present
invention, the term "method of decontaminating plasma" refers to a method
which is capable
of removing and/or inactivating a significant portion of at least one
infectious agent found in
plasma. Typically, the present methods for decontaminating plasma are capable
of achieving
a log reduction factor or log kill of at least 4, preferably at least S, more
preferably at least 6,
for at least one infectious agent found in plasma.
2o The present methods are also capable of affecting the decontamination of
plasma,
while minimizing the damage to plasma proteins. The amount of protein damage
will depend
on the particular protein in question, the particular embodiment of the
decontamination
method used, and, to some extent the source and prior handling of the plasma.
However, the
present methods are capable of achieving the above-noted log kills of at least
one infectious
agent while causing protein damage of less than
20 %, less that 10 %, and even on the order of a few percent, as determined by
the accepted
clinical laboratory method used to quantify the given protein. For example, to
determine the
fibrinogen concentration, a known amount of thrombin is added to a known
amount of
plasma, and then the elapsed time for clot formation is measured and compared
to a
3o calibrated standard. In a modern hematology laboratory, this and similar
tests for other
proteins are routinely performed by automated equipment, thus providing an
accurate,
17
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documented means of determining the degree of protein damage.
Examples of the types of infectious agents which may be removed and/or
inactivated
by the present methods include parasites, bacteria, fungi, and viruses, and
possibly prions.
Of these agents, parasites pose significant threats mainly in tropical
climates. The
greatest such risk is malaria, which is spread by all four different species
of Plasmodium, but
principally by Plasmodium malariae. Another parasitic risk is Trypanosoma
cruzi, which
causes Chaga's Disease, a serious problem in Central and South America. In the
United
States, two species of Babesi protozoans, which cause Babesiosis, can be
transmitted by
transfusion, although the more common route is tick bite. Leishmania infantum
is also a
t 0 parasite which may be found in blood products.
Bacteria also present a continuous threat in transfusion. For this reason, the
CDC and
leading members of the blood community have recently launched the Bacon
(Bacterial
Contamination) study to determine the risks of transfusion related infections.
Of particular
interest are Yerisinia enterocolitica, Escherichia coli, Citrobacter freundii,
as well as
15 Bartonella and Brucella species.
Fungal infections are a continuous and escalating problem in medical care,
particularly for those patients with compromised immune systems due to cancer
therapy,
HIV, etc. Although the terms fungus and mold are often interchanged, one
convention is that
mold refers to the typically woolly appearance of a growing fungus. Following
this
20 convention, a yeast is then a particular minute fungus, especially of the
Saccharomycetaceae
family. There are many such opportunistic infections, some of the more common
agents of
clinical concern being Candida albicans and Candida stellatoidea, as well as
Cryptococcus
neoformans.
Finally, the main viruses of concern are the various strains of hepatitis (A
through E,
25 and G), HIV (human immunodeficiency virus), HTLV (human t-cell lymphotropic
virus)
types I and II, CMV (cytomegalovirus), EBV (Epstein-Barr virus) and parvovirus
B19.
In most cases, the above contaminants are of concern mainly in transfusion
from one
patient to another. It is also possible, however, to treat the blood of any
individual for such
contaminants, and then transfuse this blood back to the patient. For example,
such an
3o approach lowers the burden of HIV circulating in the blood of AIDS
patients. Such an
approach could also be applied to the treatment of non-infectious agents in
the blood, such as
1s
CA 02481144 2004-10-04
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non-Hodgkins lymphoma (see, "Theratechnologies Enrolls 1 st Patient In
Theralux Trial,"
Wall Street Journal, 1/29/01).
Beyond the blood industry, there are also several applications in other
biologics. In
particular, there is great emerging interest in biotechnology and related
technologies. The
underlying problems will be discussed at the upcoming joint Parenteral Drug
Association
(PDA) and FDA Viral Clearance Forum, PDA Conference Files, Fall 2001. For
example,
one problem facing this industry is that a murine hybridoma would likely be
contaminated
with murine retroviruses, whatever they may be found to be. The possibility of
a resulting
cross-species contamination is of great concern, given that swine flu in the
past has caused
to devastating pandemics. Highly effective decontamination measures are
therefore essential
for the emerging biotechnologies.
In addition to biologics, there are also other applications for
decontamination
technologies. One major area is food science, which requires effective
decontamination of a
variety of contaminants, notably bacteria such as Salmonella. Effective
decontamination of
food products thus improves safety and extends shelf life.
There are also other possibilities of using improved decontamination
techniques
where the material to be treated is not normally considered to be a
contaminant. Specifically,
recent advances in in vitro fertilization (IVF) have raised the possibility of
taking an oocyte
from a female donor, and removing the nucleus along with most of the genetic
material. The
2o genetic material from a second female is then inserted into the oocyte,
which is then fertilized
with the intent of producing a viable pregnancy. The problem with this
approach is that not
all of the donor genetic material can be removed, leaving primarily some
mitochondrial
DNA. As a result, the resulting infant has a genetic contribution from three
"parents,"
causing a great deal of practical and ethical concerns. Using a
decontamination technique to
disrupt all of the donor genetic material, while leaving the other proteins
essentially intact,
would eliminate such concerns.
Finally, the unique equipment described in the following can also be used for
purposes other than decontamination. In particular, the ozone treatment unit
is also useful for
adding gasses to liquids in general. For example, in the case of medical
applications, the
device can be used to oxygenate blood during cardiopulmonary bypass. For food
and/or
industrial applications, the ozone treatment unit can be used to add carbon
dioxide to aqueous
19
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solutions; other similar applications are also possible.
I. Thus, in a first main embodiment, the present invention provides a method
for
decontaminating plasma which comprises:
(a) treating plasma with ultrasonic energy.
The terms "ultrasonic energy" and "ultrasound" refer to sonic waves with
frequencies
in the range of 20 kHz, the upper limit of human hearing, to several hundred
MHz.
Several different techniques can be used to generate ultrasound, but the most
common
approach is to apply electrical impulses to a piezoelectric crystal. The
activated crystal then
expands and contracts along its primary axes to yield pressure pulses or sonic
waves
(ultrasound). In addition, ultrasonic vibrations may also be generated by
other conventional
means, in particular by electromagnetic, electrostrictive, or magnetostrictive
devices. Such
devices are described in published PCT Patent Application WO 92/20420, which
is
incorporated herein by reference.
Because of its relatively high frequency range, ultrasound has many
applications in
industry and medicine. In particular, ultrasound has many unique and
beneficial applications
in the treatment of liquids.
Of these applications, the most common and most significant involve
cavitation.
Cavitation is a localized vaporization that occurs when the low pressure part
of the ultrasonic
2o wave becomes less than the vapor pressure of the liquid. Under these
conditions, the local
temperatures and pressures become extremely high as the cavitation bubbles
grow and then
collapse.
These extreme conditions are used for many practical applications, including
the
rupture of biological cell walls. This capability thus has immediate use in
decontamination,
particularly for parasites. Due to wavelength restrictions, smaller
contaminants such as
bacteria, fungi and viruses can also be treated, but to a lesser degree. The
treated liquids thus
include all non-cellular protein containing liquids. In particular, in the
blood industry, the
treated liquids are plasma or sera, but not red blood cells or platelets.
The overall arrangement for this first embodiment is therefore much like that
used for
3o cell disruption. Specifically, three separate components are required: a
source of ultrasound,
a target, and some means of coupling the source to the target. The limiting
factor here is that
CA 02481144 2004-10-04
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the high frequency waves of ultrasound do not propagate well in gasses such as
air, and
therefore require a carrier medium such as a rigid metal or a liquid. In the
case of metal,
waveguides are commonly made of aluminum. With careful shaping into geometries
called
horns, these waveguides can produce waves of high amplitudes and energies,
resulting in
efficient transfer of the ultrasonic energy into the target.
Another similarity between the decontamination technology in this first
embodiment
and the existing cell disruption technology is that both units operate in the
lower range of
ultrasound frequencies. The underlying physical principle here is that, for
water and dilute
aqueous systems, the power required to produce cavitation increases
dramatically above 100
1 o kHz. Of course, some cavitation occurs at even higher frequencies and the
present method
may utilize such higher frequencies of ultrasonic energy. In particular, it is
anticipated that
higher frequencies will be used as more equipment in the several hundred kHz
to the MHz
frequency, or "megasonic" range, becomes readily available.
In this first embodiment of the present method, plasma is therefore treated
with
ultrasonic energy having a frequency sufficient to result in cavitation of the
plasma. Thus,
the plasma is suitably treated with ultrasonic energy having a frequency of 20
kHz to 10
MHz, preferably 20 kHz to 1 MHz, more preferably 20 kHz to 500 kHz, even more
preferably 20 kHz to 100 kHz.
Although there are thus some similarities between the decontamination
technology
2o described in this first embodiment and conventional cell wall rupturing
equipment, there are,
however, some significant differences. Specifically, it is important for
effectiveness and for
regulatory compliance that the ultrasonic frequency is tightly maintained and
that cavitation
actually occurs in each and every use of the equipment. To ensure that these
conditions are
met, the process must be monitored, preferably by the use of a hydrophone and
supporting
electronics (model bx-208/308, ppb, Inc., San Diego, CA).
Another significant difference between the new decontamination and
conventional
cell disruption equipment is that the decontamination unit must carefully
preserve the desired
protein components, to a level well beyond the degree of protection required
for cell
disruption devices.
3o The first problem in the protection of these proteins is limiting the
strong chemical
reactions that are induced by ultrasonic cavitation. One such reaction is the
breaking of long
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chain organic compounds by the severe agitation due to bubble growth and
collapse during
cavitation. This breaking may be a significant concern for the large,
relatively delicate
proteins involved in the clotting process, as described by El'piner (I. E.
El'piner, Ultrasound
Physical, Chemical and Biological Effects, Consultants Bureau, New York, p.
217, 1964).
In the present method, such damage of the plasma proteins may be managed by
two
techniques. First, the treatment times are kept short, i.e., less than 5
minutes, preferably less
than 2 minutes, more preferably less than 30 seconds. Second, the intensity
levels are kept
low, i.e., 0.1 to 50 W/cm2, preferably 0.5 to 10 W/cm2, and more preferably 1
to 6 W/cmZ. Of
course, in practice these techniques must be adjusted to specific protein
solutions and
contaminants.
Another reaction that is quite damaging to protein solutions undergoing sonic
treatment is the formation of free radicals in the liquid due to the extreme
temperature and
pressure changes of cavitation (V. Misik and P. Riesz, "Detection of primary
free radical
species in aqueous sonochemistry by EPR spectroscopy," in Sonochemistry and
~ 5 Sonoluminescence, edited by L. A. Crum, T. J. Mason, J. L. Reisse and K.
S. Suslick, NATO
ASI Series C, Kluwer Academic Publishers, Dordrecht, pp. 225-236, 1999). The
preferred
method to limit these radicals is to reduce the ultrasonic treatment
intensities and exposure
times, as described above to prevent chain breakage.
In addition to the liquid, however, free radicals can also form at the surface
of the
treated liquid. Of these radicals, the most damaging ones are those formed
from oxygen.
Accordingly, in a preferred subembodiment of this first main embodiment, the
ultrasonic energy is applied after the gas above the plasma has been replaced
with an inert
atmosphere. In this case, "inert" does not include the noble gasses, because
such monatomic
species have too few degrees of freedom to disperse the ultrasonic energy (S.
Y. Wang, in
Symposium on Biological Effects and Characterizations of Ultrasound Sources,
edited by
D.G. Hazzard, et al, US Dept. HEW (FDA) 78-8948, US Government Printing
Office, p.196,
1977). Instead, suitable inert gasses must be polyatomic, notably carbon
dioxide. In practice,
forming a carbon dioxide gas layer simply amounts to dropping a pellet of dry
ice into the
solution to be treated (Hish Intensity Ultrasonic Processor User's Guide,
Sonics & Materials,
3o Inc. Newton, CT, 1999). The evolved gas then displaces the oxygen, and
without oxygen, no
oxygen radicals can form.
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During this process, some of the dissolved gasses are also displaced by carbon
dioxide. Part of this displacement occurs by a concentration gradient in the
immediate
vicinity of the pellet, but most of this displacement is due to transport from
the enriched
carbon dioxide gas layer above the liquid surface. In either case, the net
result is preferential
removal of oxygen, which again is beneficial because oxygen radicals are quite
damaging to
proteins. Note that the overall process is thus similar to the helium sparging
technique
commonly used in hplc, but helium cannot be used here because of the above
described
formation of noble gas radicals.
Beyond controlling the formation of free radicals, another major problem in
limiting
1 o protein damage is the control of excess heat, primarily from the source of
the ultrasound. To
achieve this control, some means of cooling must be provided. A preferred
means of limiting
the source heat is to apply a water flow to the ultrasound source and/or horn.
A preferred
means to cool the target is immersion in a water bath, which also yields
strong acoustic
coupling to the ultrasound horn and source.
15 With these techniques, it is thus possible to maintain the target at any
selected
temperature. This temperature, however, depends on concerns that are often
conflicting.
Specifically, proteins are typically heat sensitive, particularly clotting
factors such as Factor
VIII. For maximum protection, the temperature should therefore be kept
relatively low,
within the FDA specified range of 2 to 10 °C. On the other hand,
cavitation in water or dilute
2o aqueous systems is most effective at about 50°C (J. Blitz,
Ultrasonics: Methods and
Applications, The Butterworths Group, London, pp. 133-4, 1971). At this
temperature, a
minimum amount of energy is required to induce bubble formation, and with less
energy
there is less protein chain breakage and less radical formation. In addition,
there is less
dissolved gas at higher temperatures, thereby further reducing the formation
of the oxygen
25 radicals that are most damaging, as noted above.
For these reasons, the unit should be operated at about 50°C, if the
target can
withstand such elevated temperatures. In particular, for those proteins that
can tolerate even
higher temperatures, use of the highest possible range provides thermal
inactivation of
pathogens, which is an additional safety measure. In this case, temperatures
slightly greater
3o than 50°C may result in some decrease in cavitation efficiency, but
this is more than
compensated by the resulting improvement in thermal inactivation.
Alternatively, even
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higher decontamination temperatures can be used as a separate step, with
cavitation done in
the lower, 50°C range.
For less robust materials, the liquid should be kept cold until immediately
before
treatment, at which time rapid heating techniques should be applied to small
samples to raise
the operating temperature to no more than 50°C. At this time, the
ultrasound should be
applied for as short a duration and intensity as possible, with the sample
then rapidly cooled
back to storage temperatures.
Finally, for those samples that cannot withstand even minimal times at
elevated
temperatures, the sonic treatment should be performed at the highest allowable
temperature,
t o followed by rapid cooling to remove any residual heat from the ultrasound
source or from the
cavitation process.
All three of these processes thus require some means of effective heat
transfer. For
those materials that can withstand elevated temperatures; there are several
heating options to
achieve such temperatures in practice. For robust materials that can withstand
prolonged
heating, the entire source bag or container can be warmed and maintained at
the desired
elevated temperature. Water bath immersion, microwaves, air blast, or any
other convenient
technique can be employed for this warming.
For rapid heating of more temperature sensitive materials, the volume to be
treated is
first broken into smaller units, or into a continuous, low volume per time
flow. These smaller
units or flows are then passed through a separate bag with a large surface
area where they are
subjected to heat transfer from any convenient source, such as from a water
bath, microwave,
air blast, etc.
A similar approach is used for rapid cooling. In this case, however, suitable
cooling
mechanisms are water bath immersion, or contact with plates that are cooled by
gas
expansion or Peltier effects.
It should also be understood that the method of the first main embodiment may
be
carried out in either a batch-wise, semi-continuous, or continuous fashion.
For batch-wise
decontamination, single bag units may be individually treated with the
ultrasonic energy.
Alternatively, in a semi-continuous operation, single units or individual
small volumes of
3o plasma may flow through one or more stations or stages in which they are
treated with
ultrasonic energy. In this mode of operation, each individual unit or volume
is held at each
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station or stage for processing, and is then passed in bulk to the next
station or stage. Yet
another alternative is a continuous operation, in which the plasma may flow
without
interruption through the stations or stages. Both the semi-continuous and
continuous modes
are applicable to fractionation or to other processes involving very large
pools of material to
be treated.
To achieve all of these different modes of operation in practice, a special
treatment
chamber is required.
The first condition on this chamber is that the inlet tube or tubes must not
harbor any
pathogens. The underlying problem can be seen in conventional blood bags, in
which the
o inlet tube passes through a port in the top seam of such containers. As
such, any untreated
fluid that remains in this tube can subsequently contaminate the treated
fluid. This is a
particular problem for ultrasound and the other treatment technologies
described below
because after such treatment processes are terminated, no residual material
remains in the
fluid to prevent any recurrence of the pathogens.
To prevent this problem, the inlet tube may be heat sealed close to the bag,
but this
approach still leaves the port, which is relatively hard to seal. Since such
ports are also rather
narrow, the ultrasonic waves are thus effectively attenuated within a few tube
diameters of
the tube orifice, thereby leaving little or no treatment of any pathogens
farther up the length
of the tube.
A simple alternative for batch treatment is to heat seal the bag itself, below
the tube
orifice. For effective sealing, this procedure must be carried out when the
liquid has already
been forced out of the sealing zone by external compression of the bag.
For semi-continuous processes, a modified compression approach is preferred.
In this
case, the treatment chamber is clamped tightly just below the tube orifice, as
described above,
z5 but in this case, no heat seal is used. Instead, the fluid in the chamber
is treated and then
drained before the next batch of fluid is allowed to enter by releasing the
clamp. To ensure
complete treatment of the fluid volume, specifically the fluid near the clamp,
the face of the
clamp jaws are made of aluminum or stainless steel, thereby preventing any
dampening of the
waves at the clamp. On the other hand, solid metal clamp jaws would allow the
sound waves
3o to propagate through the clamp and into the input reservoir, thus possibly
causing excessive
sound treatment. To prevent this potential problem, the backs of the clamp
jaws are coated
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with rubber or other sound insulation.
Note that continuous units require no such modifications because the flow is
progressively treated.
The second condition on the treatment chamber is that the liquid layer must be
relatively thin. There are several advantages of such thin liquid layers.
First, a thin layer is
necessary to ensure uniform temperatures and uniform cooling. In addition,
thin layers also
allow the evolved gas bubbles in the liquid to rise quickly to the surface.
This is important
because rapid bubble rising reduces the time that the ultrasound can induce
strong surface
oscillations of the bubbles or strong slip-streaming around the bubbles,
thereby reducing
t o protein damage. Furthermore, rapid bubble rising also prevents
agglomeration of the bubbles
to excessive size. Compared to such large bubbles, smaller bubbles are
preferable because
they provide more uniform treatment, and have less surface oscillation and
slip-streaming.
In practice, the plasma is therefore preferably formed into a thin film having
a
thickness of 2 to 20 mm, preferably 2 to 10 mm, and more preferably then 2 to
4 mm, at least
during some part of the application of the ultrasonic energy. Preferably, the
plasma is formed
into such a thin film prior to the commencement of the application of the
ultrasonic energy
and maintained in such a thin film during the entire application of the
ultrasonic energy.
On the other hand, to obtain adequate treatment volumes, these thin layers
must be
relatively broad. In the case of batch or semi-continuous mode, the broad
surface can be of
2o any desired shape, such as a circle, square, etc., as long as the total
resulting volume can be
treated uniformly by the ultrasound. The limiting factor here is that
ultrasonic waves
typically do not produce uniform exposures in containers of water. For this
reason, the
ultrasonic cleaning industry has developed a number of ways to avoid "hot" and
"cold" spots,
primarily by using a mixture of frequencies over a narrow bandwidth and by
building
treatment tanks to dimensions that avoid resonant standing waves. These
approaches can be
used directly for batch and semi-continuous decontamination units, although
the liquid layer
in decontamination devices is much more shallow than that commonly used for
ultrasonic
cleaners.
These approaches, however, must be modified to match the unique geometry
required
of continuous flow systems. The goal here is to ensure uniform treatment of
the flowing
fluid. The problem is that moving fluids can follow several different flow
patterns. In
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common experience, such as in rivers, slow moving pipe flows, etc., laminar
flow develops
such that the center of the fluid moves rapidly, while the fluid near the
boundaries is
essentially stationary. Although quite simple to develop, such flows must be
avoided in
decontamination work because the fluid near the chamber walls would thus
receive excessive
treatment, while the fluid in the center would be essentially untreated.
One means of avoiding this problem is to use a turbulent flow so that eddy
currents
result in thorough bulk mixing of the fluid. Unfortunately, this approach is
not appropriate
for protein decontamination for several reasons. First, the Reynolds number Re
= (pVd/~)
where p is the density, V is the velocity, d is the diameter and ~, is the
viscosity, must be
1o about 1000 for open top flow channels. As such, extremely high flow
velocities must be used
to achieve turbulence, but it is quite difficult to obtain such velocities in
practice. Moreover,
even if such velocities could be achieved, a very long processor would be
required to produce
acceptable treatment times. Finally, even if a high velocity, long residence
time chamber
could be built, the resulting, prolonged turbulence would damage delicate
proteins, thus
15 limiting the range of utility of such a device. For these reasons,
turbulent mixing is not
appropriate for most protein decontamination work.
The alternative is to use a plug flow, in which all of the fluid moves in bulk
through
the processor. To generate such a flow, the fluid entering through an inlet
tube is first spread
out through an expanding section called a diffuser. At the exit of this
diffuser, a rectangular
2o geometry is used to provide the plug flow region. For example, this region
can be 30 cm
wide, 60 cm long, with a fluid depth of 0.4 cm. At the end of this rectangular
component, a
converging section, which is essentially a reversed diffuser, is then used to
guide the flow
into an exit tube. This simple geometry is used in ultraviolet flow cells and
similar, common
laboratory equipment.
25 For decontamination applications, the ultrasonic sources are placed
directly beneath
the rectangular section. With this approach, the ultrasound can not only cause
decontamination, but can also reduce the effective viscosity of the fluid.
This reduction in
viscosity is important because lower viscosities reduce the tendency of the
plug flow to
become laminar, which would otherwise occur over several chamber flow
diameters. The net
30 result of this geometry is therefore quite uniform ultrasonic treatment of
the fluid.
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II. In a second main embodiment, the present invention provides a method for
decontaminating plasma which comprises:
(a') a step for the treatment of plasma with ultrasonic energy.
In this second main embodiment, the step (a') "for the treatment of plasma
with ultrasonic
energy" may be carried out in the same way as the step "(a) treating plasma
with ultrasonic
energy" is carried out in the context of the first main embodiment.
III. In a third main embodiment, the present invention provides a method for
decontaminating a fluid, which comprises:
(a) treating a fluid with ultrasonic energy, while degassing the fluid.
Thus, in this third main embodiment, a vacuum is applied to the fluid during
the
application of the ultrasonic energy.
In this third main embodiment, the ultrasonic energy may be applied to the
fluid using
the same equipment described above in the context of the first and second
embodiments,
except for the means of controlling the free radicals. Specifically, in one
preferred
embodiment of the first and second main embodiments, the ultrasonic energy is
applied after
the gas above the plasma has been replaced with an inert atmosphere. While
quite effective,
this approach unfortunately suffers from the material costs for sterile
consumables, and the
problems of introducing these materials without also allowing contaminants
into the system.
2o An alternative approach is to apply a vacuum to remove the gasses above the
liquid
being treated with ultrasound (see, Hieh Intensity Ultrasonic Processor User's
Guide, Sonics
& Materials, Inc. Newton, CT, 1999). In this third main embodiment, the liquid
being
decontaminated may be any of fluids discussed above. In a preferred
embodiment, the fluid
is a protein-containing biological fluid, such as plasma. The discussion below
explains the
method in the context of plasma, but it is to be understood that the method
may be applied to
any of the fluids discussed above.
The gas above the fluid is effectively removed by applying a vacuum of about 2
to
100 mbar, preferably about 10 to 80 mbar, more preferably 20 to 60 mbar, to
the gas above
the fluid. The limiting factor here is the evaporation of the solvent: at low
enough pressures,
the liquid will boil uncontrollably. Since different liquids may require
different levels of
vacuum, it is preferred that the apparatus be configured such that the level
of vacuum can be
28
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varied.
The vacuum may be applied by means of a vacuum pump. To avoid oil
contamination, the vacuum pump should use a scroll, or other dry, evacuation
method.
It is preferred that the vacuum be applied to the gas above the fluid, e.g.,
plasma, at
s least at the time of commencement of the application of ultrasonic energy to
the plasma.
More preferably, the vacuum is applied to the gas above the plasma prior to
the
commencement of the application of ultrasonic energy to the plasma. Even more
preferably,
the vacuum is applied to the gas above the plasma: (1) prior to the
commencement of the
application of ultrasonic energy to the plasma; and (2) and during the
application of at least a
1 o portion of the ultrasonic energy to the plasma. Of course, ultrasonic
energy may be continued
to be applied after cessation of exposure to the vacuum.
Beyond eliminating the formation of oxygen radicals at the liquid-gas
interface, this
vacuum technique also has additional benefits in the decontamination of
protein solutions.
The main such benefit is that the applied vacuum reduces the vapor pressure
above
1 s the liquid and thereby reduces the energy required to induce cavitation.
With less applied
energy, there is less undesirable protein damage, as would otherwise occur as
described
above in the first and second embodiments.
Another benefit of applying a vacuum to the decontamination system is rapid,
effective degassing of the liquid upon ultrasound treatment. Generally
considered to be the
2o simplest application of ultrasound (T.J. Mason, "Industrial Applications of
Sonochemistry
and Power Ultrasonics," in Sonochemistry and Sonoluminescence edited by L. A.
Crum, T.J.
Mason, J.L. Reisse and K.S. Suslick, NATO ASI Series C, Kluwer Academic
Publishers,
Dordrecht, p. 385, 1999), degassing is used in industries ranging from soda
and beer
production (Marks' Standard Handbook for Mechanical En ice, Tenth Edition;
McGraw-
2s Hill, New York, 12-121-12-123, 1996) to hplc oil cleaning. In these and
similar
applications, the goal is to remove at least some of the gasses dissolved in a
liquid product.
It is important to note that there are actually two sources of gas involved in
the
sonification of a liquid. A general discussion is provided in U.S. Patent No.
4,597,876. The
first gasses to be evolved are the dissolved gasses, in a process referred to
as "rectified
30 diffusion" or "gaseous cavitation." In this case, the dissolved gasses are
simply trapped in
progressively large bubbles because the gasses are forced out of solution more
rapidly than
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they can diffuse back into the liquid. Conversely, the process more often
called "cavitation"
or "vaporous cavitation" in the sonochemistry literature refers to the
formation of bubbles
from the sonified liquid phase itself. In terms of energy, rectified diffusion
requires only
strong vibrations, as readily demonstrated by shaking a soda can. Much greater
energy,
s however, is required to vaporize a liquid to yield what is conventionally
called cavitation. As
a consequence, sonochemical systems and various ultrasonic cleaners are first
"degassed" by
extended operation and/or the use of various additives (soaps) before actual
processing;
otherwise, the dissolved gasses "soften" the sound waves and thus decrease the
performance
of the ultrasonic device.
1o Of course, neither long operating times nor soaps are acceptable for
decontamination
work. As a result, applying sound waves of sufficient energy to plasma yields
a combination
of rectified diffusion and cavitation of water. Furthermore, rectified
diffusion can occur from
the dissolved gasses towards the bubbles formed by cavitation. As described in
the first and
second embodiments, however, it is desirable to keep the ultrasound exposure
as low as
1 s possible to limit protein damage. Under these conditions, relatively
little gas evolution
occurs from either source.
Conversely, applying a vacuum reduces the vapor pressure and thus greatly
accelerates the growth of gas bubbles from both sources. In addition, the
vacuum also
removes any gas bubbles that reach the surface. The net result is a liquid
with little dissolved
2o gasses; in particular, the liquid thus has little dissolved oxygen. This
reduced oxygen
concentration results in fewer oxygen radicals due to cavitation, and
therefore less protein
damage occurs during cavitation.
An enhancement of this process is to use low intensity ultrasound and vacuum
to
degas the liquid before the higher intensity, cavitation inducing ultrasound
is applied. The
2s advantage of this approach is that the dissolved oxygen is thus largely
removed before any
cavitation occurs, thus minimizing the protein damage.
Because the dissolved gas bubbles tend to collect along the walls of the
containment
vessel and around interior points of reduced wave action, some existing
ultrasonic degassing
devices (Polaris Degasser, Polaris Instruments Ltd., Cambridge, UK) use a
pulsed ultrasound
30 driver to allow time for the evolved gasses to escape. A further
enhancement of this approach
is to use progressively longer, higher intensity pulses. The advantage of this
approach is that
CA 02481144 2004-10-04
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as the degassing process continues, the remaining gasses are more difficult to
remove. In
addition, the progressive elimination of oxygen allows more power to be
applied without
radical damage to the proteins.
The net result is that the application of a vacuum allows the initial use of
degassing
intensities of one fourth or less of the intensities required for atmospheric
pressure cavitation.
As the degassing continues, progressively higher intensities can be used. At
the completion
of the degassing process, intensities of more than twice those used for
atmospheric cavitation
can be used, without significant protein damage from oxygen radicals.
In practice, hydrophone monitoring is used to separate the different steps in
this
1o sequence. Specifically, hydrophones record a slight hissing or "frying"
sound as the liquid
degasses under sonification, followed by a sharp "popping" sound as vapor
formation and
collapse occurs (A.A. Atchley and L.A. Crum, "Acoustic Cavitation and Bubble
Dynamics,"
in Ultrasound: Its Chemical Physical and Biological Effects, edited by K.S.
Suslick, VCH
Publishers, Inc., NY, pp. 19-20, 1988). The difference in these two signals is
so distinct that
it can be recognized by automated equipment, thus providing the basis for
activating the
different levels of ultrasound described above.
A final additional benefit of vacuum operation is improved feeding of the
liquid into
the system. In particular, protein solutions such as blood products are easily
damaged by
pumping, whether by piston or peristaltic arrangements. A vacuum system avoids
this
2o problem by drawing in the fluids under a body force. In this regard, a
"body force" refers to
an action on each component of the entire fluid stream, much like gravity.
Gravity feeding,
however, requires a sufficient height to provide adequate flows, and this
height can
sometimes be difficult to arrange in a laboratory setting.
In such cases, vacuum feeding provides a useful alternative. To implement such
a
system, valves are placed between the source bag or container and the vacuum
chamber.
Upon activation, these valves thus allow the fluid to enter the processor with
minimum
transfer damage. To prevent excessive fluid velocities, a flow restrictor is
placed in the fluid
path.
In addition, these valves can be arranged to match or control the flow through
the heat
3o transfer devices described in embodiments one and two. To achieve this
effect in practice,
the heat transfer unit described earlier is placed directly beneath the source
bag or container.
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This arrangement provides supplemental gravity feeding, as well as complete
draining of the
input bag for maximum yield. Likewise, the output of the warming unit is
placed directly
over the inlet of the ultrasonic processing chamber. Two valves are thus used
for operation,
one valve on each side of the warming bag. Opening the valve between the
source bag and
s the warming bag allows the warming bag to fill to capacity. This valve is
then closed, and
the fluid is warmed. Next, the valve between the warming bag and the
ultrasound chamber is
opened, allowing the fluid to enter this chamber under the influence of both
gravity and/or the
applied vacuum.
There are thus several benefits of vacuum operation; however, vacuum operation
also
1o imposes several special requirements. In particular, effective
decontamination requires that
the vacuum operations must be performed under sterile conditions. One possible
solution to
this problem is extensive cleaning and decontamination of conventional vacuum
hardware.
Unfortunately, this approach is expensive and time-consuming, and is thus only
of use in very
large processors.
~ 5 A preferred approach is to use a special disposable that can be easily
changed
between applications. One possible approach is to use a disposable chamber
that can
withstand a 1 atmosphere vacuum. Such a device, however, would require a great
deal of
material and would therefore be quite expensive. A preferred alternative is to
use a thin
disposable plastic liner inside a conventional vacuum chamber made of metal,
preferably
2o stainless steel.
In practice, this disposable amounts to a tent arrangement, with an inlet and
outlet at
opposite sides of the base. At the apex of the tent, a connecting tube
provides vacuum access,
which thus equalizes the inner and outer pressures. For sterility, an FDA
approved filter
prevents entry or exit of any pathogens through this tube. To prevent any
liquid
25 contamination, a plastic cover is attached to this filter; this cover is
opened before use of the
unit and closed afterward. Alternatively, a connecting hose with a sterile
coupling device
(SCD) can also be used for direct vacuum connection. SCDs are also used for
the inlet and
exits. The entire disposable can thus be sterilized by gamma radiation,
autoclave, gas
treatment, or any other conventional sterilization technique.
3o As for materials, the tent can be made of either rigid or flexible plastic.
Rigid plastic
tents would be clamped to the ultrasound driver for highly effective sound
transfer and great
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structural integrity. Unfortunately, such an arrangement would be relatively
expensive and
would require a significant amount of storage space. An alternative is a
flexible tent with
mounting hooks at the corners to maintain the proper shape. This cheaper
arrangement is
preferable. If desired, the tent can be immersed in a liquid bath to improve
acoustic coupling
and heat transfer.
With or without this bath, however, all vacuum systems pose a potential risk
of
aspiration when treating liquids. The standard means of avoiding this problem
is to use a
vacuum trap to capture any aspirated liquids before they are drawn into the
pump. While this
approach can be used for decontamination work, a preferred technique is to use
separate traps
for the plasma container and the vacuum chamber.
Under this arrangement, any contaminated liquid aspirated from the plasma
container
is thus captured in a separate chamber for disposal. For least expense, this
trap is placed
beyond the sterile filter so that the trap can be used for multiple cycles. To
save space in the
vacuum chamber, the trap is located externally. Under this approach, the
connection to the
plasma chamber is made through a molded insert placed in a recess of the
vacuum chamber
door seal. Routing the vacuum hose, and any feed lines, through this insert
thus allows the
disposable to be mounted and dismounted quickly and easily.
The vacuum chamber trap in this approach thus captures any immersion liquid,
and
also provides an additional safety measure should the plasma container fail.
2o The vacuum chamber trap also provides a convenient mounting location for
the
vacuum gauge that is required to monitor the process. Specifically, this gauge
must be
mounted on the pump side of the trap and sterile filters for protection from
any aspirated
material.
With this arrangement, the system can be operated in batch, semi-continuous,
or
continuous mode, as described in the previous embodiments.
In batch mode, the system is evacuated simply by closing the vacuum chamber
and
turning on the vacuum pump. If the system is not pre-filled, the vacuum then
draws the fluid
to be treated into the processing chamber. Next, a vacuum sensor and relay
activate the
ultrasound at the selected vacuum level. Degassing and cavitation are then
performed as
3o described above, with the process continuing for a preset time. At the
conclusion of this
process, the vacuum is released, and the processed material is removed. After
fitting a new
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disposable unit, the system is then ready for another operation.
Semi-continuous operation is performed in a like fashion, except that the
filling is
done in steps from a large reservoir, and the disposable is not changed at
each cycle. In this
case, the processed material is collected in a second reservoir at the end of
each cycle.
Finally, after draining the input reservoir, there will be some residual
material left in the
processor. To achieve maximum yield, with minimal residual material to pose a
biohazard
waste product, the treatment unit is then tilted slightly to drain the
residual product. This
action can be driven by a pneumatic piston, an electric motor, a solenoid,
etc.
Continuous operation shares these features, but also requires additional
modifications.
1o The first problem is that a continuous flow precludes the use of variable
intensity and time
pulses of ultrasound on a single batch, as described above. Instead, the
ultrasound treatment
may be achieved by passing the plasma through sequential pools, each pool
having its own
ultrasound sources acting at successively higher powers and longer times. Plug
flow, as
described above in embodiment I, is used in each pool.
Because water is an excellent conductor of ultrasound, however, the pools must
be
acoustically isolated. This is achieved by having the flow cascade from one
pool to the next,
with sufficient space so that the fluid thins out to sheets. Next, these
sheets flow over a saw
tooth pattern of streamers cut from the outlet plate, thus forming a series of
ligaments. Under
the action of gravity and ultrasound, these ligaments then break up into
multiple droplets.
2o The space between these droplets cannot propagate ultrasound, thus
providing the necessary
acoustic isolation between treatment pools.
Once so treated, it is then necessary to remove the fluid without releasing
the vacuum.
One means of achieving this removal is to use a peristaltic pump, which is
quite useful for
those fluids that can withstand the action of such pumps. The limiting factor
here is that
2s conventional peristaltic pumps do not work well in vacuum due to heat
buildup and lubricant
degassing. It is therefore necessary to use either an external driver with an
access port
through the vacuum chamber wall, or a sealed peristaltic pump.
Another means of removal is to collect the treated fluid in a vessel that is
alternately
exposed to vacuum and atmospheric environments by a valve and vacuum pump
3o arrangement. Under this approach, opening the valve at the exit of the
treatment chamber
allows the treated fluid to flow into a collection bag that is also in an
evacuated chamber.
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When this bag is full, the treatment chamber valve is closed, and the vacuum
in the collection
chamber is released. After the collection chamber reaches atmospheric
pressure, the outlet
valve is then released. When the collection bag is completely drained, the
exit valve is
closed, and the collection chamber is pumped back to vacuum conditions. The
process is
then repeated as necessary. This approach thus provides minimum protein damage
during
fluid transfer.
IV. In a fourth main embodiment, the present invention provides a method for
decontaminating a fluid, which comprises:
to (a') a step for the treatment of a fluid with ultrasonic energy, while
degassing the
fluid.
In this fourth embodiment, the step (a') "for the treatment of a fluid with
ultrasonic energy,
while degassing the fluid" may be carried out in the same way as step "(a)
treating a fluid
with ultrasonic energy, while degassing the fluid" is carried out in the
context of the third
main embodiment.
V. In a fifth main embodiment, the present invention provides a method for
decontaminating a fluid, which comprises:
(a) simultaneously treating a fluid with at least two different frequencies of
2o ultrasonic energy.
As described above, ultrasound is quite beneficial in the decontamination of
liquids,
but such treatment has four main problems: protein damage by excessive
cavitation, protein
damage by excessive heat, long processing time, and low effectiveness.
To overcome these problems, it is necessary to improve the delivery of the
ultrasound
to the liquid being treated. The limiting factor here is the interaction of
the ultrasound with
the bubbles in the liquid, which arise from either dissolved gasses or from
vaporous
cavitation of the liquid itself, as described above. From wave mechanics, the
effectiveness of
the interaction between these bubbles and the sound waves depends principally
on the
intensity and the frequency of the ultrasound. As discussed above, higher
intensities promote
3o cavitation of the liquid, while lower intensities promote degassification.
Depending on the
size of the bubble, however, it is also possible for the sound waves to cause
the gas bubbles to
CA 02481144 2004-10-04
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be absorbed entirely back into the liquid. In this case, the growing bubbles
are said to be
unstable.
This size dependence arises because the sound waves induce motions in the
shape of
the bubble wall, as well as motions of the bubble through the liquid. These
motions are
particularly strong when the frequency of the applied sound matches the
resonant frequency
of the bubble, as discussed in U.S. Patent No. 4,597,876. In this patent, a
sweep over
multiple frequencies is proposed so that the growing bubbles are always
subjected to
resonance conditions. In practice, however, bubbles of multiple sizes are
continuously
generated, so that no one frequency is ideal at any time. As such, the
expensive electronics
t 0 and the difficulty in resonance matching of the ultrasound source and the
treatment vessel
over a broad range of frequencies make this approach undesirable.
Another approach is to use two different frequencies simultaneously (see, CP
Zhu, R
Feng, YY Zhao, "Sonochemical effect of a bifrequency radiation," Chinese
Science Bulletin,
vol. 25, No. 2 (Jan), pp. 142-145 (2000)). The advantage of this approach is
that the
combined exposure of two separate sources of greatly different frequencies is
significantly
greater than the sum of the two sources acting alone. The importance of this
result in the
context of the present invention is that the use of multiple frequencies
provides a means of
achieving the same level of cavitation, but with less power applied to the
system. With less
input power, there is less sample heating, and less intense shear and
oscillation around the
bubbles. As a result, the material suffers less damage during sonification,
while also
providing the option of increased total power to improve decontamination
and/or to shorten
the processing times.
An important condition of such multiple source applications is that the sound
must be
applied in orthogonal directions to prevent simple superposition. For this
reason, Zhu et al
used two sources arranged at right angles. For decontamination, this is
preferentially
extended to three dimensions, so that the waves propagate along the
conventional x, y, and z-
axes. Note, of course, that such arrangements can be compromised by multiple
reflections, so
that it is therefore necessary to design the exposure chamber appropriately.
This can be done
using the standard conventions of acoustics, with the addition of wave scatter
at the induced
3o bubbles.
A second consideration in multiple source arrangements is that the frequencies
must
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also be sufficiently great to prevent beating or even prolonged superposition
of extrema. For
this reason, Zhu et al used frequencies in the low tens of kHz range to the
MHz range. The
general relationship is that the waves must be separated by at least an order
of magnitude in
frequency, and preferably also separated in addition by a small constant scale
factor. As
such, suitable separations can be on the order of 15 or 20 or so. Using this
arrangement for a
3-dimensional system, the lowest frequency band is on the order of 20 to 100
kHz, the next
band is on the order of 500 kHz to 1.5 MHz, and the highest band is on the
order of 10 MHz.
As long as the frequency separation is at least an order of magnitude, these
ranges are not
extremely critical; the main limitation in this procedure is simply the
availability of
l0 commercial generating equipment.
While the above work was developed for cavitation, this approach also has
significant
benefits for degassing. For example, it is known that bubble growth under
rectified diffusion
is greatly accelerated under conditions that favor microstreaming and
asymmetric bubble
geometries. It is also known that the Bjerknes force strongly separates
bubbles relative to
their resonance size. Furthermore, under traveling wave conditions, bubbles
can translate
rapidly through a sonified volume. Combined with the diffusion shell
limitations of rectified
diffusion, dissolved gasses are thus released quite rapidly under exposure to
multiple sound
sources.
In practice, this can be achieved using the same geometry and frequencies
required
zo for multiple frequency cavitation. Only the intensity needs to be lowered.
A useful enhancement, however, is to use cavitation power for the highest
frequency,
with or without pulsing. The evolved gas pockets are quite small, and thus
provide
nucleation points for subsequent growth by rectified diffusion under the
influence of the
lower frequency sources.
Finally, it is also possible to extend the above techniques to the reverse
process of
adding a gas to a liquid. This aspect will be discussed below in the ozone
treatment
embodiments.
The other conditions and parameters for application of the ultrasonic energy
to the
fluid may be carried out as described above in the context of the first,
second, third, and
3o fourth main embodiments.
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VI. In a sixth main embodiment, the present invention provides a method for
decontaminating a fluid, which comprises:
(a') a step for the simultaneous treatment of a fluid with at least two
different
frequencies of ultrasonic energy.
In this sixth embodiment, the step (a') "for the simultaneous treatment of a
fluid with at least
two different frequencies of ultrasonic energy" may be carried out in the same
ways as step
"(a) simultaneously treating a fluid with at least two different frequencies
of ultrasonic
energy" is carried out in the context of the fifth main embodiment.
1o VII. In a seventh main embodiment, the present invention provides a method
for
decontaminating a fluid, which comprises:
(a) treating a fluid with ultrasonic energy to obtain a de-oxygenated fluid;
and
(b) irradiating said de-oxygenated fluid.
In this seventh main embodiment, the ultrasonic energy may be applied to the
fluid in
the same way and by using the same apparatus discussed above in the context of
the first,
second, third, fourth, fifth, and sixth main embodiments. In this seventh main
embodiment,
the main role of the ultrasonic energy is to affect degassing of the fluid
prior to exposure to
the radiation.
The purpose of this irradiation is to achieve more effective decontamination
than can
2o be achieved by using the previously described ultrasound techniques alone,
while still
protecting the treated liquids from excessive damage. In terms of prior art,
U.S. Patent No.
3,362,823 describes using ultraviolet light to improve decontamination after
ultrasound
treatment. Later, U.S. Patent Number 4,597,876 describes using ultraviolet
light on liquids
that have been treated with ultrasound and vacuum for degassing purposes.
These patents,
however, do not address the unique problems of protein protection, as
described in the
present invention.
As described above, the major limitation in applying radiation, such as UV,
gamma,
or x-rays, for decontamination is the formation of free radicals of oxygen.
Also as described
above, one possible solution to this problem is to use some type of scavenging
agent, but
3o these agents are expensive and often toxic, and they must therefore be
removed before the
product can be used.
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To avoid this problem, the present invention uses a degassing technology
before
irradiating the liquid. In particular, this technology is directed towards the
removal of
oxygen. Without dissolved oxygen, no oxygen radicals can form under subsequent
irradiation, thereby sparing the proteins being decontaminated.
There are several alternative means of degassing liquids in general industrial
practice.
For example, freezing and boiling are commonly used for degassing, but both of
these
processes cause severe protein damage. It is also possible to use tubes or
membranes as
separation media, with or without a vacuum assist. Because these media are
currently
expensive and clog readily, however, their use is somewhat restricted for most
protein
to solutions. There are also several mechanical devices, such as static mixing
nozzles
(Upchurch Scientific, Oak Harbor, WA) or rotor based systems (Walter P. Nold
Company,
Natick, MA). The mechanical action of such devices, however, is quite damaging
to delicate
materials such as proteins. Alternatively, the oxygen alone can be displaced
by other gasses,
but this is expensive and time consuming. For these reasons, vacuum ultrasonic
degassing
15 techniques have been developed, particularly in terms of treating small
volumes of expensive
reagents (Polaris Degasser, Polaris Instruments Ltd., Cambridge, UK).
As described in embodiments I-VII above, however, applying ultrasound to
protein
systems results in some undesirable damage to the material being treated. In
particular,
embodiment III describes the protein damage expected during degassing prior to
ultrasound
20 decontamination.
This raises the question of just how much oxygen removal is necessary prior to
irradiation. In this regard, chemical quenching agents can reduce, but not
eliminate, radical
damage. The underlying physical principle is that the incoming photon splits
the dissolved
oxygen molecule into two radicals, but these radicals may not be in immediate
contact with a
25 quenching agent. The radicals can therefore contact and damage some protein
molecules
before eventually being inactivated. It should also be noted that in addition
to radicals, there
are other products of irradiated oxygen that are less reactive, but still
damaging. Called
"reactive oxygen species," these high energy oxygen forms are bound with other
molecules,
notably hydrogen. Depending on the type of the quenching agent, and the type
of the oxygen
3o species, quenching agents may have little or no inactivating effects on
such species. The net
result is that quenching agents do not eliminate all oxygen effects, leaving
some radical
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damage and some damage due to other reactive oxygen species. Because quenching
agents
are known to be effective even with these limitations, it is therefore not
necessary that the
new degassing technology remove all of the dissolved oxygen to be effective in
protecting the
proteins during irradiation.
In practice, the actual amount of residual dissolved oxygen must be determined
on an
individual basis. One factor to be considered in this determination is the
damage that the
degassing process itself inflicts upon the proteins. When performed under the
procedure
described above, however, most materials suffer little or no damage during
degassing, at least
for moderate oxygen removal. At progressively lower oxygen concentrations,
however, there
1o is the possibility of some protein damage, especially for labile clotting
factors. Conversely,
there is less protein damage due to radical formation at such lower oxygen
concentrations.
Finally, progressively more time and expense are required to reach
progressively lower
concentrations. The net result is that there are several competing factors to
be considered in
any practical application.
Applying these considerations to blood products, it is generally helpful to
keep
process losses at less than 10%, and preferably less than 5%, which is more or
less the
accuracy of the measuring instruments. Before processing, blood products have
normal
oxygen levels on the order of several ppm; the actual concentration depends on
the individual
product, the temperature, the type of storage bag, the length of storage, etc.
With the
2o previously described degassing techniques, the dissolved oxygen
concentration can easily be
decreased to about 1 to 2 ppm in 5 minutes or less, depending on the starting
temperature and
oxygen concentration. To achieve concentrations in the hundreds of ppb range,
however, the
processing time increases to about 30 minutes. As will be described more fully
below, it is
possible to treat the degassed material with several different radiations. The
result of such
irradiation is about 50% protein damage for untreated plasma. The protein
damage is less
than 10% for the 1 to 2 ppm samples, and there is progressively less damage,
down to the
limit of machine accuracy, in the ppb dissolved oxygen levels.
Of course, at dissolved oxygen concentrations in the ppb range, only very few
oxygen
radicals are formed under irradiation. Under such conditions, it is possible
that these few
oxygen radicals would be most damaging to the most sensitive components, which
are
typically the pathogens themselves. If so, the residual oxygen may thus
actually have a slight
CA 02481144 2004-10-04
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benefit in terms of decontamination.
With or without such an effect, the target dissolved oxygen range for blood
products
is preferably 10 to 3000 ppb, more preferably 100 to 2500 ppb, and most
preferably 500 to
2000 ppb.
Incidentally, it should be noted that in the determination of these limits,
conventional
electric resistance-based dissolved oxygen meters are not accurate because the
samples are
too small and there is inadequate flow to ensure representative reactions at
the electrodes.
This flow limitation is particularly important in the ppb range, where a
significant amount of
liquid must be tested in order to obtain enough oxygen for an accurate result;
otherwise, false
1o low readings will occur as the local environment is depleted of the few
oxygen molecules
present. Because of these limitations, an optical absorption meter (Model VVR,
CHEMetrics
Calverton, VA) with appropriate dilution factors should therefore be used to
determine the
actual dissolved oxygen concentrations.
Having thus determined the means and the appropriate level of degassing, the
15 remaining concern is the radiation to be used for the subsequent
decontamination.
Specifically, it is necessary to select the type of radiation, the required
dosage of this
radiation, and the means of applying this radiation to the material to be
treated.
In this regard, gamma radiation is commonly used in the decontamination
industry,
typically from Cobalt-60 or Cesium-137 sources. In either case, the required
dosages are
2o known for most pathogens, but for general decontamination work, a dosage
must be selected
that will treat most pathogens, without causing excessive protein damage. An
appropriate
test virus must therefore be selected; the conditions that inactivate this
virus are then
considered to be adequate for other pathogens as well.
A particularly useful test subject is parvovirus. This small, non-enveloped
virus is
25 quite difficult to inactivate, thus assuring destruction of less robust
viruses such as HIV. In
its porcine form, parvovirus is harmless to humans, and is therefore easy to
handle in the
laboratory. Furthermore, because of its potential damage to a developing human
fetus, and
because of its ease of transmission by transfusion, the human form of this
virus is of clinical
significance. For these reasons, parvovirus is therefore commonly used as a
benchmark for
30 inactivation technologies (SI Miekka et al, "New methods for inactivation
of lipid-enveloped
and non-enveloped viruses," Haemophilia 1998 Jul;4(4):402-8).
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On the basis of this benchmark, the appropriate dosage range for gamma
radiation of
blood products is between 1 to 100 kGy (kiloGray), more preferably 2 to 60
kGy, and even
more preferably 4 to 40 kGy.
In practice, it is quite easy to obtain such dosages with gamma radiation
because the
high energy photons are quite penetrating, and can thus be readily used
without concern for
shadowing or incomplete local exposures. In the present invention, this
capability allows
great flexibility in the design of the exposure chamber. One option is to
irradiate the liquid
while it is still in the vacuum chamber. In this case, the radiation source is
placed outside the
sterile tent arrangement described above. As such, the source can be placed
inside the
1o vacuum chamber, or outside the vacuum chamber with access through a thin,
low-absorption
window.
Another option is to irradiate the liquid after the chamber vacuum has been
released.
In this case, a valve is placed on the tube leading to the processing tent.
Closing this valve
after the tent and vacuum chamber have been evacuated and sonified allows the
chamber to
~ s be returned to atmospheric conditions, while still maintaining a vacuum on
the processed
material. As such, the flexible bag then simply collapses upon the essentially
incompressible
fluid being treated. The advantages of this approach include very simple
shielding and
control arrangements. The only constraint is that the treatment bag must be
relatively
impermeable to oxygen so that the irradiation is completed before the
dissolved oxygen
2o concentration rises to unacceptable levels by diffusion from the
surrounding air.
Alternatively, the irradiation must be completed before significant amounts of
oxygen pass
through less impermeable bags.
In either of the above options, batch and semi-continuous operations can be
readily
achieved by simply irradiating the target for a specified time, with
continuous monitoring of
25 the applied radiation by conventional detectors and recorders. For
continuous operation,
however, the previously described plug flow must be constrained in a closed
channel
arranged so that the fluid is continuously rising in height. This constraint
ensures that all of
the fluid has sufficient residence time in the exposure chamber to receive
full treatment;
otherwise, the fluid could flow out of the channel too rapidly under the force
of gravity. The
3o driving force for the upward flow can be a pump, or a gravity head from the
higher-placed
vacuum chamber.
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The type of force is particularly important for the cellular components of
blood that
are readily damaged by most mechanical pumps, as noted above. In this regard,
gravity
feeding thus provides little or no such damage. For this reason, the present
invention is well
suited for the leukoreduction of cellular blood components. In addition,
leukoreduction by
gamma irradiation avoids the problems of filter clogging, long processing
times, high
expense, and the related difficulties that complicate existing, filter-based
technologies.
Furthermore, an additional advantage in the present invention is that
degassing prior to
leukoreduction prevents oxygen radical attack on the walls of the leukocytes,
and thus
prevents the leakage of the cellular contents that are quite detrimental to
erythrocytes. Note
in this application that although cellular blood components require oxygen,
these cells can
survive in an oxygen-depleted environment long enough (several minutes) for
decontamination. Oxygen can then be supplied to the fluid after the
irradiation is completed.
The above advantages of gamma exposure are particularly significant in terms
of the recent
recommendation for universal leukoreduction (Wall Street Journal, "Federal
Panel Hears
Debate On Filtering All Donated Blood," 1/25/2001).
Unfortunately, one potential problem in the above decontamination processes is
the
formation of aggregates, particularly from albumin, under gamma irradiation.
These
aggregates are undesirable because they not only reduce the amount of usable
protein, but
they also require a separate, time-consuming step for removal. In the present
invention, such
aggregates are therefore destroyed by ultrasound. The underlying principle
here is that
ultrasound can be used to agglomerate or disperse solids in a liquid,
depending on how the
sound is applied. In the present invention, the irradiation chamber is
therefore sonified
uniformly along the chamber length during irradiation, thereby disrupting any
small
aggregates as soon as they form.
Another potential problem with gamma irradiation is the formation of reactive
species
from molecules other than oxygen. In particular, gamma radiation has
sufficient energy to
dissociate and/or excite water molecules. To reduce this effect, protein
concentrates can be
used. PCT Application number WO 0016872 describes a procedure for generating
such
concentrates. Because concentrates contain relatively little water by
definition, the number of
3o reactive water species is reduced proportionately, compared to the original
protein solutions.
Being an electromagnetic radiation of the same energy as gamma radiation, x-
rays can
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also achieve all of the above benefits. Unlike the nuclear decay sources used
for gamma
irradiation, x-rays are instead generated by high voltage, electron
acceleration sources.
Because of the energy requirements and maintenance costs of such accelerators,
however,
gamma radiation is currently the preferred of these two radiations for
decontamination work.
Unfortunately, both gamma and x-ray sources are not only relatively expensive,
but
they also require strict shielding, licensure and other constraints.
For these reasons, UV sources are more commonly used for decontamination.
There
are four Types of UV radiation, which in terms of increasing energy are UVA,
UVB, UVC,
and VUV. UVA and UVB are the relatively weak radiations associated with sun
exposure.
Being too weak to induce radical formation, these radiations unfortunately
require some type
of light-activated chemical to induce decontamination. As described above,
however, the
intent of this invention is to avoid the costs of any such chemicals, so these
radiations are not
useful here by themselves. Conversely, VUV, or vacuum ultraviolet, is much
more energetic,
being on the border of soft x-rays in terms of energy. VUV, however, is
difficult to generate
and is absorbed so readily that it is not practical for decontamination work.
UVC is therefore the preferred form of ultraviolet radiation in the present
invention,
particularly the UVC produced by mercury sources. These sources are useful
because they
emit light mostly at about 254 nm. Because these sources thus have relatively
low emissions
at wavelengths less than the 185 nm threshold required for ozone generation,
they produce
little of this undesirable gas. More importantly, however, this wavelength is
in the middle of
a local minimum of absorption that exists in the range of 250 to 260 nm for
most proteins, but
both DNA and RNA have a local absorption maximum in this range. For this
reason,
mercury lamp emissions thus selectively attack the DNA and RNA found in most
contaminants, while sparing the other proteins in the material being treated.
In particular, this
selectivity is most useful for treating blood components because, as discussed
earlier, most
blood components lack the genetic materials DNA and RNA.
The exception is that leukocytes do contain these materials, but in this case,
UVC
irradiation is still of benefit in terms of leukoreduction. Specifically, in
this application, UVC
has all of the clinical advantages described above for gamma rays, as well as
the advantage of
3o selective protection of the surrounding proteins.
Having thus selected UVC as the ultraviolet radiation of choice, appropriate
dosages
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must then be determined. There is a great deal of data on UVC dosages in terms
of water
purification and food processing, but protein solutions have unique
requirements, notably
regarding the types of contaminants.
In terms of blood products, parvovirus is of particular interest, as described
above.
s Specifically, H. Sugawara has recently summarized the current state of the
art of UVC
inactivation of this virus in regard to plasma products (H. Sugawara et al,
"Inactivation of
parvovirus B19 in coagulation factor concentrates by UVC radiation: assessment
by an in
vitro infectivity assay using CFU-E derived from peripheral CD34+ cells,"
Transfusion 2001
Apr;41 (4):456-61 ).
1o The net result of this and similar studies is that the UVC irradiation
should preferably
be on the order of 1 to 10,000 J/mz, more preferably 10 to 5,000 J/m2, and
even more
preferably 100 to 3,000 J/mz.
Having thus determined the appropriate dosages, the next concern is how to
obtain
this irradiation with minimum protein damage. The problems to be addressed are
uniformity
t s of exposure, excessive heat, the formation of aggregates, and the recovery
of pathogens.
Beginning with the uniformity of the exposure, several dimensions must be
considered. First, the treatment chamber must be of uniform thickness, as
discussed above
for the ultrasound system. Also as discussed for ultrasound systems, moving
systems must
have plug flow as described in first main embodiment.
2o Unlike ultrasound systems, however, the depth of the treatment chamber is
critical for
UVC systems. The underlying problem is that the optical absorption in the
treated liquid
follows an exponential curve according to Beer's Law. As such, the exposure on
one side of
the treatment chamber can be much less than the exposure on the other side of
the chamber,
even for relatively thin units. In the present invention, the UVC irradiation
is therefore
2s preferably applied from both sides of the treatment chamber. Under this
arrangement, the
exposure sum from the two opposing sources is thus nearly constant for
moderate target
widths.
In addition to the liquid, the walls of the treatment chamber can also absorb
UVC.
For this reason, these walls must also be of uniform thickness, and of UVC
transparent
3o material, preferably fused quartz.
Of course, uniform exposure also requires at least some uniformity in the UVC
source
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itself. In this regard, spot lamps and tubes generate only relatively small
zones of uniform
exposure. Conversely, grid lamps, which consist of long, thin, tightly coiled
tubes, yield
much more uniform exposures. Such lamps (Model LF 1805, UVItec, Cambridge, UK)
are
therefore used in the present invention.
In addition to the above shape uniformity, the lamps must also have reasonable
uniformity over time. The fundamental problem here is that all UV equipment is
subject to
"solarization," which is essentially degradation due to the high energy of the
generated
photons. As a consequence, the applied dose can be inadequate for
decontamination if a
simple timing procedure is used to control an aged lamp. It is therefore
preferable to use an
1o integrating monitor (Model RX 003, UVItec, Cambridge, UK) to ensure that
the specified
exposures are actually maintained over the lifetime of the lamp. Furthermore,
placement of
such a monitor beside each lamp thus ensures that a bank of lamps functions as
intended. In
this case, at least one monitor should also be placed opposite at least one
lamp, beyond the
liquid being treated, to ensure that the appropriate absorption levels are
maintained; this is a
15 particularly important consideration for blood products, in which the
optical properties often
differ greatly from sample to sample.
Unfortunately, all UVC lamps produce significant amounts of heat, and while
grid
lamps produce relatively little heat compared to their quite high UVC
intensity, their heat is
still a problem. Part of this heat can be controlled by fan-assisted
convection over the lamps.
2o This approach, however, does not limit the infrared component from the
lamps, which is
absorbed directly within the target. For this reason, a flow or spray of water
is therefore used
to cool the target, since water is a very poor absorber of UVC light. To
prevent this cooling
water or spray, or any leaking contaminated fluid, from falling into the lamps
and causing
electrical problems or breakage, the treatment module and lamps should be
oriented
25 vertically. This geometry also aids the maintenance of controlled flow for
continuous
systems, as discussed earlier.
Even with uniform UVC exposure and two cooling mechanisms, there is
nevertheless
a possibility of forming protein aggregates at isolated points in the treated
liquid. As
described above for gamma irradiation, however, these aggregates can be
eliminated by
3o exposure to ultrasound.
The final problem with UVC treatment is that after the sources have been
turned off,
46
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there can be some recovery of the pathogens, apparently by an inherent repair
mechanism.
Recent work with discharge lamps, however, (see, WH Cover, "Effect of Broad
Spectrum
Pulsed Light (BSPL) on Platelet Function," Cambridge Healthtech Eighth Annual
Symposium on Blood Product Safety, February 4-7, 2002) indicates that other
wavelengths of
light can inhibit such recovery. Unfortunately, discharge lamps also produce a
great deal of
heat. Since mixtures of UVB and UVA are known to be more effective than either
acting
alone, and since the UV wavelengths are the most energetic in the emission of
discharge
lamps, a preferred approach is to include UVA and/or UVB sources in the
treatment protocol
to suppress pathogen repair. Since these wavelengths do not induce radical
formation, their
t o inclusion thus causes no intrinsic protein damage, and their infrared
contribution can be
readily handled by the existing cooling system. In addition, any polymer that
is transparent
to tJVC is also transparent to UVA and UVB, so the design considerations
reduce to only
building a system capable of UVC treatment.
With the above considerations, UVC systems can be built for a variety of
operating
conditions and contaminated liquids. The overall approach is similar to that
described above
for gamma irradiation systems, and can thus be used for batch, semi-
continuous, and
continuous operation. The major difference is the limited penetration
capability of UVC
versus gamma irradiation, which leads to several concerns in regard to bag
design.
The primary consequence of this limited UVC penetration range is that the
disposable
2o treatment bags must be made of UVC transparent material. In particular,
Teflon~ and
similar fluoropolymers are quite transparent to UVC, and are thus the
preferred materials;
however, they are somewhat expensive and permeable to oxygen. For these
reasons, thin
films of other polymers, such as ethyl vinyl acetate, can be laminated with
Teflon~ to
provide a reasonably cheap, strong, oxygen impermeable, UVC transparent
container. Also,
performing the exposure rapidly ensures that the decontamination is complete
before
significant amounts of oxygen can diffuse into the system.
Another problem in terms of treatment bags is that Teflon~ and other
fluoropolymers are difficult to seal with the inclusions that are required for
sterile blood
product ports and vacuum access. This problem is of little consequence when
the degassing
3o and UVC exposure processes are done in the same bag. On the other hand,
when these
processes are performed in separate bags, the degassing step should be done in
any cheap,
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easily sealed bag material such as PVC, while the UVC exposure should be done
in a higher
cost, harder to seal, but UVC transparent, bag.
Yet another bag problem is that the access ports and connecting tubes are
typically
not UVC transparent, and can thus shield the pathogens from UVC exposure. The
solution to
this problem is similar to that described in embodiment I for ultrasound feed
ports, except in
this case any clamping must be done by UVC transparent materials to prevent
shading
effects. For example, the clamps can be made of fused quartz, but for greater
mechanical
strength, polymers such as Teflon~ AF are preferred. Yet another alternative
is to use
stainless steel or other metallic pincers, tapered to reduce end shadows. In
this case, the
l0 clamping should be done in a section of the bag that is fully illuminated,
and also elevated to
drain the fluid away from the clamping location. Under this arrangement, the
available light
will be minimally attenuated, thereby providing maximum treatment at the clamp
location.
The final bag problem is that for relatively large treatment volumes, the
thickness
must remain small, and thus the surface area must be large. Beyond the matter
of costs, this
restriction also causes substantial hydrostatic pressures, particularly for
the vertically oriented
systems described earlier. One alternative is to use very heavy quartz plates
to contain the
treatment bags, but this approach is expensive and causes undesirable
attenuation of the UVC
light. It is therefore preferred that the center sections of the bags be
joined front to back, thus
reducing the loading on the fused quartz plates. In addition, it is also
preferred that the
2o boundary seal of the treatment bags include holes to match registration
pins. These pins can
be either permanently mounted in the periphery of the treatment chamber, or
mounted in a
plastic frame that allows for rapid insertion and removal of the bag assembly.
These considerations are particularly important for erythrocyte treatment. In
this
application, the strong attenuation of UVC by the heme groups in the
erythrocytes restricts
the fluid path in the treatment chamber to a thickness of about 30 to
60~microns. Even though
this layer is thus very thin, the associated hydrostatic pressures are
nevertheless still quite
high. The joints in the center are thus not only useful in this application,
but they also
provide a means of mixing the flowing liquid. This mixing is achieved by
staggering several
consecutive joints, thereby splitting the fluid flow accordingly.
3o Unfortunately, as described earlier, such flow mixing is relatively weak
except under
the turbulent conditions that are quite detrimental to blood proteins. As
such, whatever
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mixing is available by the addition of flow restrictions is inadequate to
prevent the shading
effect among multiple erythrocytes. For this reason, U.S. Patent 6,219,584
describes
vibrating the treatment chamber.
In the present invention, ultrasound is used to provide extremely effective
vibration.
The underlying principle is that ultrasound can agglomerate or disperse solids
in a liquid, as
noted earlier in the discussion of aggregate treatment. For maximum
effectiveness in the
present invention, the vibrating ultrasound is provided by a horn in direct
contact with the
treatment bag. Specifically, this horn should be in contact with the bottom of
the treatment
bag, thus ensuring adequate vibration for even partially filled bags. This
approach thereby
i o avoids vibrating the entire treatment chamber, which would be difficult
due to problems in
impedance matching, as well as possible chipping of the expensive fused quartz
plates.
As noted in US patent 5,997,812, the addition of ultrasound to a liquid being
irradiated by UV not only improves mixing, but also improves the "killing
effect of UV
radiation."
VIII. In an eighth main embodiment, the present invention provides a method
for
decontaminating a fluid, which comprises:
(a') a step for the treatment of a fluid with ultrasonic energy to obtain a de-
oxygenated fluid; and
(b') a step for the irradiation of said de-oxygenated fluid.
In this eighth main embodiment, the step (a') "for the treatment of a fluid
with ultrasonic
energy to obtain a de-oxygenated fluid" may be carried out in the same ways
that step "(a)
treating a fluid with ultrasonic energy to obtain a de-oxygenated fluid" is
carried out in the
context of the seventh main embodiment. In addition, the step (b') "for the
irradiation of said
de-oxygenated fluid" may be carried out in the same ways that step "(b)
irradiating said de-
oxygenated fluid" is carried out in the context of the seventh main
embodiment.
IX. The inventor has further discovered, in a ninth main embodiment, that such
fluids
may be effectively decontaminated by a method involving:
(a) treating a fluid with ultrasonic energy to obtain a de-oxygenated fluid;
and
(b) contacting said de-oxygenated fluid with a pulsed electric field.
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In this ninth main embodiment, the de-oxygenation of the fluid may be carried
out as
described above. After the fluid has been de-oxygenated, it is then contacted
with a pulsed
electric field (PEF).
The underlying concept of PEF is to use short pulses (tens of microseconds) of
very
high voltage (tens of kV) electric fields to decontaminate temperature
sensitive materials.
This approach is commonly used in the food industry, particularly for
bacteria, parasites, etc.
The limiting factor is that the target pathogen must be sufficiently large to
establish a voltage
gradient. While this limitation effectively excludes pathogens on the size of
viruses or
smaller, many pathogens can nevertheless be treated by PEF.
1o A major limitation of PEF is that the sample may break down during
treatment. In
particular, dissolved gasses readily cause breakdown. For this reason, Q.H.
Zang has
described the benefits of degassing apple cider prior to PEF treatment
http://www.fst.ohio-
state.edu/hS/pef/s1d027.htm slide 27/91 (1998). Under this approach, many more
pulses can
be applied to the sample before electrical breakdown, thus improving
decontamination
without product degradation.
This embodiment relies on a synergistic effect between the application of
ultrasonic
energy and PEF. The underlying principle of PEF is that the various fields can
act on
charged species, or even upon polar molecules.
Constant electric and magnetic fields are the simplest to analyze and
implement.
2o Beginning with electric fields, there is a long history of applying a
current through a
contaminated liquid to affect some kind of cleaning treatment. The overall
approach is
simply to put two electrodes on opposite sides of a pool of liquid and then
apply electricity.
The essential problem of this approach is that electrochemical reactions,
primarily at
the electrodes, can contaminate the product. This is a critical concern for
biological
materials, such as plasma, that will be used for medical treatment.
A new means of avoiding this problem is to use a salt bridge across a sterile
filter to
couple the electrodes to the fluid to be treated. In a further enhancement, a
thin tube leads
from the filter and salt bridge, extending to the sides of the treatment bag.
For further
protection, a flow restriction is placed at the juncture of the tube and bag.
After treatment,
this tube is then heat sealed at the flow restriction, and the filter and salt
bridge may then be
discarded.
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Under this arrangement, undesired compounds are first trapped at the leading
edge of
the salt bridge. Any residual compounds that escape this trap are then caught
in the
connecting tubes before they reach the bulk of the fluid.
An immediate extension of the constant electric field is the pulsed electric
field, or
s PEF. PEF typically involves very high voltages, on the order of 20kV, but
very short
duration. In particular, PEF and ozone are known to have a synergistic effect
(R Unal, JG
Kim, and AE Yousef, "Inactivation of Escherichia coli O1 57:H7, Listeria
monocytogenes,
and Latobacillus leichmannii by combinations of ozone and pulsed electric
field," J. Food
Prot., Jun;64(6), pp. 777-782 (2001)). In this embodiment, PEF is therefore
combined with
1o the above salt bridge and tube arrangement.
Like constant electric fields, strong magnetic fields have also been used for
several
decades in decontamination work. Recently, strong magnetic fields have been
combined with
UV irradiation (US Patent No. 5,997,812). This technique, however, does not
apply well for
biological systems without magnetically susceptible materials.
Is The most advanced form of electric and magnetic treatment is of course the
electromagnetic field, as noted above for UV, gamma and x-rays. Synergistic
effects with
ozone have also been noted (MW Byun et al, "Gamma irradiation and ozone
treatment for
inactivation of Escherichia coli 0157:H7 in culture media," J. Food Prot.,
Jun; 61(6), pp.
728-730 ( 1998)). The net result is that the new technology has multiple
opportunities for
2o synergistic effects. In particular, ultrasound and PEF can be applied
either together or
separately during either or both the UV and the ozone steps described below.
In terms of plasma treatment in the new technology, the preferred location to
apply
PEF is thus immediately after the degassing step. As such, PEF can be done
before, during,
or after UVC or gamma irradiation (as described below). In particular, it
should also be
25 noted that the improved speed of ultrasonic vacuum degassing is of immense
use in the PEF
treatment of foodstuffs.
X. The inventor has also discovered, in a tenth main embodiment, that such a
fluid :nay
be effectively decontaminated by a method involving:
30 (a' ) a step for the treatment of a fluid with ultrasonic energy to obtain
a de-
oxygenated fluid; and
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(b') a step for contacting said de-oxygenated fluid with a pulsed electric
field.
In this tenth main embodiment, the step (a') "for the treatment of a fluid
with ultrasonic
energy to obtain a de-oxygenated fluid" may be carried out in the same ways
that step "(a)
treating a fluid with ultrasonic energy to obtain a de-oxygenated fluid" is
carried out in the
context of the ninth main embodiment. In addition, the step (b') "for
contacting said de-
oxygenated fluid with a pulsed electric field" may be carried out in the same
ways that step
"(b) contacting said de-oxygenated fluid with a pulsed electric field" is
carried out in the
context of the ninth main embodiment.
XI. In an eleventh main embodiment, the present invention provides a method
for
decontaminating a fluid, which comprises:
(a) treating a fluid with ultrasonic energy to obtain a de-oxygenated fluid;
and
(b) contacting said de-oxygenated fluid with ozone.
As described earlier, decontamination is best achieved by applying multiple,
independent processes. Under this approach, the pathogens that escape one
decontamination
technique may not escape a second, or third, technique etc. In addition, any
given technique
can destroy only a limited number of pathogens before it also causes a
significant amount of
protein damage, so it is preferable to use multiple techniques at partial
power instead of one
technique carried to extreme limits. It is therefore desirable to integrate
the above
2o technologies with yet another independent technique. A particularly useful
such technique is
ozone exposure.
Ozone is a triatomic molecule of oxygen, while the common form of oxygen is
diatomic. As a result, ozone is an unstable molecule and is thus extremely
reactive. In
particular, this high reactivity makes ozone an extremely strong
decontamination agent. The
underlying mechanism is that ozone rapidly attacks the complicated protein
structures that
pathogens require to propagate, thus causing rapid inactivation. An additional
benefit is that
after it has reacted, ozone then reverts to non-toxic molecules that are
naturally present. As
such, ozone and its products do not have to be removed from the treated
material, thus saving
a separate, expensive, and time-consuming step that is typically required for
other
decontaminating agents.
For these reasons, ozone has been used for many years in a variety of
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decontamination devices. Some of these applications involve treatment of a
given area or
volume by gas exposure, such as a room or a device inside an enclosure. While
these
applications are quite numerous, the main concern of the present invention is
liquid
treatment, whereby the ozone is absorbed into an aqueous solution. Such
aqueous solutions,
in turn, have many particular applications. In this regard, the most common
use of ozone
decontamination is for processing water, including both potable water
treatment and pollution
control. While some aspects of the present invention are applicable to such
processes,
however, the main application here is the decontamination of biological
products, particularly
blood products. But even in this somewhat restricted discipline, several such
devices have
already been disclosed.
For example, U.S. Patent No. 4,632,980 discloses an ozone blood treatment
device, in
particular a technique for controlling damage to blood products while
preferentially attacking
enveloped viruses. There are, however, several problems with this patent,
beginning with the
restriction to enveloped viruses. Specifically, while enveloped viruses such
as HIV were of
primary concern in the early 1980's when this patent was under development,
the subsequent
development of advanced viral testing and the emergence of more non-enveloped
viruses
have greatly changed the needs of the blood industry. Another problem is that
although this
patent mentions pH, the pH of blood and blood products depends strongly on the
choice of
anticoagulant, and as discussed later, the pH strongly affects the behavior of
dissolved ozone.
Another significant problem is that the disclosed device uses a glass roller
chamber, but
since the clotting sequence can be initiated within glass vessels, such
materials should
therefore not be used for blood products. Furthermore, rollers of any material
are inherently
slow. Finally, the glass vessel and the associated seals would be expensive,
difficult to store,
and difficult to destroy once used.
Another ozone device for blood treatment is disclosed in U.S. Patent No.
5,709,992.
The main feature of this patent is a method for protecting red blood cells
from ozone damage
by adding reducing enzymes. As discussed below, however, red blood cells
already have
some intrinsic protection. Furthermore, as noted many times above, in blood
work the added
materials are commonly removed before use of the treated material, at
considerable time and
3o expense. Finally, the 48 hour processing time is simply too long to be
accepted in normal
blood bank operation.
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An alternative approach is described in U.S. Patent No. 5,882,591, which
discloses a
spraying system. The advantage of this system is that the finely divided spray
promotes rapid
deactivation. There are, however, several possible problems with this
approach. In
particular, while the inactivation of the contaminants in the small droplets
is indeed quite
rapid, the overall process of converting a large volume of fluid into a spray
is not rapid. As
such, the total process is too slow to be used in a blood bank environment.
Another concern
is the mechanical damage due to the spraying process, which increases as the
droplet size
decreases. Finally, there is also the concern of the confinement of the spray
itself: aerosols of
potentially contaminated blood products are usually avoided because of the
danger of
infection. While a number of traps could be developed, they would be expensive
and not
completely effective. In a large blood processing facility, the cumulative air
loading would
thus be quite dangerous.
All of the above techniques are essentially in vitro applications, in which
the treated
material is collected for later use. However, ozone can also be used for XCT,
or
extracorporeal treatments, as disclosed in U.S. Patent No. 6,027,688. In this
device, blood is
withdrawn from the patient, treated, and then re-infused, with the intent of
reducing the HIV
burden. One problem is that this device is quite complicated and would thus be
expensive to
buy and operate. In addition, this device also has a glass treatment tube,
which, as discussed
above, could cause severe clotting, and thus lead to pulmonary embolism and
death. Finally,
even with long processing times, the disclosed 99% (or log 2) viral reduction
is quite small
compared to the log 6 or 7 that is desired.
The net result of the above and similar works is that ozone is a quite
effective
decontamination agent for protein solutions, but a major limitation is the
overall speed of this
process. It is therefore necessary to develop a faster ozone treatment
technique. This
development can be realized using basic ideal gas laws.
The first such consideration is the concentration of the input ozone gas:
higher
concentrations are preferable because they yield more collisions among the
reacting species,
and thus yield more product in less time. The limiting factor here is that
gaseous ozone
concentrations of greater than 20% are explosive. At lesser concentrations, a
number of
practical concerns limit the effective concentrations that can be achieved.
The main such
concern is that ozone is so reactive that it cannot be stored for prolonged
time periods.
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Instead, ozone is typically generated at the site where it is to be used.
There are three major means of ozone generation: ultraviolet light exposure,
corona
discharge and chemical reaction (Handbook of Ozone Technology and A~nlications
Volume
One, R. G. Rice and A. Netzer, Eds., Ann Arbor Science, The Butterworth Group,
Kent,
s England, 1982).
Ultraviolet light sources function by first splitting diatomic oxygen
molecules into
singlet oxygen, which then reacts with other diatomic molecules to form
triatomic ozone.
Although this process is the origin of the earth's ozone layer, ultraviolet
exposure is
inefficient. The underlying problem is that while certain frequencies of light
are quite
effective in generating ozone, other frequencies are almost as effective at
dissociating the
formed ozone. As a result of these competing processes, UV units are limited
to
concentrations of less than 1%. Although UV sources are quite clean and easy
to control, this
low ozone concentration limits their use in decontamination work.
An alternative ozone generation technique is corona discharge. In this
process,
~ 5 oxygen is passed through a channel bound by high voltage electrodes. The
resulting
discharge ruptures the diatomic molecules, and some of the resulting, high
energy single
oxygen molecules react with some of the neighboring oxygen molecules to form
ozone.
Typical yields are in the range of 1 to 15% by volume ozone.
Unfortunately, there are several problems with corona discharge systems. One
such
2o problem is that the feed gas may also contain nitrogen, water vapor, or
other gasses. If so,
there is a possibility that molecules other than oxygen and ozone may
contaminate the
product. Medical applications of discharge systems therefore typically use
high grade oxygen
as a feedstock, but this entails additional costs. In addition to this gaseous
contamination,
there is also the possibility that degradation of the electrodes may introduce
solid
25 contaminants into the product; therefore, expensive filters are required.
Furthermore, the
eroding electrodes also produce electromagnetic noise, which is undesirable in
a medical
environment. Yet another problem with discharge systems is that the resulting
gas is so hot
and dry that it can damage the proteins being treated. Finally, electric
discharges are difficult
to control, particularly at partial load operation. For these reasons, corona
discharge units can
30 be used for decontamination work, but a great deal of conditioning is
necessary.
An alternative approach is to generate ozone by various chemical reactions.
For
CA 02481144 2004-10-04
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example, U.S. Patent No. 5,709,992 discloses one such technique for adding
activated
ceramic particles directly into the pool to be treated. A much more promising
approach,
however, is to use an electrochemical technique. For example, U.S. Patent No.
5,989,407 by
Lynntech, Inc. (which is incorporated herein by reference) discloses a device
that produces
s ozone at concentrations of 10 to 1 S%. This device works on the principles
of electrolysis of
water, thereby avoiding the cost of expensive medical grade oxygen as a
feedstock while also
avoiding the problem of electromagnetic noise. In addition, the ozone produced
by this
device is self pressurizing, relatively cool, and fully humidified.
Because of these advantages, electrochemical units are currently the preferred
ozone
1 o sources for the present invention. In the following, several unique
features of electrochemical
ozone generators are described in detail, along with the modifications and
extensions that are
necessary that are necessary to exploit these features in practice.
Beginning with self pressurization, the underlying ideal gas law here is that
all
gasses, including ozone, are much more soluble at elevated pressures. As such,
an increase in
15 pressure therefore results in more ozone in solution, and thus more rapid,
more effective
treatment.
To achieve these benefits, the ozone must be either generated at elevated
pressure or
generated at lower pressure and then compressed. Because compressing ozone is
difficult
and expensive, the above-noted ability to generate ozone at pressure is a
major advantage of
20 electrochemical ozone units.
Unfortunately, the available electrochemical units are not yet capable of
generating
ozone at pressures beyond about 50 psi. It is therefore necessary to compress
the gas to reach
higher pressures. The preferred means of compressing ozone is a diaphragm pump
(BA
series, Fluitron, Ivyland, PA). Diaphragm pumps are useful because they have
no seals that
2s can be destroyed by ozone contact, and the entire flow path can be
constructed of easy to
clean, chemically inert materials. The limiting factor in the design of such
compressors is
that ozone decomposes at higher temperatures. It is therefore necessary to
start with the
ozone as cold as possible, and then compress the ozone through multiple
stages. At each
stage, the temperature should not exceed about 40 °C. To achieve this
limit, the maximum
3o compression ratio in each stage can be calculated by the standard adiabatic
ideal gas laws.
Water cooling of the compression heads thus ensures that the peak temperatures
are kept well
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below the theoretical upper limit. While effective, such compressors are quite
expensive, and
will be discarded as advances are made in electrochemical generation. In the
meantime, the
output from the existing generators provides a cool, partially compressed
feedstock. For
example, the 50 psi output from an electrochemical ozone generator can be fed
directly into a
two stage diaphragm pump, with each stage operating at a pressure ratio of
1.7:1. The
resulting gas is therefore at a pressure of about 150 psi, with a peak
temperature of less than
35 °C.
Of course, once generated, the ozone must be applied to the material to be
treated.
For blood work, one option is simply to add the pressurized ozone into a
sterile blood bag
1o system. The difficulty here is that conventional bags are not designed to
handle such
pressures. Although new bags could be built, they would be much more expensive
than
conventional units. Even then, should a bag rupture during treatment, it would
then spray
potentially contaminated plasma throughout the laboratory. Finally, from a
practical
standpoint, generating sufficient ozone to pressurize the bag would be
expensive and time-
rs consuming, and would waste much of the valuable ozone that could otherwise
be used for
attacking the contaminants.
For these reasons, a new exposure system is necessary. This system consists of
a
pressure cell, which is driven by a standard air compressor. By pressurizing
this cell with air
to the same pressure as the ozone, the pressure on both sides of the treatment
bags is
20 equalized. Cheap bags can thus be used, there is no risk of rupture, and
the ozone
requirements are greatly reduced. With conventional 110 VAC compression
equipment,
pressures up to about I O atmospheres (about 150 psig) can be achieved easily,
and if desired
even higher pressures can be generated by 220 VAC equipment.
During the time that ozone is not required, such as during bag changes or
ultrasonic
25 vacuum degassing, it is desirable to maintain the ozone source at pressure
so that processing
can be continued immediately when necessary. With conventional gasses, the
pressure is
typically maintained in a simple storage tank. Ozone, however, degrades so
rapidly that this
is not an option. Furthermore, electrolytic units, such as the Lynntech
device, must be
operated continuously for best output.
3o For these reasons, the use of a bypass circuit is preferred. The first part
of this circuit
is a solenoid valve placed at the outlet of the ozone generator. When
activated, this valve
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diverts the ozone around the treatment vessel and through a check valve that
maintains the
desired pressure. The output of this valve is then joined through a y-
connection with the
treatment chamber output. The resulting combined flows then proceed to a
destruct unit that
converts the ozone back to oxygen before venting. Although not necessary for
operation of
the decontamination unit per se, such destruct units ensure that the
decontamination process
does not contribute to low level ozone pollution.
Finally, for electrolytic units, it is necessary to balance the pressure
loading on the
generator cell. Specifically, electrolytic units produce ozone and oxygen on
one side of this
cell, and hydrogen on the opposite side. In practice, the hydrogen back
pressure can be
t 0 maintained simply by a check valve. Downstream processing can then be done
at
approximately atmospheric pressure, using a simple drain trap for water and an
optional
hydrogen destruct unit. This equipment can thus be conveniently arranged
parallel to the
ozone bypass circuit.
While the above system has been described for individual or batch units, the
previously described feeding and emptying procedures for the vacuum operation
embodiments can be readily modified to accommodate continuous flows. The only
significant change is that the described pressure differences must be
reversed.
After pressure, the next concerns are temperature and humidity, which are
inter-
dependent. The potential temperature problem here is that the gas may be so
hot or cold that
it damages the proteins. In addition, the humidity can be so low that the
proteins could be
excessively dried, or so high that the proteins could be diluted with excess
moisture.
For precise control of the ozone temperature, a Pettier system is desirable
(Model
TLC-1400, TECA, Inc., Chicago, IL). Alternatively, conventional refrigeration
and heating
devices can also be used if proper controls are provided (Model RTE, Neslab,
Portsmouth,
NH). Both systems provide a source of either heated or cooled water.
Connecting a heat
exchanger to these devices therefore provides a simple means of regulating the
ozone
temperature.
A particularly simple arrangement is to use a Teflon~ or similar plastic tube
to
connect the ozone source to the treatment unit. Teflon~ is desirable in these
applications
because it is quite resistant to attack by ozone. Although Teflon~ is somewhat
permeable to
gasses, these losses are not excessive. If desired, however, lower
permeability forms of
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Teflon, notably Teflon~ PFA, can also be used. Another alternative is to use a
Teflon~
laminate with a low permeability plastic.
Placing loops of the selected tubing in the controlled temperature bath thus
provides
the desired ozone heating or cooling. Alternatively, metal heat exchangers
could also be
used, but in this case it is necessary to protect the metal surface from
attack by the highly
reactive ozone. An effective protective layer is Teflon. For even faster heat
transfer,
stainless steel tubing can be used. In particular, tubing that has been
treated with nitric acid
rapidly forms an inert layer that resists further corrosion, without greatly
reducing the heat
flow (Stainless steel tubing: Nitric acid treated, Upchurch Scientific, Oak
Harbor, WA).
1 o Immediately downstream of the heat exchanger, a water trap is used to
collect and
remove any condensates in high humidity systems. The problem here is that the
condensed
water must be removed without losing the system pressure. As such, the first
part of the
water trap is a small pressure vessel. This vessel is connected to a scale,
float, optical or
electrical sensor to determine the level of the water in the trap. When the
vessel becomes
filled, a solenoid valve is then actuated to release the pressurized liquid
into a drain. For
complete draining a "flip-flop" circuit is used to keep the valve actuated
during the entire
draining process, with the state of the circuit reversed by a switch placed at
the closing
location. This valve should have Teflon~ flow surfaces to resist attack by the
ozone. Also,
for minimal energy consumption, this valve should be "normally closed."
Downstream of
2o this valve, a flow restriction must be placed in the outlet tube to prevent
excessive spraying at
elevated pressures. An alternative approach is to use a peristaltic pump, if
it is necessary to
transport the liquid to a higher level than the ozone pressure can support. In
either case, the
drain must be closed before the trap is completely empty; otherwise, there
will be some
leakage of the ozone gas.
In the case of low humidity generators, a device to increase the humidity to
the
required level replaces the water trap. In this case, a source of high purity
water is required,
as well as some means to vaporize this water at low temperatures. Sonic
humidifiers are well
suited for this application. Finally, it is also possible to use a heated
vaporization system if
the entering ozone is sufficiently cool, or can be cooled after water
addition.
3o With these combined features, the ozone is thus delivered to the treatment
chamber at
the proper concentration, pressure, temperature and humidity. However, while
these
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conditions are quite effective in decontamination, it is possible to
accelerate the process even
more by incorporating the previously described degassing technology.
The underlying principle here is, again, a matter of basic ideal gas behavior.
Specifically, each gas has its own characteristic solubility in a given
liquid; furthermore,
Fick's first law describes the diffusion of this gas in the liquid, while
Henry's law describes
the concentration of this gas in the liquid, relative to the partial pressure
of this gas above the
liquid.
Under normal conditions, water or a dilute aqueous solution thus has an oxygen
concentration of about 35%, and a nitrogen concentration of about 63%; these
values differ
1o from the respective 21% and 78% values in air because oxygen is more
soluble than nitrogen.
When subsequently exposed to a saturated mixture of 15% ozone and 85% oxygen,
the
nitrogen concentration then decreases as the liquid takes up ozone and oxygen.
Ozone,
however, is about 13 times more soluble than oxygen, so the uptake of oxygen
is more rapid.
On the other hand, ozone reacts with the liquid in which it is dissolved.
Thus, some of the
oxygen in the ozone combines with some of the other components in the
solution, and some
of the remaining oxygen reverts to the normal, diatomic form. In either case,
the incoming
ozone eventually completely reacts to a lower energy form, leaving a
decontaminated liquid
that is enriched with oxygen, and depleted from other gasses.
Although the above sequence thus describes the events that occur in
conventional
ozone decontamination units, in the present invention an additional factor
must be
considered. Specifically, conventional liquids already contain some dissolved
gasses that
must be displaced when a new gas is introduced. Conversely, a degassed liquid
has no such
gasses present, and thus the intermolecular spaces that would otherwise be
occupied by gas
molecules are instead vacant. As a result, when the pressurized ozone is
introduced, it
essentially acts upon a liquid under partial vacuum, and the resulting uptake
is therefore much
more rapid than would occur under simple diffusion through a normal liquid.
Furthermore,
this rapid absorption allows the ozone to penetrate more deeply within the
liquid before
reacting or being displaced, thereby yielding a more uniform distribution of
ozone within the
liquid being treated. Compared to conventional liquids, the immediate benefits
of degassing
3o prior to ozone exposure thus include higher processing speeds and more
thorough
decontamination.
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To obtain even greater improvements, the above sequence is cycled in the
present
invention. The underlying phenomena have been previously proposed for
increasing the
concentration of oxygen in water (see, Samuel Glasstone, Textbook of Physical
Chemistry,
Van Nostrand, New York, p.699, 1946). The basic concept here is to use the
higher water
solubility of oxygen versus nitrogen to differentiate these gasses. Using
partial heating io
drive out the nitrogen, several such cycles would eventually yield residual
oxygen
concentrations approaching 90%. In practice, of course, oxygen can be more
rapidly and
cheaply produced by cryogenic pumping.
Thus, while not practical for oxygen generation, this cycling approach is
however
1 o quite useful in the present invention. The main modification is to use the
above vacuum and
ultrasound degassing system, thus sparing the cost and protein damage of
heating. Since the
relative solubility of ozone to oxygen is about 13:1, which is much greater
than the above
noted values for oxygen versus nitrogen, the concentration proceeds extremely
rapidly. In
particular, the concentration quickly reaches levels that are well beyond
those that are
15 obtained under normal circumstances.
The immediate concern is just how high these concentrations can reach.
Unfortunately, there is no definitive answer here for two main reasons. First,
because these
concentrations are well beyond those that can be maintained in a steady state,
they do not last
long enough for accurate measurement.
20 The second problem in trying to determine the concentration limit is that
ozone reacts
with the liquid in which it is dissolved. For example, ozone has a half life
in once-distilled
water of about 20 minutes, but a half life of 80 minutes or more in water that
has had
multiple distillations. Furthermore, small amounts of acids or neutral salts
increase the
solubility of ozone and extend the half life of the solution. Conversely,
alkalis decrease the
25 solubility of ozone (see, Atherton Seidell, Solubilities of Inorganic and
Or~~anic Compounds,
Van Nostrand, New York, p. 473, 1919).
The immediate result is that even small amounts of contaminants greatly affect
the
behavior of dissolved ozone. This is of particular concern in the present
invention because
the salt-buffered acid-base systems that are characteristic of biological
systems can thus
3o strongly affect the speed and degree of decontamination. Furthermore, in
the case of blood,
the effects of anticoagulants must also be considered because there are many
different types
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of these agents in common use today, including various citrates, EDTA,
heparin, etc., and
each of these agents has its own unique chemistry.
The net result is that upper concentration limits cannot be strictly
established because
of the transient nature of such concentrations, combined with highly variable
reaction rates
that are specific to individual cases. Nevertheless, some practical guidelines
can be
established. For example, for human gaseous exposure, 0.1 ppm can be tolerated
over an
eight hour period.
For decontamination work, gaseous concentrations can be on the order of
hundreds or
even thousands of parts per million. Conventional liquid concentrations are on
the order of
l0 0.3 to 10 mg/L. This range is also the base for the present invention, but
the peak transient
concentrations are on the order of 100 to 200 mg/L.
On the other hand, ozone is quite toxic at these higher concentrations, and
can rapidly
damage delicate proteins. For this reason, the present invention utilizes the
previously
described degassing equipment to remove the excess ozone as soon as the
decontamination is
completed.
Finally, the above arguments hold for ozone treatment systems in general. For
example, increased pressure will always force more ozone into solution. In
actual practice,
however, the behavior of any ozone decontamination system depends strongly on
how the
ozone is introduced into the liquid.
2o In the ozone industry, the process of introducing ozone into a liquid is
called
"contacting," and the devices used for this process are called "contactors."
To be effective,
the contactor must be designed to match the properties of the fluid being
treated. For
example, in the preparation of drinking water or in the treatment of toxic
wastes, high levels
of turbulence and shear can be tolerated without concern for damaging the
liquid being
processed; furthermore, the contactors can be fabricated from any reasonably
strong
construction material. As noted above, however, protein systems, notably those
involving
blood products, must be handled much more carefully, and plastics that will
not induce the
clotting sequence must be used instead of materials such as glass.
For example, these concerns are particularly important in the treatment of
blood
3o platelets, which are quite easily damaged by mechanical or thermal stress.
In terms of
contactor design, a unique factor is that platelets are processed in small
volumes, on the order
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of 50 milliliters or so, depending on the equipment and the donor.
Because of this relatively small volume, the entire donation can be spread
over the
treatment chamber at one time. A particularly preferred chamber for the
contacting of
platelets is shown in Figure 11; the treatment chamber consists of a
rectangular or similarly
s shaped block 1101 with staggered, opposed shelves in the shape of sharp
wedges 1102. With
the chamber in the horizontal position, the liquid enters a trough or inlet
port 1103 along one
side. After filling this trough, the chamber is then rotated upwards to about
80 degrees, at
which point the fluid flows over the first shelf 1102a towards the opposing
wall. Because the
shelf does not actually touch the opposing wall, however, the fluid drops down
to the next
l0 shelf 1102b and the flow then reverses. Meanwhile, ozone is introduced
through ports 1104.
Note that this arrangement is unlike the above cited patents because the flow
reversal
thoroughly mixes the material at each step, with the top layer becoming
largely the bottom
layer and vice versa.
The rotation continues until all of the fluid is emptied from the inlet
trough, which
15 occurs at about 90 degrees. The entire arrangement is then rotated back
into its original
position, and then on to -90 degrees to repeat the process from the opposite
direction. During
these movements, ozone is continuously fed into one side of the treatment
chamber, and spent
gas removed from the opposite side. Because the motion of the chamber is thus
essentially
two reversing half turns, the gas connections can be conventional flexible
hoses. This
2o arrangement thereby saves the costs and installation problems of the sealed
bearings, etc., that
are required for the continuous rotation units described earlier.
Additional enhancements of this device include an ultrasonic driver to improve
the
rate of fluid flow and to aid in the mixing of the ozone; a pressurized
treatment cell; and an
ultrasonic degassing option with vacuum assist. The benefits of each of these
components
25 have been discussed earlier, but even with these enhancements, this device
cannot handle
larger volumes of fluid effectively. It is therefore necessary to modify this
device to treat
plasma and other larger volume, heat-sensitive solutions.
One such approach is shown in Figure 5. This device employs an ultrasonic
spray
nozzle at the top of an enclosed chamber. Unlike the previously described
spray system (US
3o Patent No. 5,882,591), this device has no electrostatic fields, but it does
incorporate elevated
pressures, direct ultrasonic processing of the sprayed fluid, and other
enhancements described
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more fully below.
Unfortunately, while the ultrasound greatly reduces the shear on the liquid
being
sprayed, this process can still damage delicate proteins and cell suspensions.
Also as noted
earlier for other spray applications (US Patent No. 5,882,591), a fine spray
of contaminated
biological materials should be avoided whenever possible. It is therefore
necessary to
develop yet another contactor for general blood work, and similar
applications.
The key feature of this contactor is that the fluid to be treated flows
through an
enclosed channel. Ozone is then fed into the liquid through a series of small
holes in the
channel wall.
l0 While this arrangement thus has some similarities to conventional
gas/liquid bubbling
devices, there are however significant differences. Specifically, the ability
to rotate the
chamber aids in the bulk mixing of the fluid, thereby providing more uniform
treatment. This
mixing is further aided by the ultrasound, but ultrasound also has other
important effects in
the present application.
Historically, the use of ultrasound to aid ozone contacting has been discussed
by W.S.
Masschelein (see, "Handbook of Ozone Technoloay and Applications Volume One,"
R. G.
Rice and A. Netzer, eds., Ann Arbor Science, The Butterworth Group, Kent,
England, p. 180,
1982; See also, C. Nebel, P.C. Unangst and R.D. Gottschling, "An Evaluation of
Various
Mixing Devices for Dispensing Ozone in Water," Water Sew. Works Ref. No. R-6
(1973)).
2o In particular, it is noted that reversing the gas and liquid channels of a
conventional ultrasonic
nozzle produces a finely divided bubble distribution. Finally, as noted
earlier, U.S. Patent
4,597,876 describes ultrasonic resonance effects on ozone bubbles. In
particular, it is known
that ultrasound will drive small bubbles into the liquid, but larger bubbles
will grow to the
point that they can be removed.
In the present invention, ultrasound is coupled with a unique contactor to
force as
much ozone into the solution as possible. Specifically, the contactor in the
present invention
is driven directly by ultrasound. Furthermore, this ultrasound is delivered by
a high
amplitude horn, so that the oscillations are large in displacement. In
addition, this
displacement is larger than the diameter of the holes through which the ozone
flows. The net
result is that the ultrasound shears off extremely small bubbles into the
surrounding liquid.
Being much smaller than resonance, or even stable, size these small bubbles
are then forced
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into the liquid rapidly under the action of ultrasound.
Under the above degassing, pressurization, and contacting procedures, it is
thus
possible to drive quite large amounts of ozone into the treated liquid. For
effective
decontamination, however, it is necessary to measure the amount of dissolved
ozone.
Because ozone is highly reactive, this measurement must be accurate to avoid
over treatment.
For feedback purposes, this measurement must be made in real time. Finally,
for protein
solutions, this measurement must be made under sterile conditions.
Optical measurement techniques satisfy all of these conditions. In particular,
the
absorption of UV light is a particularly useful measurement technique (Ocean
Optics Model
2000, Dunedin, FL). To achieve this measurement in practice, a UV transparent
window is
provided in the ozone treatment path. Like the UV exposure bags, Teflon~ is
ideal, but
expensive; lower quality plastics may be used if a very bright UV source is
available.
The major limitation in this technology is the presence of gas bubbles.
Although
present as a result of decontamination, these bubbles are a significant
problem in the
measuring process because they are optically quite different from the
concentrated ozone
solution. To minimize this problem, the measurement cell may be made with a
wide top and
a narrow bottom. Under this geometry, the gas bubbles rise to the surface,
leaving only
liquid in the path of the measuring UV light beam.
The details for achieving the above processes in practice are described more
fully in
2o the following embodiments.
XII. In a twelfth main embodiment, the present invention provides a method for
decontaminating plasma by:
(a') a step for the treatment of a fluid with ultrasonic energy to obtain a de-
2s oxygenated fluid; and
(b') a step for the treatment of said de-oxygenated fluid with ozone.
In this twelfth main embodiment, the step (a') "for the treatment of a fluid
with ultrasonic
energy to obtain a de-oxygenated fluid" may be carried out in the same ways
that step "(a)
treating a fluid with ultrasonic energy to obtain a de-oxygenated fluid" is
carried out in the
30 context of the eleventh main embodiment. In addition, the step (b') "for
the treatment of said
de-oxygenated fluid with ozone" may be carried out in the same ways that step
"(b)
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contacting said de-oxygenated fluid with ozone" is carried out in the context
of the eleventh
main embodiment.
XIII. In a thirteenth main embodiment, the present invention provides a method
for
decontaminating a fluid by:
(a) mixing a fluid with ozone, to obtain an ozone-containing, fluid; and
(b) treating said ozone-containing fluid with ultrasonic energy.
In this thirteenth embodiment, the treatment of the fluid with the ultrasonic
energy may be
carried out in the same ways and by using the same apparatus as described
above in the
t o context of the first, second, third, fourth, fifth, sixth, seventh,
eighth, ninth, tenth, eleventh,
and twelfth main embodiments.
In this thirteenth main embodiment, ultrasonic energy is used to enhance the
decontamination effect of ozone. As noted above, ozone treatment is a standard
decontamination technique. While quite effective, however, ozonation
unfortunately suffers
from relatively long treatment times. As also noted above, it is useful to
combine techniques
to yield more complete decontamination than can be achieved by using any one
technique
acting alone. This thirteenth main embodiment of the present invention
therefore uses
ultrasonic energy to accelerate the ozone decontamination process, and to
improve the overall
effectiveness of the combined system.
It is already known that ultrasound is effective in enhancing the speed of
bacterial
decontamination (see, W.S. Masschelein, "Handbook of Ozone Technology and
Applications, Volume One," R. G. Rice and A. Netzer, eds., Ann Arbor Science,
The
Butterworth Group, Kent, England, p. 180, 1982). In addition, it is also known
that there is a
synergistic effect between ozone and ultrasound (see, Burleson GR; Murray TM;
Pollard M
2s "Inactivation of viruses and bacteria by ozone, with and without
sonication," Appl Microbiol
1975 Mar;29(3):340-4).
The apparent mechanism behind these effects is that ultrasound is known to
improve
chemical reactivities, particularly those involving free radicals (V. Misik
and P. Riesz,
"Detection of primary free radical species in aqueous sonochemistry by EPR
spectroscopy."
3o in Sonochemistry and Sonoluminescence, edited by L. A. Crum, T. J. Mason,
J. L. Reisse and
K. S. Suslick, NATO ASI Series C, Kluwer Academic Publishers, Dordrecht, pp.
225-236,
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1999). In addition, it is also possible that some of the observed enhancements
could also be
due to improved mixing.
In the present invention, the unique aspects of applying ultrasound to a
liquid
containing dissolved ozone is that the above conditions and techniques are
applied to protect
the proteins in solution during the decontamination process. In the following,
this
embodiment will be described in the context of plasma, and amounts to first
mixing the ozone
with the plasma. Next, the ozone-containing plasma is then treated with
ultrasonic energy.
In this embodiment, the plasma is treated with ultrasonic energy as described
above.
Of course, it is to be understood that the term "treating said ozone-
containing fluid
o with ultrasonic energy" does not require that the application of ultrasonic
energy to the
ozone-containing fluid commence after the introduction of ozone into the fluid
has ceased.
To the contrary, this term means that the application of ultrasonic energy to
the ozone-
containing fluid may commence: (1) prior to the commencement of the
introduction of ozone
into the fluid; (2) at the time the introduction of ozone into the fluid is
commenced; (3) after
the introduction of ozone into the fluid has commenced; or (4) after the
introduction of ozone
into the fluid has ceased. In fact, in an especially preferred sub-embodiment,
the ultrasonic
energy is applied to the fluid during the entire time that the ozone is
introduced into the fluid.
XIV. In a fourteenth main embodiment, the present invention provides a method
for
decontaminating a fluid by:
(a' ) a step for mixing a fluid with ozone, to obtain an ozone-containing
fluid; and
(b') a step for the treatment of said ozone-containing fluid with ultrasonic
energy.
In this fourteenth main embodiment, the step (a') "for mixing a fluid with
ozone, to obtain an
ozone-containing fluid" may be carried out in the same ways that step "(a)
mixing a fluid
with ozone, to obtain an ozone-containing fluid" is carried out in the context
of the thirteenth
main embodiment. In addition, the step (b') "for the treatment of said ozone-
containing fluid
with ultrasonic energy" may be carried out in the same ways that step "(b)
treating said
ozone-containing fluid with ultrasonic energy" is carried out in the context
of the thirteenth
main embodiment.
XV. In a fifteenth main embodiment, the present invention provides a method
for
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decontaminating a fluid by:
(a) treating a fluid with ultrasonic energy to obtain a de-oxygenated fluid;
(b) contacting said de-oxygenated fluid with ozone, to obtain an ozone-
containing
fluid; and
(c) treating said ozone-containing fluid with ultrasonic energy.
The fifteenth main embodiment is essentially a combination of the eleventh and
thirteenth main embodiments. Thus, in this fifteenth main embodiment, the
fluid is first
degassed using ultrasonic energy as discussed above. The degassed fluid is
then contacted
with ozone, and the ozone-containing fluid is treated with ultrasonic energy
to enhance the
1 o reactivity of the ozone, as described in the ninth main embodiment.
XVI. In a sixteenth main embodiment, the present invention provides a method
for
decontaminating fluid by:
(a') a step for the treatment of a fluid with ultrasonic energy to obtain a de-
15 oxygenated fluid;
(b') a step for the treatment of said de-oxygenated fluid, to obtain an ozone-
containing fluid; and
(c') a step for the treatment of said ozone-containing fluid with ultrasonic
energy.
In this sixteenth main embodiment, the step (a') "for the treatment of a fluid
with ultrasonic
2o energy to obtain a de-oxygenated fluid" may be carried out in the same ways
that step "(a)
treating a fluid with ultrasonic energy to obtain a de-oxygenated fluid" is
carried out in the
context of the fifteenth main embodiment. In addition, the step (b') "for the
treatment of said
de-oxygenated fluid, to obtain an ozone-containing fluid" may be carried out
in the same
ways that step "(b) contacting said de-oxygenated fluid with ozone, to obtain
an ozone-
25 containing fluid" is carried out in the context of the fifteenth main
embodiment. Lastly, the
step (c') "for the treatment of said ozone-containing fluid with ultrasonic
energy" may be
carried out in the same ways that step "(c) treating said ozone-containing
fluid with ultrasonic
energy" is carried out in the context of the fifteenth main embodiment.
3o XVII. In a seventeenth main embodiment, the present invention provides a
method for
decontaminating a fluid by:
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(a) treating a fluid with ultrasonic energy to obtain a de-oxygenated fluid;
(b) irradiating said de-oxygenated fluid, to obtain an irradiated fluid; and
(c) contacting said irradiated fluid with ozone, to obtain an ozone-containing
fluid.
This seventeenth main embodiment represents a combination of the irradiation
and
ozone-treatment embodiments described above. Thus, this main embodiment
represents a
combination of the fifth and seventh main embodiment, and the steps (a), (b),
and (c) may be
carried out in the same ways and with the same apparatus described above. As
noted earlier,
combined decontamination processes are quite attractive because they produce
very high log
1 o reduction rates.
In this embodiment, the ozone-treatment decontamination may either precede or
follow the irradiation decontamination. However, even though ultrasonic
degassing is very
effective, it is generally not desired to add extra dissolved oxygen species
in the ozone
process before subsequently removing them in the irradiation process.
Furthermore,
performing the ozone-treatment decontamination after the irradiation
decontamination would
allow any residual ozone additional time to react with the pathogens, which
would thus
improve the overall kill effectiveness. For these two reasons, it is preferred
that the
irradiation decontamination precede the ozone-treatment decontamination.
Accordingly, in a
preferred embodiment utilizing plasma as the fluid, this method comprises:
2o (a") treating plasma with ultrasonic energy to obtain deoxygenated plasma;
(b") irradiating said deoxygenated plasma, to obtain irradiated plasma; and
(c") mixing said plasma with ozone, to obtain ozone-containing plasma.
XVIII. In an eighteenth main embodiment, the present invention provides a
method for
decontaminating a fluid by:
(a') a step for the treatment of a fluid with ultrasonic energy to obtain a de-
oxygenated fluid;
(b') a step for the irradiation of said de-oxygenated fluid, to obtain an
irradiated
fluid; and
3o (c') a step for the treatment of said irradiated fluid, to obtain an ozone-
containing
fluid.
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In this eighteenth main embodiment, the step (a') "for the treatment of a
fluid with ultrasonic
energy to obtain a de-oxygenated fluid" may be carried out in the same ways
that step "(a)
treating a fluid with ultrasonic energy to obtain a de-oxygenated fluid" is
carried out in the
context of the seventeenth main embodiment. In addition, the step (b') "for
the irradiation of
said de-oxygenated fluid, to obtain an irradiated fluid" may be carried out in
the same ways
that step "(b) irradiating said de-oxygenated fluid, to obtain an irradiated
fluid" is carried out
in the context of the seventeenth main embodiment. Lastly, the step (c') "for
the treatment of
said irradiated fluid, to obtain an ozone-containing fluid" may be carried out
in the same
ways that step "(c) contacting said irradiated fluid with ozone, to obtain an
ozone-containing
fluid" is carried out in the context of the seventeenth main embodiment.
XIX. In a nineteenth main embodiment, the present invention provides a method
for
decontaminating a fluid by:
(a) treating a fluid with ultrasonic energy to obtain a de-oxygenated fluid;
(b) irradiating said de-oxygenated fluid, to obtain an irradiated fluid;
(c) contacting said irradiated fluid with ozone, to obtain an ozone-containing
fluid; and
(d) treating said ozone-containing fluid with ultrasonic energy.
This embodiment represents another combination of the irradiation and ozone-
treatment embodiments described above. Thus, this nineteenth main embodiment
represents
a combination of the fifth and thirteenth main embodiments, and the steps (a),
(b), (c), and (d)
may be carried out in the same ways and with the same apparatus described
above.
As noted earlier, combined decontamination processes are quite attractive
because
they produce very high log kill rates. In this embodiment, the ozone-treatment
decontamination may either precede or follow the irradiation decontamination.
However,
even though ultrasonic degassing is very effective, it is generally not
desired to add extra
dissolved oxygen species in the ozone process before subsequently removing
them in the
irradiation process. Furthermore, performing the ozone-treatment
decontamination after the
irradiation decontamination would allow any residual ozone additional time to
react with the
3o pathogens, which would thus improve the overall kill effectiveness. For
these two reasons, it
is preferred that the irradiation decontamination precede the ozone-treatment
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decontamination. Accordingly, in a preferred embodiment utilizing plasma as
the fluid, this
method comprises:
(a") treating plasma with ultrasonic energy to obtain deoxygenated plasma;
(b") irradiating said deoxygenated plasma, to obtain irradiated plasma;
(c") mixing said plasma with ozone, to obtain ozone-containing plasma; and
(d") treating the ozone-containing plasma with ultrasonic energy.
XX. In a twentieth main embodiment, the present invention provides a method
for
decontaminating a fluid by:
to (a') a step for the treatment of a fluid with ultrasonic energy to obtain a
de-
oxygenated fluid;
(b') a step for the irradiation of said de-oxygenated fluid, to obtain an
irradiated
fluid;
(c') a step for the treatment of said irradiated fluid, to obtain an ozone-
containing
~ 5 fluid; and
(d') a step for the treatment of said ozone-containing fluid with ultrasonic
energy.
In this twentieth main embodiment, the step (a') '"for the treatment of a
fluid with ultrasonic
energy to obtain a de-oxygenated fluid" may be carried out in the same ways
that step "(a)
treating fluid with ultrasonic energy to obtain a de-oxygenated fluid" is
carried out in the
2o context of the nineteenth main embodiment. In addition, the step (b') "for
the irradiation of
said de-oxygenated fluid, to obtain an irradiated fluid" may be carried out in
the same ways
that step "(b) irradiating said de-oxygenated fluid, to obtain an irradiated
fluid" is carried out
in the context of the nineteenth main embodiment. The step (c') "for the
treatment of said
irradiated fluid, to obtain an ozone-containing fluid" may be carried out in
the same ways that
25 step "(c) contacting said irradiated fluid with ozone, to obtain an ozone-
containing fluid" is
carried out in the context of the nineteenth main embodiment. Lastly, step
(d') "for the
treatment of said ozone-containing fluid with ultrasonic energy" may be
carried out in the
same ways that step "(d) treating said ozone-containing fluid with ultrasonic
energy" is
carried out in the context of the nineteenth main embodiment.
XXI. In a twenty-first main embodiment, the present invention provides an
apparatus for
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decontaminating a fluid, comprising:
( 1 ) a chamber for containing a fluid;
(2) a vacuum source coupled to the chamber; and
(3) a source of ultrasonic energy coupled to the chamber,
wherein said chamber comprises (i) a flat panel, (ii) an inlet, and (iii) an
outlet; and wherein
said flat panel of said chamber and said inlet are dimensioned such that a
fluid flowing
through said inlet and across said flat panel to said outlet will form a thin
film and travel in
plug flow at least during some portion of its flow across said flat panel.
Any surface of the chamber which comes into contact with the plasma should be
l0 constructed of materials that will have no have no deleterious effect on
the fluid, especially
when the fluid is plasma. Suitable materials for those portions of the chamber
which come
into contact with the plasma are as specified by FDA for contact with blood.
Although not an
absolute requirement, it is preferred that at least a portion of the chamber
be constructed of a
transparent material to permit visual inspection of the decontamination
process.
In any case, PVC is currently widely used, and there are various polyolefin
bags
under development. The main concern with these new materials is that the
plasticizer may
leach out over time. For the present methods, however, the contact time is
quite short. On
the other hand, the sonification may accelerate the leaching process. However,
because tests
to date show no measurable degradation, there appears to be no unique
restrictions for the
present method and apparatus.
The chamber is configured to contain a flat panel or plane at the bottom.
Although
there is no particular limitation on the size of the flat panel, there are two
general types of
sizes. First, for individual units from an apheresis donation of about 600 ml,
the flat panel
would be approximately 25 by 25 cm. On the other hand, continuous, large scale
units for
pool processing would have planar sections on the order of several meters.
The chamber also contains an inlet and an outlet. The inlet is preferably
located near
the bottom of the chamber and extends along the width of one end of the flat
panel. The inlet
is preferably a divergent spreader to assist in forming the plasma into a thin
film as it flcws
across the flat panel at the bottom of the chamber. The height of the inlet is
preferably
dimensioned such that plasma forms a thin film. The exact thickness of the
film is not by
itself critical. All that is required is that the gas bubbles reach the
surface relatively quickly.
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In the case of very durable proteins, this is not even a consideration. For
less durable proteins
and cells, a thickness of 2 to 20 mm, preferably 2 to 10 mm, and more
preferably then 2 to 4
mm may be used. This is by no means precise, and it is possible the thickness
may be varied
by simply changing the vacuum settings, power, etc. and then tuning to a
different range.
The creation of plug flow is well known (John A. Roberson, Clayton T. Crowe,
Engineering Fluid Mechanics, Third Edition, Houghton Mifflin Company, NY,
1985).
The dimensions of inlet and the flat panel are preferably adjusted such that
the plasma
flows across the flat panel in plug flow. Thus, the ratio of the length of the
flat panel to the
width of the inlet is less than twenty, preferably less than fifteen, more
preferably less than
0 about ten.
In a preferred embodiment, the inlet is connected to a device for controlling
the flow
rate of plasma across the flat panel. The flow of the fluid may be controlled
as follows. In
terms of blood, the treatment range includes plasma, as well as platelets and
erythrocytes (red
blood cells).
15 First, all blood applications should include a means to remove the white
blood cells
(leukocytes). While leukocytes are obviously useful in the donor, transfusion
of these cells
can result in a number of adverse immune reactions. Even worse, these cells
also present an
opportunity to transmit diseases, notably nvCJD (new variant Creutzfeldt-Jacob
Disease).
For this reason, these cells should either be destroyed, or preferably,
removed. A simple
2o approach is to use one of the many FDA approved filters, one example being
those of Pall
Corporation (New York).
Next, plasma should be heated to about 53 °C for one hour. This
procedure alone
kills many viruses. Another advantage of this heating is that the dissolved
oxygen drops
rapidly at such elevated temperatures. Another advantage is that cavitation is
much easier at
25 elevated temperatures. The actual heating method is not critical, as long
as it is reasonably
fast. There are several blood, plasma, and IV solution warmers on the market
capable of
providing the necessary heating.
Of course, some components of plasma (notably Factor V) are labile, and will
not
tolerate such treatment; likewise, platelets and rbe's cannot be heated this
way. For these
30 cases, the bulk of the material will be maintained at the lower
temperature, and heat will be
applied only as the liquid enters the degassing unit.
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The fluid is then cooled immediately after degassing, thereby minimizing the
total
heat exposure. Of course, there is also the option of no heat at all.
The net effect is at this point, the fluid to be treated is in a bag, which
may or may not
be heated. The next task is to get this fluid into the degassing unit. As
described earlier, one
option is a peristaltic pump. While quite effective for robust materials such
as plasma, rbc's
and platelets would however suffer severe degradation because only finely
spaced rollers on a
very thin tube can achieve the required low, steady flow rate. This
arrangement,
unfortunately, would cause excessive pumping damage to the entrained cells.
Furthermore,
the vacuum on the discharge side would exacerbate the pumping damage.
1o For these reasons, cellular systems will use a body force system for fluid
flow.
Specifically, the suction from the vacuum system will provide the overall
driving force. To
prevent the fluid from being drawn in too rapidly, the flow will be retarded
by several
techniques. One option is to use a very narrow tube, thus causing frictional
losses. Another
option is a flow restriction, such as a pin hole in an occluding membrane. A
third option is to
place the inlet bag below the degassing unit, so that the suction must
overcome gravity. A
fourth option is to include the bag inside a partial vacuum system, so that
the pressure
difference between the degassing and feed side can be controlled. A fifth
option is a variable
screw arrangement, which can be tightened or loosened as necessary to control
the flow
through the connecting tube. All of these approaches, as well as other
standard metering
techniques, can be applied.
The only remaining concern is how to control the process in practice. The
problem
here is that the vacuum must be established, the ultrasound made ready, the UV
lamps
warmed, etc., before the liquid is drawn into the system. The necessary
control can be
achieved by placing a shut off valve on the feed tube. For complete
automation, this valve
may be controlled electronically.
In another preferred embodiment, the inlet is configured to be connected to
the outlet
of an individual apheresis donation unit. In this embodiment, the device for
controlling the
flow rate of the plasma may be contained within the individual apheresis
donation unit itself
or located between the inlet and the individual apheresis donation unit.
Alternatively, the
inlet may be configured to be easily connected and disconnected to any plasma
container,
such as a plasma bag.
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The outlet is also preferably located near the bottom of the chamber at the
end of the
flat panel opposite that of the inlet. In any event, the outlet is positioned
such that the plasma
flowing across the flat panel will exit the chamber through the outlet after
having traversed
the flat panel.
In one preferred embodiment, the outlet is configured so as to be easily
connected and
disconnected to a container for receiving the decontaminated plasma. Such a
container may
range in size from many hundreds or even thousands of liters for apparatus
used for the
continuous decontamination of large pools of plasma units to as small as a few
hundreds or
even tens of ml for apparatus used to decontaminate individual units.
to In another preferred embodiment, the chamber includes a second outlet which
is in
communication with a vacuum source, such as a vacuum pump. The second outlet
is
preferably located near the top of the chamber or at least above the top of
the plasma layer,
such that plasma is not sucked into the second outlet when a vacuum is applied
to the
chamber through the second outlet. Preferably, the vacuum source can provide a
vacuum to
15 the space above the thin film of plasma in the chamber of 2 to 100 mbar,
preferably about 10
to 80 mbar, more preferably 20 to 60 mbar.
In another preferred embodiment, the apparatus comprises a liquid trap with a
sterile
filter located between the second inlet and the vacuum source.
The source of ultrasonic energy may be any which is capable of generating
ultrasonic
2o energy having the desired frequency and intensity. Such ultrasound
generators include those
described above.
The source of the ultrasonic energy is coupled to the chamber such that the
desired
intensity and frequency of ultrasonic energy may be applied to the thin film
of plasma
flowing across the flat panel. In a preferred embodiment, the apparatus
comprises an
25 ultrasound driver located beneath the flat panel. In a particularly
preferred embodiment, the
apparatus comprises a water jacket located between the ultrasound driver and
the flat panel.
In another particularly preferred embodiment, the apparatus comprises a
resonator plate
located between the ultrasound driver and the water jacket. The water jacket
is preferably
connected to a cooling and circulation system such that cooled water
circulates through the
3o water jacket when the ultrasonic energy is being applied to the plasma.
The present apparatus may further comprise additional sensors and data loggers
to
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ensure regulatory compliance. Such additional sensors may include a hydrophone
to ensure
adequate cavitation or degassing, thermocouples to ensure adequate temperature
maintenance, digital scales on the input and output bags to ensure proper flow
rates as
functions of time, and bar code readers and data printers to maintain a
traceable path. Direct
radical detection and recording are also possible. The hydrophone and
thermocouples should
be located in the chamber such that they are in communication or contact with
the thin film of
plasma as it flows across the flat panel.
The apparatus may be constructed such that all of the components are permanent
or
semi-permanent, i.e., such that all or most of the components are intended to
be used
to repeatedly for the processing of large amounts of plasma. Alternatively,
the apparatus may
be divided into a permanent or semi-permanent subunit and a disposable
subunit. In this
embodiment, the permanent or semi-permanent subunit is constructed such that
all or most of
the components are intended to be used repeatedly for the processing of large
amounts of
plasma.
The permanent or semi-permanent subunit may comprise:
( 1 ) a source of ultrasonic energy; and
(2) a region designed to accept a chamber,
wherein said source of ultrasonic energy is coupled to said region designed to
accept said
chamber such that ultrasonic energy can be applied to a liquid in a chamber
when said
2o chamber is placed in said region.
The permanent or semi-permanent subunit may further comprise other fixed
hardware, including a peristaltic pump, a water jacket, and a vacuum pump. The
peristaltic
pump is positioned such that it can be used to control the flow rate of plasma
through the
disposable unit. The water jacket is positioned such that it will be between
the ultrasound
2s driver and the chamber when the chamber is placed in the region designed to
accept it. The
vacuum pump is placed such that it can supply a vacuum to the gas above a thin
film of
plasma flowing through the chamber when the chamber is placed in the region
designed to
accept it. The permanent or semi-permanent subunit may further optionally
comprise a
resonator plate which is positioned such that it will be located between the
water jacket and
30 the ultrasound driver.
The disposable subunit may comprise:
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( 1 ) a chamber,
wherein said chamber has a flat panel, an inlet, and an outlet, and wherein
said flat panel of
said chamber and said inlet are dimensioned such that plasma flowing through
said inlet and
across said flat panel to said outlet will form a thin film and travel in plug
flow.
The disposable unit may further comprise a second outlet which may be
connected to
the vacuum pump of the permanent or semi-permanent subunit for supplying a
vacuum to the
gas above the plasma.
The use of a preferred embodiment of the present apparatus will now be
described in
more detail by referring to Figure 3. Figure 3 shows a decontamination system
30 designed
to for use in a method in which the plasma is decontaminated by the
application of ultrasonic
energy without application of UVC radiation or subsequent ozone treatment. The
plasma
enters the system from a plasma bag 31 or other source on the left, with the
flow rate of the
plasma controlled by a peristaltic pump 32. The plasma flow then crosses a
divergent
spreader 33, thus yielding a uniform plug flow of a thin film of plasma across
the flat panel at
the bottom of chamber 34, toward the collection bag 311. (Means for achieving
plug flow of
the plasma are also shown in Figure 4. The plasma enters the unit from the
left, the flow
controlled by a peristaltic pump 42. This flow then crosses a divergent
spreader 43, thus
yielding a thin film of the plasma, which flows across the flat panel at the
bottom of the main
chamber 44 in plug flow.) Plug flow is achieved by keeping the planar section
short relative
20. to the entrance zone, which guarantees continuous plug flow in this
design, using the general
fluid mechanics rule that a flow section of about 20 times the inlet width is
required to
develop laminar flow under non-turbulent, low Reynolds numbers. The flow rate
of the
plasma across the flat panel is as described above.
The ultrasonic energy is applied to the plasma by means of the ultrasound
driver 35,
which is coupled to the flat panel at the bottom of chamber 34 via a resonator
plate 36. Thus,
as the plasma flows across the flat panel, it is sonified from below. The
sonification is driven
by an ultrasonic driver 35 acting on a metal plate 36 which is resonance
coupled for efficient
energy transfer.
The temperature of the plasma flowing across the flat panel is controlled by
the water
jacket 37. The water jacket 37 between resonator plate 36 and the flat panel
prevents excess
heat from the ultrasonic driver 35 from reaching the plasma; water is an
excellent sound
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transmission medium, and any losses of ultrasonic energy are thus
insignificant. After
flowing across the flat panel, the decontaminated plasma then exits the
chamber via outlet
310 and enters the collection bag 311.
The gas above the plasma in chamber 34 and gas evolved from the plasma during
application of the sonic energy to the plasma are removed from the chamber by
the vacuum
pump 38. Biological materials such as infectious agents are captured by the
filter trap 39 to
prevent contamination of the vacuum pump 38.
The entire process may be carried out under refrigeration, and the entire
apparatus 30
or at least one or more of the starting plasma bag 31, chamber 34, and
collection bag 311 may
o be contained in one or more refrigeration units.
Apparatus 30 in Figure 3 may be constructed as a complete permanent or semi-
permanent unit, with only the starting plasma and collecting plasma bags being
disposable or
consumable subunits. Alternatively, and preferably, apparatus 30 in Figure 3
is constructed
as a permanent or semi-permanent subunit and a disposable or consumable
subunit. In the
context of apparatus 30 of Figure 3, pump 32, ultrasound driver 35, resonator
plate 36, water
jacket 37, and vacuum pump 38, may be part of the permanent or semi-permanent
subunit,
while starting plasma bag 31, inlet 33, chamber 34, outlet 310, and collection
bag 311 may be
part of one or more disposable or consumable units. The vacuum line including
the filter trap
39 may be part of either the permanent or semi-permanent subunit or a
disposable or
2o consumable subunit.
To keep costs down, the disposable or consumable units, with the exception of
the
collection bag, may be preferably blow molded from inexpensive plastics. In
this regard, it
should be rioted that the rigorous conditions that apply to the plastics in
plasma bags need not
be met in the remainder of the disposable unit, because it will never be
subjected to freezing,
transport, or long term storage. The collection bag, however, preferably
should meet these
standards. Accordingly, it is preferred to use a conventional plasma bag.
Preferably, for
compatibility with existing practices, any disposable parts of the present
apparatus should
have no metal parts so the disposables or consumables can be incinerated.
When the chamber is part of a disposable or consumable unit, the walls of the
chamber can be made of quite thin and/or flexible material. In other words,
the disposable
chamber may be a bag or liner for the region which is designed to accept it.
When the
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chamber is a bag or flexible liner, it may be made to hold a desired shape or
to conform to the
shape of the region designed to accept it, by applying a slight vacuum to draw
the liner out to
the required dimensions under differential pressure, thus allowing the use of
a very cheap
treatment bag as the chamber.
In another preferred embodiment, the disposable bag used as the chamber
further
comprises a virus tight filter at one end of the bag to equilibrate the
pressures inside and
outside of the bag during vacuum processing. This FDA approved component also
allows for
easier mounting of the bag inside the region designed to accept the chamber.
In another
preferred embodiment, the disposable bag used as the chamber further comprises
grommets at
1o the inlet and outlet tubes to prevent them from collapsing during the
application of the
vacuum.
In another preferred embodiment, the chamber has a roughened inner surface. A
roughened inner surface allows the evolved gas bubbles to travel up to local
spikes on the bag
liner. From these points, the ultrasonic vibrations can dislodge the bubbles
relatively easily.
15 For comparison, bubbles flattened along one side of a smooth bag surface
are more difficult
to remove, even with agitation.
The apparatus may further comprise certain safety features, including
electrical
shielding, splash guards, and particularly a commercial ultrasound shielding
enclosure.
In another preferred variation, the present apparatus may further comprise a
device or
2o means for detecting when a particular amount of fluid has been processed.
For example,
when individual units are being processed into storage containers, it may be
preferred to
include a scale to detect when the storage container is full. Alternatively,
an optical device
which measures the level of fluid in the container may also be used.
It may also be preferred to include a scale to measure the amount of fluid in
the input
25 container or bag. Specifically, mounting the input bag on a scale (with
computer output)
provides a means to measure the flow rate, given the time from the digital
controller. This
flow rate, in turn, provides information which may be used to control the
opening or closing
of the valve system.
3o XXII. In a twenty-second main embodiment, the present invention provides an
apparatus for
decontaminating a fluid, comprising:
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(1') a means for containing said fluid;
(2') a means for contacting said fluid with a vacuum; and
(3') a means for introducing ultrasonic energy into said means for containing
said
fluid,
wherein said means for containing said fluid comprises (i) a means for the
introduction of
said fluid into said containing means, (ii) a means for said fluid to flow
through said
containing means, and (iii) a means for the removal of said fluid from said
containing means;
and wherein said containing means is dimensioned such that a fluid flowing
through said
containing means will form a thin film and travel in plug flow at least during
some portion of
l0 its flow through said containing means.
In this twenty-second main embodiment, the "means for containing said fluid"
may
be the same as described for the "chamber for containing a fluid" described
above in the
context of the twenty-first main embodiment; the "means for contacting said
fluid with a
vacuum" may be the same as the "vacuum source coupled to the chamber"
described above in
the context of the twenty-first main embodiment; and the "means for
introducing ultrasonic
energy into said means for containing said fluid" may be the same as the
"source of ultrasonic
energy coupled to the chamber" described above in the context of the twenty-
first main
embodiment. In addition, all the optional and preferred components described
above in the
context of the twenty-first main embodiment may also be present in this twenty-
second main
embodiment.
XXIII. In a twenty-third main embodiment, the present invention provides an
apparatus for
decontaminating a fluid, comprising:
( 1 ) a chamber for containing a fluid;
(2) a vacuum source coupled to the chamber;
(3) a source of ultrasonic energy coupled to such chamber; and
(4) a source of UV, gamma, or x-ray radiation.
In this twenty-third main embodiment, the "chamber for containing a fluid" may
be the same
as the "chamber for containing a fluid" described above in the context of the
twenty-first
main embodiment; the "vacuum source coupled to the chamber" may be the same as
the
"vacuum source coupled to the chamber" described above in the context of the
twenty-first
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main embodiment; and the "source of ultrasonic energy coupled to such chamber"
may be the
same as the "source of ultrasonic energy coupled to the chamber" described
above in the
context of the twenty-first main embodiment.
As noted above, the chamber and source of ultrasonic energy in this embodiment
may
be the same as described above in the context of the twenty-first main
embodiment.
However, if the source of UV, gamma, or x-ray radiation is placed such that
the UV, gamma,
or x-ray radiation must pass through a portion of the chamber wall to reach
the plasma, then
at least that portion of the chamber wall must be sufficiently transparent to
the radiation so
that the desired degree of decontamination is achieved.
t o The principle difference between the apparatus of this embodiment and that
of the
embodiment described above is the presence of the UV, gamma, or x-ray
radiation source.
The source of UV, gamma or x-ray radiation may be any that is capable of
generating
radiation of the desired frequency and intensity. Suitable sources of UV
include those
described above. As for gamma radiation, Cobalt-60 and Cesium-137 are the most
common
medical application sources. X-rays may be generated by standard, high
voltage, electron
accelerating sources.
In another preferred embodiment, the apparatus contains a dissolved-oxygen
meter
inside chamber. The dissolved-oxygen meter is located such that it can detect
the oxygen
content in a thin film of plasma flowing across the flat panel.
This apparatus may also be constructed such that all of the components are
permanent
or semi-permanent, i. e., such that all or most of the components are intended
to be used
repeatedly for the processing of large amounts of plasma. Alternatively, the
apparatus may
be divided into a permanent or semi-permanent subunit and a disposable
subunit. In this
embodiment, the permanent or semi-permanent subunit is constructed such that
all or most of
the components are intended to be used repeatedly for the processing of large
amounts of a
fluid, such as plasma.
The permanent or semi-permanent subunit comprises:
( 1 ) a source of ultrasonic energy;
(2) a source of UV, gamma, or x-ray radiation; and
(3) a region designed to accept a chamber,
wherein said source of ultrasonic energy is coupled to said region designed to
accept said
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chamber such that ultrasonic energy can be applied to a liquid in a chamber
when said
chamber is placed in said region and wherein said source of UV, gamma, or x-
ray radiation is
positioned such that UV, gamma, or x-ray radiation can be applied to a liquid
in a chamber
when said chamber is placed in said region
The permanent or semi-permanent subunit may further comprise other fixed
hardware, including a peristaltic pump, a water jacket, and a vacuum pump. The
peristaltic
pump is positioned such that it can be used to control the flow rate of plasma
through the
disposable unit. The water jacket is positioned such that it will be between
the ultrasound
driver and the chamber when the chamber is placed in the region designed to
accept it. The
1 o vacuum pump is placed such that it can supply a vacuum to the gas above a
thin film of
plasma flowing through the chamber when the chamber is placed in the region
designed to
accept it. The permanent or semi-permanent subunit may further optionally
comprise a
resonator plate which is positioned such that it will be located between the
water jacket and
the ultrasound driver.
15 The disposable subunit of this embodiment is essentially the same as that
described
above, with the proviso that at least one portion of the chamber wall must be
constructed of
material which is sufficiently UV-, gamma-, and/or x-ray-transparent, so that
the plasma can
be effectively decontaminated by the UV, gamma, and/or x-ray- radiation.
2o XXIV. In a twenty-fourth main embodiment, the present invention provides an
apparatus for
decontaminating a fluid, comprising:
(1') a means for containing said fluid;
(2') a means for contacting said fluid with a vacuum;
(3') a means for introducing ultrasonic energy into said means for containing
said
25 fluid; and
(4') a means for the treatment of said fluid with UV, gamma, or x-ray
radiation.
In this twenty-fourth main embodiment, the "means for containing said fluid"
may be
the same as the "chamber for containing a fluid" described above in the
context of the twenty-
first and twenty-third main embodiments; the "means for contacting said fluid
with a
30 vacuum" may be the same as the "vacuum source coupled to the chamber"
described above in
the context of the twenty-first and twenty-third main embodiments; the "means
for
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introducing ultrasonic energy into said means for containing said fluid" may
be the same as
the "source of ultrasonic energy coupled to such chamber" described in the
context of twenty-
first and twenty-third main embodiments; and the "means for the treatment of
said fluid with
UV, gamma, or x-ray radiation" may be the same as the "source of UV, gamma, or
x-ray
radiation" described above in the context of the twenty-third main embodiment.
In addition,
all the optional and preferred components described above in the context of
the twenty-first
main embodiment may also be present in this twenty-fourth main embodiment.
XXV. In a twenty-fifth main embodiment, the present invention provides an
apparatus for
1o decontaminating a fluid, comprising:
( 1 ) a chamber for containing a fluid;
(2) a vacuum source coupled to the chamber;
(3) a source of ultrasonic energy coupled to such chamber; and
(4) a source of ozone,
1 s wherein said chamber comprises: (i) an inlet for introducing ozone from
the source of ozone;
(ii) an inlet for introducing plasma; and (iii) a device for mixing ozone from
the source of
ozone with a fluid.
The ozone may be generated as described above, in the context of the
thirteenth
through twentieth main embodiments. Having thus generated the ozone, the next
concern is
2o how to apply it to the fluid.
In the present preferred embodiment, two alternative methods or contactors may
be
used.
Accordingly, in a preferred embodiment the ozone is mixed with the fluid with
a
contactor which comprises:
2s ( 1 ) a substrate which has a lower surface and an upper surface and which
has a
plurality of passage-ways connecting said lower surface with said upper
surface;
(2) a source of ultrasonic energy coupled to said substrate, such that said
ultrasonic energy is introduced into the fluid by the vibration of said
substrate;
30 (3) a source of ozone connected to said lower surface of said substrate.
In this preferred contactor, the ozone is introduced into the fluid by passing
through the same
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substrate which couples the source of ultrasonic energy to the fluid. The
ozone passes
through the passage-ways in the substrate and is introduced into the fluid in
the form of
bubbles. The size of the bubbles may be controlled, at least in part, by
controlling the size of
the openings of the passage-ways to the fluid. Suitably, the openings are
circular in shape
with the diameters of the openings of the passage-ways having a size of 25 to
1000 microns,
preferably 50 to 500 microns, depending on the ultrasonic frequency range. The
size of the
ozone bubbles introduced into the fluid is also influenced, in part, by the
frequency and
amplitude of the vibration of the substrate. Typically, the substrate will
vibrate at a frequency
of 20 to 250 kHz, preferably 20 to 100 kHz, with an amplitude greater than the
diameter of
the openings.
The first part of the ozone treatment system is the plasma input reservoir,
which is in
direct contact with heat transfer plates for cooling. As noted in a previous
section, cooled
liquids are much more receptive to gasses. The overall geometry of the
reservoir is a
cylinder, decreasing in size towards the base. At the bottom of this cylinder,
the reservoir
becomes rectangular in cross section. This rectangular cross section matches
the inlet of the
ozone nozzle. This nozzle is shaped like a "V" with small (several micron)
holes on both
sides of each planar section. These holes connect to an ozone source.
Ultrasound is applied
normal to the plane of the "V" along the direction of the bottom channel.
On the opposite side of the nozzle, a similar reservoir is placed to collect
the treated
2o fluid. The height of this second reservoir, however is less than the height
of the first so that
the liquid flows under gravity; alternatively, the fluid can be pumped.
With this arrangement, the ozone enters the liquid already divided into
"ligaments."
The direct action of the ultrasound on these gaseous ligaments is immediate
disruption into
bubbles. It should be noted in particular that the motion of the ultrasonic
horn is on the order
of a mm, which is much greater than the ozone orifice diameters. As such, the
fine gas
bubbles are typically spread over a wide area. Also, this motion allows many
orifices to be
spaced close together, with subsequent rows staggered, to yield a quite
uniform distribution.
In practice, fewer orifices are placed on the inlet side because the incoming
downward flow
tends to force the rising bubbles together, leading to undesirable larger
sizes. Conversely,
buoyancy on the exit side has the opposite effect, so more gas can be
introduced here.
There are several benefits of such an arrangement. First, the fluid in the
reservoir is
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immediately exposed to some gas, thus improving the overall treatment time.
Second, the
requirement that all of the liquid must pass through the nozzle ensures
uniform treatment.
Third, the continued ozone treatment on the exit side also extends the total
treatment time,
under good mixing conditions. Fourth, the small, micron-sized bubbles are much
less than
the optimum for resonance for a typical 20 kHz source, and are therefore
rapidly driven into
solution by the applied ultrasound, as discussed earlier. Finally, the low
amplitude source
improves mixing and diffusion, without excessive bubble growth or protein
damage due to
cavitation.
Allowing the narrow sides of the "V" to flex slightly under ultrasonic motion
can
o further enhance this mixing. In this case, the flexing allows the
essentially incompressible
fluid to move more readily relative to the orifices of the nozzle. In
addition, a single driver at
the base of the "V" is more cost effective than a pair of drivers on each
side.
After ozone treatment, the liquid is then collected into a second reservoir,
as
described above. From here, the liquid is then pumped by a peristaltic unit
through a heater
~ 5 into a vacuum/ultrasound degasser as described earlier. As described
earlier, the fluid is then
partially degassed, preferentially removing the oxygen while leaving the
ozone. After
degassing at this slightly elevated temperature, the fluid is then recycled
into the starting
reservoir.
The entire process can be repeated as many times as desired. In this process,
the
20 overall intent is to achieve a high concentration of ozone rapidly. For
optimum time
utilization, part of the fluid can be in the degassing component while the
remainder of the
fluid is in the ozone nozzle component. Some of the material is thus
continuously being
processed, thereby decreasing the overall system time requirements.
The ozone flow rate into the fluid depends on the pressure applied to the
lower
25 surface of the substrate and on the size and density of the passage-ways.
As noted above, the
size of the passage-ways, in part, determines the size of the bubbles
introduced into the fluid.
Also as noted above, the size of the bubbles is important because bubbles
greater than a
critical size are stable and grow so large that they escape the liquid, while
bubbles smaller
than this critical size are unstable and are driven back into the solution by
the ultrasound.
3o Because the critical size limit depends on the frequency of the ultrasound,
all bubbles less
than the critical size are suitable.
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Thus, the amount of ozone introduced into the fluid is typically controlled by
varying
the pressure of ozone applied to the lower surface of the substrate and by
careful selection of
the size and the density of passage-ways in the substrate. Typically, the
ozone is applied to
the lower surface of the substrate at a flow velocity of 1 to 10 mm/sec,
preferably 1 to S
mm/sec. The critical factor limiting the flow velocity on the ozone is the
exit pressure, after
the ozone leaves the passage-ways. Specifically, it is desirable that the gas
be moving
slowly, on the order of less than 1 cm/sec, along with negligible residual
pressure, to prevent
damage to the delicate proteins and/or any cells. For example, passing the
output of the
above described ozone generator through 400 holes each of 75 micron diameter
yields a
o maximum velocity of about 0.6 cm/sec. In actual practice, the flow velocity
is much slower
due to pressure losses, as desired. Using 100 holes per square cm, distributed
as described
above, yields a total surface area of 4 cm2.
In one particularly preferred sub-embodiment, the substrate is part of a v-
shaped
trough, with one "leg" of the "v" taller than the other. The inside surface of
the tall "leg"
corresponds to the upper surface of the substrate described above, and the
outside surface of
the tall "leg" corresponds to the lower surface of the substrate described
above. The fluid
flows down the inside surface of the tall "leg" (the upper surface of the
substrate), where it is
effectively contacted with the ozone, to the bottom and then up and over the
short "leg."
In another particularly preferred sub-embodiment, the substrate forms part of
a hollow
2o apparatus which has an approximate U shape. In this preferred sub-
embodiment, the fluid
flows from an inlet (preferably after degassing and even more preferably after
exposure to
UV, gamma, and/or x-ray radiation). In one embodiment, the outside member of
the hollow
"U" corresponds to the substrate, and its inside surface corresponds to the
upper surface of
the substrate, while its outside surface corresponds to the lower surface of
the substrate. In
another embodiment, both the inside and outside members of the hollow "U"
correspond to
the substrate, with both inside surfaces corresponding to the upper surface of
the substrate
and both outside surfaces corresponding to the lower surface of the substrate.
XXVI. In a twenty-sixth main embodiment, the present invention provides an
apparatus for
decontaminating a fluid, comprising:
(1') a means for containing said fluid;
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(2') means for contacting said fluid with a vacuum;
(3') a means for introducing ultrasonic energy into said means for containing
said
fluid; and
(4') a means for generating ozone,
wherein said means for containing said fluid comprises: (i) a means for the
introduction of
ozone from said means for generating ozone into said containing means; (ii) a
means for the
introduction of said fluid into said containing means; and (iii) a means for
mixing said ozone
from said means for generating ozone with said fluid in said containing means.
In this twenty-sixth main embodiment, the "means for containing said fluid"
may be
o the same as the "chamber for containing a fluid" described above in the
context of the twenty-
fifth main embodiment; the "means for contacting said fluid with a vacuum" may
be the same
as the "vacuum source coupled to the chamber" described above in the context
of the twenty-
fifth main embodiment; the "means for introducing ultrasonic energy into said
means for
containing said fluid" may be the same as the "source of ultrasonic energy
coupled to such
t 5 chamber" described above in the context of the twenty-fifth main
embodiment; and the
"means for generating ozone" may be the same as the "source of ozone"
described above in
the context of the twenty-fifth main embodiment. Moreover, the "(i) a means
for the
introduction of ozone from said means for generating ozone into said
containing means" and
the "(iii) a means for mixing said ozone from said means for generating ozone
with said fluid
2o in said containing means" may together form any of the ozone contactors
described above in
the context of the twenty-fifth main embodiment.
XXVII. In a twenty-seventh main embodiment, the present invention provides an
apparatus for decontaminating a fluid, comprising:
25 ( 1 ) a chamber for containing a fluid;
(2) a vacuum source coupled to the chamber;
(3) a source of ultrasonic energy coupled to such chamber;
(4) a source of UV, gamma, or x-ray radiation; and
(S) a source of ozone,
30 wherein said chamber comprises: (i) an inlet for introducing ozone from the
source of ozone;
(ii) an inlet for introducing a fluid; and (iii) a device for mixing ozone
from the source of
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ozone with a fluid.
In this twenty-seventh main embodiment: ( 1 ) the "chamber for containing a
fluid;" (2)
the "vacuum source coupled to the chamber;" (3) the "source of UV, gamma, or x-
ray
radiation;" (4) the "source of ultrasonic energy coupled to such chamber;" and
(5) the "source
of ozone" may be any of the corresponding elements described above in the
twenty-first,
twenty-third, and twenty-fifth main embodiments. Moreover, the "device for
mixing ozone
from the source of ozone with a fluid" may be any of the ozone contactors
described above in
the context of the twenty-fifth main embodiment.
Thus, the apparatus of the twenty-seventh main embodiment is designed for
l0 implementation of a process in which the fluid is first degassed, then
exposed to UV, gamma,
or x-ray radiation, and then treated with ozone, i.e., the methods of main
embodiments
seventeen through twenty.
XXVIII. In a twenty-eighth main embodiment, the present invention provides an
apparatus for decontaminating a fluid, comprising:
(1') a means for containing said fluid;
(2') a means for contacting said fluid with a vacuum;
(3') a means for introducing ultrasonic energy into said means for containing
said
fluid;
(4') a means for the treatment of said fluid with UV, gamma, or x-ray
radiation;
and
(S') a means for generating ozone,
wherein said means for containing said fluid comprises: (i) a means for the
introduction of
ozone from said means for generating ozone into said means for containing;
(ii) a means for
the introduction of said fluid into said means for containing; and (iii) a
means for mixing said
ozone from said means for generating ozone with said fluid in said means for
containing.
In this twenty-eighth main embodiment, the "means for containing said fluid"
may be
the same as the "chamber for containing a fluid" described above in the
context of the twenty-
first, twenty-third, twenty-fifth, and twenty-seventh main embodiments; the
"means for
3o contacting said fluid with a vacuum" may be the same as the "vacuum source
coupled to the
chamber" described above in the context of the twenty-first, twenty-third,
twenty-fifth, and
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twenty-seventh main embodiments; the "means for introducing ultrasonic energy
into said
means for containing said fluid" may be the same as the "source of ultrasonic
energy coupled
to such chamber" described above in the context of the twenty-first, twenty-
third, twenty-
fifth, and twenty-seventh main embodiments; the "means for the treatment of
said fluid with
UV, gamma, or x-ray radiation" may be the same as the "source of UV, gamma, or
x-ray
radiation" described above in the context of the seventh main embodiment; and
the "means
for generating ozone" may be the same as the "source of ozone" described above
in the
context of the twenty-fifth and twenty-seventh main embodiments. Moreover, the
"(i) a
means for the introduction of ozone from said means for generating ozone into
said means for
1 o containing" and "(iii) a means for mixing said ozone from said means for
generating ozone
with said fluid in said means for containing" may together form any of the
contactors
described above.
XXIX. In a twenty-ninth main embodiment, the present invention provides an
apparatus for
contacting ozone with a liquid, which comprises:
( 1 ) a substrate which has a lower surface and an upper surface and which has
a
plurality of passage-ways connecting said lower surface with said upper
surface;
(2) a source of ultrasonic energy coupled to said substrate, such that said
2o ultrasonic energy is introduced into a liquid by the vibration of said
substrate;
(3) a source of ozone connected to said lower surface of said substrate.
This twenty-ninth main embodiment corresponds substantially to the contactor
shown in
Figure 8, which is described in detail below.
The basic principles behind the ozone contactor could also be applied to
adding other
gasses to liquids. Specifically, the underlying principle is to degas the
liquid first, and then
add the desired gasses immediately afterward, using a sonic assist contactor.
Finally, partial
degassing to remove reacted products and/or undesired species should
then be done.
Of course, the most common application liquid is water, but this could be
extended to
3o include aqueous solutions, or even other liquids. The gasses could include
everything from
ozone to carbon monoxide or dioxide to various nitrogen compounds, etc. As
such, the end
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product would not necessarily be something for sterilization, but instead
could include
various feedstocks for the chemical industry.
XXX. In a thirtieth main embodiment, the present invention provides an
apparatus for
contacting a gas, e.g., ozone, with a fluid, said apparatus comprising:
(1) a rotatable chamber;
(2) a source of a gas connected to said chamber; and
(3) a source of ultrasonic energy coupled to said chamber,
wherein said chamber comprises an a fluid inlet;
1 o wherein said chamber comprises a first sidewall and a second sidewall and
said first
and second sidewalls are positioned opposite to each other;
wherein said chamber further comprises a plurality of partitions, and said
partitions
are attached to said first and second sidewalls in an alternating arrangement,
and each
partition attached to said first sidewall projects toward said second
sidewall, and each
is partition attached to said second sidewall projects toward said first
sidewall, such that said
plurality of partitions forms a plurality of shelves;
wherein said inlet is positioned in said chamber such that a fluid entering
said
chamber through said inlet occupies a first shelf;
wherein said chamber is capable of rotating such that on rotation of 90 to -90
° of said
2o chamber fluid which occupies said first shelf will flow to a second shelf;
wherein said source of gas is connected to said chamber to permit mixing of a
gas
with a fluid in said chamber; and
wherein said source of ultrasonic energy is coupled to at least one of said
partition, to
permit application of ultrasonic energy to fluid which occupies a shelf formed
by said at least
2s one partition.
The apparatus of this thirtieth main embodiment corresponds to that depicted
in
Figure 11 and which is described below. The contactor of this thirtieth main
embodiment is
especially useful for contacting ozone with fluids which contain platelets,
and its use and
operation and described in detail in connection with the description of Figure
11. However, it
3o is noted that the discussions above which relate to the materials used for
the chamber, the
source of ozone, and the source of ultrasonic energy apply to this thirtieth
main embodiment.
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Of course it is to be undPrstooa~that any~and all of the steps and/or
components
described in the above-disclosed methods and apparatus may be carried out or
operated by
means of computer control. Such computer control substantially reduces the
possibility and
risk of problems and/or malfunctions resulting from human error.
XXXI. The Figures:
Various other objects, features and attendant advantages of the present
invention will
be more fully appreciated as the same becomes better understood from the
following detailed
description when considered in connection with the accompanying drawings in
which like
1 o reference characters designate like or corresponding parts throughout the
several views.
Figure 1 is a schematic flow chart of the method of the fifteenth through
eighteenth
main embodiments, which are especially preferred. In the first step shown in
Figure 1, the
fluid is subjected to a temperature preparation step. In a second step, the
fluid is degassed by
application of ultrasonic energy. In the third step shown in Figure 1, the
degassed fluid is
15 irradiated. In the fourth step shown in Figure 1, the irradiated fluid is
treated with ozone.
Although these steps are shown as progressive, this does not mean that the
technology
must perform only one function at a time. In this regard, it should be noted
that the first step
in the process (heating of the entire plasma bag) may require an hour or more.
Likewise,
degassing and UV exposure may take a half hour or so, while the ozone exposure
may take a
2o similar time, or more if multiple cycles are used. To avoid tying up the
entire machine during
these separate cycles, a clinical unit would have two or more bags being
warmed
simultaneously. As one of these bags reaches the completion of its heat
treatment, it is
degassed, etc., while the other bag continues its heat processing. Likewise,
at the end of the
process, several different bags can be undergoing ozone exposure, while other
units are being
25 heated, degassed, etc. As a result, the unit is kept processing at all
times for maximum return
on investment.
Figure 2 shows a preferred apparatus which is useful for ultrasonic degassing
as
carried out in the third and fourth main embodiments. When using the apparatus
shown in
Figure 2, the fluid enters the chamber, which is a flexible, disposable bag,
21, through an
3o inlet, 22, and eventually exits through a drain port, 23. The disposable
bag is received inside
a vacuum chamber, 24, which is equipped with water cooling and ultrasonic
drivers, 25, such
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that ultrasonic energy is introduced into the fluid. The flexible, disposable
bag, 21, is
maintained in an expanded shape by means of a vacuum applied to the outside of
the bag via
a chamber vacuum port, 26 or by fasteners mated to the fixed vacuum chamber
walls. A
vacuum is applied to the fluid inside the bag via bag vacuum port, 27. The
vacuum chamber,
24, is also equipped with temperature and mass sensors, 28 and 29, so that the
flow rate and
heating due to the introduction of ultrasonic energy may be monitored and
controlled.
Figure 3 shows a preferred apparatus of the present invention which
corresponds to
the twenty-first and twenty-second main embodiments. In the embodiment shown
in Figure
3, the fluid is introduced into the main chamber 34 through an inlet 33 from a
starting plasma
1o bag 31. The feed rate of the fluid may be controlled by pump 32. The fluid
is sonified from
below, as it flows across the planar section of the main chamber 34. The
ultrasonic energy is
provided by the ultrasound driver 35, which is coupled to the fluid via the
resonator plate 36.
The temperature of the fluid may be controlled by a water jacket 37. During
sonification, the
dissolved gasses, including oxygen may be released from the fluid and then
trapped in the
plastic housing. This housing and the planar section are a sealed unit, thus
preventing
external air from being drawn into the system. Evolved gasses may then
captured by the
vacuum pump 38. For safety, the vacuum line may incorporate a sterile coupling
and a filter
trap 39 to prevent any pathogens from contaminating the vacuum pump. The
decontaminated
fluid is then collected in the collection bag 311.
Figure 4 shows another preferred apparatus of the present invention which
corresponds to the twenty-first and twenty-second main embodiments. Figure 4
shows a
decontamination system 40 designed for use in a method in which the fluid, in
particular
plasma, is decontaminated by the application of ultrasonic energy without
application of
UVC radiation or subsequent ozone treatment. The plasma enters the system from
a plasma
bag 41 or other source on the left, with the flow rate of the plasma
controlled by a peristaltic
pump 42. The plasma flow then crosses a divergent spreader 43, thus yielding a
uniform plug
flow of a thin film of plasma across the flat panel at the bottom of chamber
44.
The ultrasonic energy is applied to the plasma by means of the ultrasound
driver 45,
which is coupled to the flat panel at the bottom of chamber 44 via a resonator
plate 46. Thus,
as the plasma flows across the flat panel, it is sonified from below. The
sonification is driven
by an ultrasonic driver 45 acting on a metal plate 46 which is resonance
coupled for efficient
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energy transfer.
The temperature of the plasma flowing across the flat panel is controlled by
the water
jacket 47. The water jacket 47 between resonator plate 46 and the flat panel
prevents heat
from the ultrasonic driver 45 from reaching the plasma; water is an excellent
sound
transmission medium, and any losses of ultrasonic energy are thus
insignificant.
The gas above the plasma in chamber 44 and the gas evolved (particularly
oxygen)
from the plasma during application of the sonic energy to the plasma are
removed from the
chamber 44 by vacuum pump 48. Chamber 44 is a sealed unit, thus preventing
external air
from being drawn into the system. For safety, the vacuum line incorporates a
sterile coupling
1 o and a filter trap 49 to prevent any pathogens from entering into and
contaminating the
vacuum pump.
After the plasma has been deoxygenated, the plasma then passes under
irradiation
source (in this case, UV lights) 410 for decontamination. Note that the water
jacket 47
extends under this section to prevent excess heating of the plasma by the UV
lights 410. In
~ 5 Figure 4, the ultrasound driver 45 also extends under the section in which
the plasma passes
under the UV lights 410. Extension of the ultrasound driver 45 under this
section provides
enhanced decontamination due to improved mixing of the plasma during UV
exposure, as
well as the dispersal of any aggregates. In an alternative arrangement, the
ultrasound
generator does not extend under the region where the plasma passes under the
UV lights 410.
2o However, even when the ultrasound generator does not extend under the
region where the
plasma passes under the UV lights 410, it is preferred that the water jacket
47 extends under
the region where the plasma passes under the UV lights 410.
After UV (or gamma ray or x-ray) exposure in this section, the flow then
enters a
converging zone (outlet) 411, which leads to a tube connected to a collection
vessel or bag
25 412 for the decontaminated product. Optionally, the flow may pass through a
converging
zone, which leads to a tube that passes through an optional peristaltic pump
and then into
collection bag 412.
The entire process may be carried out under refrigeration, and the entire
apparatus 40
or at least one or more of the starting plasma bag 41, chamber 44, and
collection bag 412 may
30 be contained in one or more refrigeration units.
Apparatus 40 in Figure 4 may be constructed as a complete permanent or semi-
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permanent unit, with only the starting plasma and collecting plasma bags being
disposable or
consumable subunits. Alternatively, and preferably, apparatus 40 in Figure 4
is constructed
as a permanent or semi-permanent subunit and a disposable or consumable
subunit. In the
context of apparatus 40 of Figure 4, pump 42, ultrasound driver 45, resonator
plate 46, water
jacket 47, and vacuum pump 48, may be part of the permanent or semi-permanent
subunit,
while starting plasma bag 41, inlet 43, chamber 44, outlet 41 l, and
collection bag 412 may be
part of one or more disposable or consumable units. The vacuum line including
the filter trap
49 may be part of either the permanent or semi-permanent subunit or a
disposable or
consumable subunit.
1 o As described above, the disposable or consumable units, with the exception
of the
collection bag, may be preferably blow molded from inexpensive plastics, while
it is
preferred to use a conventional plasma bag and the disposable parts of the
present apparatus
should have no metal parts so they can be incinerated.
When the chamber is part of a disposable or consumable unit, the chamber walls
can
1 s be made of quite thin and/or flexible material, with only a small window
for UV transmission
directly under the lamps. In other words, the disposable chamber may be a bag
or liner for
the region which is designed to accept it. When the chamber is a bag or
flexible liner, it may
be made to hold a desired shape or to conform to the shape of the region
designed to accept it,
by applying a slight vacuum to draw the liner out to the required dimensions
under
2o differential pressure, thus allowing the use of a very cheap treatment bag
as the chamber. An
additional enhancement is the presence of another window in the bottom of the
bag to allow
exposure from both sides of the fluid layer by a second source of UV, gamma,
or x-ray
radiation.
In another preferred embodiment, the disposable bag used as the chamber
further
2s comprises a virus tight filter at one end of the bag to equilibrate the
pressures inside and
outside of the bag during vacuum processing. This FDA approved component also
allows for
easier mounting of the bag inside the region designed to accept the chamber.
In another
preferred embodiment, the disposable bag used as the chamber further comprises
grommets at
the inlet and outlet tubes to prevent them from collapsing during the
application of the
30 vacuum.
In another preferred embodiment, the chamber has a roughened inner surface. A
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roughened inner surface allows the evolved gas bubbles to travel up to local
spikes on the bag
liner. From these points, the ultrasonic vibrations can dislodge the bubbles
relatively easily.
For comparison, bubbles flattened along one side of a smooth bag surface are
more difficult
to remove, even with agitation.
This apparatus may further comprise certain safety features, including
electrical
shielding, splash guards, and particularly a commercial ultrasound shielding
enclosure.
In the embodiment shown in Figure 4, the plasma is sonified from below, as it
flows
across the planar section at the bottom of the main chamber 44. During
sonification, the
dissolved gasses, including oxygen are thus released from the plasma and are
then trapped in
t o the plastic housing. This housing and the planar section are a sealed
unit, thus preventing
external air from being drawn into the system. The evolved gasses are then
captured by the
vacuum pump. For safety, the vacuum line incorporates a sterile coupling and a
filter trap to
prevent any pathogens from contaminating the vacuum pump.
Certain of the components of the apparatus shown in Figure 4, as well as
certain
15 optional components not shown in Figure 4 will now be discussed in more
detail.
1. Input reservoir, 41. The first of these components is simply a bag to
receive the
output from the degassing unit. For those materials requiring tight
temperature control, this
reservoir incorporates a heat exchanger. In extreme cases, such as platelets,
this reservoir is
preceded by a heating/cooling pack as described for the inlet to the degassing
unit. With or
2o without heat transfer capabilities, however, the main function of the
reservoir is simply to
provide a uniform pressure head for the gravity flow throw the rest of the
system. As such,
the reservoir is broad and shallow, so that there is relatively little
pressure difference between
a full reservoir and a nearly empty reservoir.
2. UVC lamps, 410. To achieve rapid and thorough processing, high intensity UV
25 lamps are necessary. Several such UVC sources have recently been marketed
(Spectronics
Corporation, Westbury NY, and UVItech, Cambridge, England). Unfortunately,
these lamps
also produce a great deal of heat, and any such heating must be controlled to
avoid protein
damage. One means of obtaining this control is simply to blow air across the
UV sources,
which is indeed one of the reasons for performing UV exposure at atmospheric,
instead of
3o vacuum, conditions. A second consideration is that the cell does not need
to be exposed to
the lamps at all times. For example, if there is a delay between batches of
fluid from the
CA 02481144 2004-10-04
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degassing unit, exposing the cell contents to the lamps would result in
excessive UV
exposure. To avoid protein damage during such times, the beam path must be
interrupted.
There are several possible ways of achieving this interruption in practice.
One option is
simply to turn off the UV lamps. This approach is quite simple, and is
preferred in those
cases in which the lamps can be restarted rapidly. For those lamps that
require a relatively
long warming time, however, power cycling is not an option. In this case, the
cell can be
removed from the beam path, but this is difficult for those applications in
which the cell is
attached to numerous mounts, tubes, etc. The preferred approach, however, is
to use a shutter
arrangement between the sources and the flow cell to block the light when
necessary.
1o In addition, there is also the problem of direct radiative heating of the
sample. In this
case, the incoming radiation generates heat in the liquid that is not readily
dissipated through
the flow cell walls. The net result is essentially a greenhouse effect, which
is a particular
problem for erythrocytes because they are optically dense and red. For cases
where this heat
is a significant problem, the flow cell is modified to include a thin layer of
cooling water or
other heat exchange liquid around the material to be treated, but constrained
to separate
channels. Finally, for protection from lamp failure or from inadvertent leaks,
the lamps must
be separated from the flow cell by a thin, UV transparent shield.
3. Output pump. At the end of the UV treatment chamber, the connecting hose
may
lead to an ozone exposure unit, rather than collection bag 412. In this case,
the essential
problem is that the ozone unit typically operates at much greater pressures
than the
atmospheric pressure that exists in the UV unit. Thus, some provision must be
made to
handle this pressure difference. As described earlier for the transition from
the vacuum
degassing unit to the UV unit, the two alternative approaches are a pressure
lock chamber or a
peristaltic pump. Again, each has the previously described advantages and
disadvantages.
4. Monitoring equipment. As for monitoring, the essential problem is that UV
systems tend to degrade over time in a process called "solarization." To
compensate for this
loss in performance, several UV manufacturers (for example, the Spectroline
part of
Spectronics Corporation, Westbury, NY) have developed automatic monitoring and
correction systems. These units monitor the actual UV emissions, and then
adjust the
exposure times accordingly. Although originally developed for cross linking UV
sensitive
reagents onto a substrate, this technology is directly transferable here.
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5. Flow regulation. The remaining concern is to control the flow through the
system.
The most important consideration here is that the liquid must be exposed to
the light long
enough for effective treatment, but not so long as to lead to excessive
protein damage. The
approach here is to control the flow as described for the inlet to the
degassing unit, using flow
restrictions, pulsed flow, mass measurements, etc. The essential consideration
here is to keep
the UV exposure unit working quickly enough to process continuously all of the
output from
the degassing unit. In this regard, the reservoir has to maintain sufficient
head to pass all
liquids rapidly, even though there may be substantial variations in viscosity
from one unit to
the next. While this holds in most cases, a control loop is also incorporated
to terminate flow
t o into the degassing unit if necessary.
System operation: The overall system operation follows more or less from the
above
component descriptions. Basically, fluid from the degassing unit enters the
feed reservoir.
Under gravity, the fluid then flows through a valve system to control the flow
rate. Next, the
fluid enters the UV chamber, which is cooled by water and air flow. Shutters
between the
lamps and the flow chamber interrupt the light when the flow stops. After
treatment, the fluid
is then pumped out to the ozone exposure unit, or to a collection bag if ozone
is not to be
used.
Like the rest of the unit, including the ozone exposure module, the first step
in the
startup process is to evacuate the fluid path with the vacuum pump. This is
necessary to
2o ensure that there is no oxygen in the system that might be absorbed by the
fluid, and thus
form oxygen radicals during UV exposure.
As for shut down, the exposure chamber should be tilted as described for the
degassing unit so that only a minimum amount of fluid is left behind. The
reason for this
effort is that the fluid is very valuable and thus must be collected as well
as possible;
furthermore, any residual material is simply a biohazard, thus presenting a
disposal problem.
As noted above, it is preferred that the UVC illumination be done from both
sides.
This provides for much more uniform exposure. In particular, this uniformity
makes red
blood cell treatment possible; otherwise, the strong absorption by hemoglobin
prevents
adequate treatment. In this case, using double side exposure allows the use of
a flow layer on
3o the order of 10 to 40 microns, more preferably in the range of 30-40
microns. At this
thickness, the variation in intensity is less than 10% even for high
hematocrit samples. Note
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that these dimensions are based on the size of erythrocytes, which are about
10 microns in
length. Although quite precise, the required level of machining is available
from specialized
companies, such as Mindrum Precision, Inc., Rancho Cucamonga, CA. This firm
specifies a
flatness tolerance of about 0.5 microns for its UV flowcells.
s To treat red blood cells effectively, the illuminator must be sonified at
low intensity to
promote uniform mixing and ensure plug flow. It should be noted that
illuminator
sonification was previously mentioned to prevent aggregation of plasma
proteins.
For easy manipulation, it is preferred that the flexible bags be mounted on a
rigid
frame that matches the processing equipment. This frame can be either reused
or discarded.
1o With or without the use of such frames, the bags should be manufactured
with registration
holes in their borders. Of course, the frame and/or the processor must have
matching pins for
these holes. This arrangement thus provides an easy way of aligning the bags
in the
processor, and also helps to prevent accidental misalignment by the operators.
To achieve the necessary precision, particularly for red blood cell treatment,
the frame
t s and/or bag assembly must be mounted in a recess inside the quartz flow
cell. As such, the
quartz thus provides rigid support after the fluid enters the treatment zone.
Also, the quartz
surfaces thus are in direct contact at the boundary, thereby ensuring tight
tolerances. To
avoid excessive pressures during the contact process, the opposing panels are
mounted on
rubber supports, which compress on contact. Note that the sonification must
therefore be
2o applied directly to the panels under this arrangement, which will otherwise
excessively damp
the sound waves.
The last problem is to control the flow of the system. In a preferred
configuration, all
of the donation sample is illuminated in one step (for example, plasma or
platelets). In this
case, all of the degassed liquid is poured into the top of the exposure
chamber in one
25 operation. The lamps are then turned on. After the end of the treatment all
of the liquid is
then drained. The only problem here is that the clamp at the exit of the tube
must not shadow
the treatment volume. This can be avoided by designing a protrusion onto the
clamp to
extend sharply beyond the clamp body. Constructing this protrusion from UVC
transparent
materials, such as Teflon~ AF or quartz, eliminates any remaining shadow
effects.
3o Figure 5 shows another preferred apparatus, which corresponds to the
apparatus of the
twenty-fifth and twenty-sixth main embodiments and is useful for carrying out
the method of
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the thirteenth through sixteenth main embodiments. In Figure 5, the fluid,
plasma, in the
illustrated case, enters the ozonation unit from the plasma bag, 51, via a
pump, 52, where it is
mixed with ozone in a mixing tip/spray nozzle, 55. The ozone enters the mixing
tip/spray
nozzle, 55, from an ozone generator, 53, passing through a filter trap, 56.
The fluid and the
s ozone are mixed in the mixing tip/spray nozzle, 55, and enter the reaction
vessel, 57, as a
spray or mist. The fluid collects at the bottom of the reaction vessel, 57, to
the fill line, S 11.
Ultrasonic energy is applied to the fluid at the bottom of the reaction
vessel, 57, via an
ultrasound driver, 58, and a water jacket, S 10, is placed between the bottom
of the reaction
vessel, 57, and the ultrasound driver, 58, to control the degree of heating.
When the treatment
o is complete the fluid is drained from the reaction vessel, 57, via a line to
a collection bag,
512.
The use of this preferred embodiment of the present apparatus to
decontaminated
plasma will now be described in more detail by referring to Figure S. Figure 5
shows a
decontamination system 50 in which plasma enters the system from a plasma bag
51 or other
15 source on the right, the flow controlled by a peristaltic pump 52. Ozone
from a conventional
generator 53 is then passed through a connecting tube 54 to the mixing
tip/spray nozzle
assembly 55. Like the vacuum line in the light exposure line, the ozone feed
tube is passed
through a filter 56 and trapped across a sterile coupling to prevent
inadvertent contamination
of the ozone generator 53. After mixing the ozone and plasma, the product is
then collected
2o in the reaction vessel 57. Like the processor tray in the light exposure
unit, this vessel sits on
an ultrasonic driver 58 coupled to a resonator plate 59 and separated from the
reaction vessel
57 by a water-driven cooling jacket 510. After sonification, the product is
then drained into a
collection vessel.
Apparatus 50 in Figure 5 may be constructed as a complete permanent or semi-
25 permanent unit, with only the starting plasma and collecting plasma bags
being disposable or
consumable subunits. Alternatively, and preferably, apparatus 50 in Figure S
is constructed
as a permanent or semi-permanent subunit and a disposable or consumable
subunit. In the
context of apparatus 50 of Figure 5, pump, 52, ozone generator, 53, ultrasound
driver, 58,
resonator plate, 59, and water jacket, 510, may be part of the permanent or
semi-permanent
3o subunit, while starting plasma bag, 51, reaction vessel, 57, and collection
bag, S 12, may be
part of one or more disposable or consumable units. The ozone line, 54,
including the filter
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trap, 56, and the mixing tip/spray nozzle, 55, may each be part of either the
permanent or
semi-permanent subunit or a disposable or consumable subunit.
In another preferred embodiment, the disposable bag used as the chamber
further
comprises a virus tight filter at one end of the bag to equilibrate the
pressures inside and
outside of the bag during vacuum processing. This FDA approved component also
allows for
easier mounting of the bag inside the region designed to accept the chamber.
In another
preferred embodiment, the disposable bag used as the chamber further comprises
grommets at
the inlet and outlet tubes to prevent them from collapsing during the
application of the
vacuum.
1 o In another preferred embodiment, the chamber has a roughened inner
surface. A
roughened inner surface allows the evolved gas bubbles to travel up to local
spikes on the bag
liner. From these points, the ultrasonic vibrations can dislodge the bubbles
relatively easily.
For comparison, bubbles flattened along one side of a smooth bag surface are
more difficult
to remove, even with agitation.
The main expense of the current disposables is the equipment required to
produce thin
layers or sprays in which the ozone can make intimate contact with the
contaminants.
Ultrasound provides several alternatives to this problem, the main benefits
being in the
mixing process itself. The basis for these effects is the ability of
ultrasound to modify the
properties of a liquid. One such effect is the tendency for ultrasound to mix
gasses into the
2o surface of a liquid if the applicator horn is not deeply immersed into the
liquid. Because the
resulting poor coupling causes reduced cavitation, such operation of
conventional ultrasound
equipment is to be avoided (High Intensity Ultrasonic Processor User's Guide,
Sonics &
Materials, Inc. Newton, CT, 1999).
Reduced cavitation and gas entrapment, however, is just what is required for
this
project. To encourage these processes, the ozone and plasma will therefore be
brought
together in a mixing chamber in which a plastic extender is placed just above
the plasma
surface. When sonified, this extender will then oscillate in and out of the
plasma, thus
entrapping small ozone pockets 20,000 times per second.
After forming this finely divided mixture, the next step is conceptually
similar to the
spraying systems, or "nebulizers" of existing ozone technologies. The problem
with these
conventional systems, however, is that they cause too much shear for plasma
proteins. The
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alternative is to use ultrasound to spray the already partially mixed plasma
and ozone
mixture, thus yielding even better mixing. Fundamentally, this process is not
unique to this
application; various ultrasonic nozzles are available commercially to produce
a fine, soft
spray. The only modification for this process is to use an extended length of
plastic tubing as
a nozzle, the end of which is free at an antinode to whip under sonification.
While this
simple arrangement is not as effective as commercial nozzles, it is quite
cheap.
Having thus developed a dispersed spray, the next concern is to contain and
process
it. These goals can be met by directing the spray into a small plastic
reaction vessel. The
spray then accumulates into pools, which coalesce into a volume of liquid.
When sonified at
o low intensity, any ozone gas bubbles disperse, aided by the enhanced
diffusion and decreased
viscosity effects of ultrasound.
Next, a short burst of more intense ultrasound is applied to the reaction
vessel. At
this time there is some radical formation, but this.can be minimized by using
a reaction vessel
shaped like an hourglass, thus yielding progressively less surface area up to
the fill line at the
midpoint. Much more important than radical formation, however, is the increase
in ozone
reaction speed. This speed is crucial not only for overall processing speed,
but also because
ultrasound is quite effective at removing ozone from aqueous solutions. The
apparent
mechanism behind this rapid purging is partly due to increased chemical
reactivity and partly
due to simple degassing. Effective decontamination thus requires rapid ozone
and pathogen
2o reactions, before the ozone is lost.
The overall arrangement for such a unit is shown in Figure 5. As done for the
light
exposure unit, the first step in this process is to use a peristaltic pump to
control the flow rate
of the plasma. Ozone from a conventional generator is then passed through a
connecting tube
to the mixing tip/spray nozzle assembly. Like the vacuum line in the light
exposure line, the
ozone feed tube is filtered and trapped across a sterile coupling to prevent
inadvertent
contamination of the ozone generator. After mixing the ozone and plasma, the
product is
then collected in the reaction vessel. Like the processor tray in the light
exposure unit, this
vessel sits on an ultrasonic driver separated by a water cooling pack. After
sonification, the
product is then drained into a collection vessel.
3o Figure 6 depicts a portion of another preferred apparatus of the present
invention,
which corresponds to the twenty-fifth and twenty-sixth main embodiments and is
useful for
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carrying out the method of the thirteenth through sixteenth main embodiments,
in continuous,
as opposed to batch-wise manner. In Figure 6, the fluid enters from an earlier
ultrasonic
degassing unit, which is not shown, and may pass through an optional cooler,
61. The fluid
then is formed into a thin film in the treatment assembly, 62, where it passes
between one,
preferably two, light sources, 63. The fluid then passes on to ozone treatment
or packaging,
64.
Figure 7 shows a preferred embodiment of the apparatus of the twenty-fifth and
twenty-sixth main embodiments, which is useful for carrying out the methods of
the
thirteenth through sixteenth main embodiments. A portion of Figure 7 also
corresponds to a
1o preferred embodiment of the contactor of the twenty-eighth main embodiment.
In Figure 7,
the fluid passes from bag 1, 71, through ozone contactor, 72, to bag 2, 73.
Bag 1, 71 is
equipped with a fill and drain port, 74, through which the fluid may be
introduced, and an
equalizer port, 75, through gas may be introduced to fill the void created by
exiting of the
fluid from the bag l, 71. Bag 1, 71, is maintained within a vacuum chamber,
76, to allow for
partial degassing, so that the spent ozone, which becomes oxygen, can be
replaced by fresh
ozone. An alternative approach to the vacuum chamber is to use very high
treatment
pressures, in excess of 150 psi. In this case, simply releasing the pressure
to ambient causes
the excess gas to evolve rapidly. A further enhancement for high pressure
operation is to
surround the fluid bags to be treated by solid blocks inside a pressure
chamber. Under this
2o approach, the air compressor is not necessary, because the blocks fill the
available residual
space in the chamber, thereby preventing the disposable bags from over
expanding and
subsequent rupture. As the fluid passes through the contactor, 72, it is
simultaneously
exposed to ozone and ultrasonic energy. The ozone is introduced into the
contactor, 72, via
an ozone inlet, 77, and into the fluid via a plurality of passage-ways in the
inner surfaces (not
shown) of the contactor, 72, while the ultrasonic energy is introduced into
the fluid via the
vibration of the inner surfaces of the contactor, 72, driven by an ultrasonic
driver, 78. After
passing through the contactor, 72, the fluid enters bag 2, 73, which is
equipped with a vent
port, 79, to vent the gas displaced by the entering fluid, and a drain port,
710, for draining the
fluid. Both bag 1 and bag 2 may be equipped with a mounting ring, 711.
For repeated treatment, such as indicated above for degassing, there are two
options.
One such option is to progress from one disposable bag to another. This is
preferred
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wherever possible.
Alternatively, the bags could be reused. Of course, such reuse raises the
question of
residual contamination. Note, however, that all parts of the system are
subject to direct ozone
gas exposure, and thus are continually being cleaned. Specifically, the
surfaces are cleaner
than the liquid passing through the system in bulk because when no bulk fluid
is present, the
surfaces are coated by at most a thin fluid layer, which is readily treated by
ozone exposure.
It is also possible to extend this reuse option even further. Specifically,
the above
description first uses a UV system followed by a separate ozone processor.
This is the option
that frees up the individual components most rapidly, i.e., one donated unit
can be exposed to
to UV, while a second unit is being treated with ozone. Another approach is to
reuse the
vacuum degasser bag from the UV unit in the ozone unit, again with enough
ozone flow to
decontaminate the bag between cycles.
Likewise, it is also possible to make the UV degasification bag from UVC
transparent
material so that a single bag suffices for both processes. The determining
factor here is
whether an individual site is more concerned with throughput or disposable
expense. For
example, a major metropolitan blood collection center would use maximum
throughput,
while an isolated military field hospital or a hospital in a lesser-developed
country would
minimize the requirements for disposables. Such considerations can be made
only on a site-
by-site analysis.
2o Figure 8 is a detailed cross-sectional view of the contactor portion of the
apparatus
shown in Figure 7. The contactor of Figure 8 is made up of 4 distinct layers,
which form
three different flow fields. First there are upper and lower outer layers, 81
and 82,
respectively. Second, there are upper and lower inner layers, 83 and 84,
respectively, which
are perforated by a plurality of channels, 85. It should be noted that the
upper and lower
2s inner layers correspond to the substrates) described above in the context
of the twenty-
seventh and twenty-eighth main embodiments, and the channels correspond to the
passage-
ways described above in the context of the twenty-seventh and twenty-eighth
main
embodiments. The lumen formed by the upper outer and inner layers, 81 and 83,
and by the
lower outer and inner layers, 82 and 84, are connected to the ozone inlet, 77,
depicted in
3o Figure 7, and permit the flow of ozone through the contactor, and are
referred to as ozone
flow fields, 86. The lumen formed by the upper and lower inner layers, 83 and
84, is
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connected to a source of the fluid such as bag l, 71, depicted in Figure 7,
and is referred to as
the liquid flow field, 87. As the fluid flows through the liquid flow field,
87, ozone is
introduced into the fluid through the channels, 85. Simultaneously, ultrasonic
energy is
introduced into the fluid by the vibration of the upper and lower inner
surfaces, 83 and 84 by
means of an ultrasound generator (not shown) which is coupled to the upper and
lower inner
surfaces, 83 and 84.
Figure 9 shows another preferred embodiment of the apparatus of the twenty-
fifth and
twenty-sixth main embodiments, which is useful for carrying out the methods of
the
thirteenth through sixteenth main embodiments. A portion of Figure 9 also
corresponds to a
l0 preferred embodiment of the contactor of the twenty-ninth main embodiment.
In Figure 9,
the fluid passes from bag 1, 91, through ozone contactor, 92, to bag 2, 93.
Bag 1, 91 is
equipped with a fill and drain port, 94, through which the fluid may be
introduced, and an
equalizer port, 95, through gas may be introduced to fill the void created by
exiting of the
fluid from the bag 1, 91. Bag 1, 91, is maintained within a vacuum chamber,
96, to allow for
partial degassing, so that the spent ozone, which becomes oxygen, can be
replaced by fresh
ozone. As the fluid passes through the contactor, 92, it is simultaneously
exposed to ozone
and ultrasonic energy. The ozone is introduced into the contactor, 92, via an
ozone inlet (not
shown) and into the fluid via a plurality of passage-ways in the inner
surfaces (not shown) of
the connector, 92, while the ultrasonic energy is introduced into the fluid
via the vibration of
the inner surfaces of the connector, 92, driven by an ultrasonic driver (not
shown). After
passing through the contactor, 92, the fluid enters bag 2, 93, which is
equipped with a vent
port, 99, to vent the gas displaced by the entering fluid, and a drain port,
910, for draining the
fluid. Both bag l and bag 2 may be equipped with a mounting ring, 911. The
flow of the
fluid from bag 1, 91, through the contactor, 92, to bag 2, 93, is assisted by
a pump, 912.
Figure 10 shows a cross-sectional view of a preferred embodiment of an ozone
contactor according to the twenty-ninth main embodiment of the present
invention. In Figure
10, the fluid flows from the inlet, 1001, through the fluid flow field, 1002,
to the outlet, 1003.
The ozone enters the ozone inlet, 1004, flows through the ozone flow field,
1005, and is
introduced into the fluid flow field, 1002, through a plurality of channels,
1006, in the walls
forming the fluid flow field, 1007. Any excess ozone exits the ozone flow
field, 1005,
through an exit, 1008. The walls which form the fluid flow field, 1007, are
made to vibrate
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by being coupled to one or more ultrasonic drivers, 1009.
Figure 11 shows a cross-sectional view of another ozone contactor which is
particularly useful for platelets. In the contactor shown in Figure 11, the
treatment chamber
consists of a rectangular or similarly shaped block 1101 with staggered,
opposed shelves in
the shape of sharp wedges 1102. With the chamber in the horizontal position,
the liquid
enters a trough or inlet port 1103 along one side. After filling this trough,
the chamber is then
rotated upwards to about 80 degrees, at which point the fluid flows over the
first shelf 1102a
towards the opposing wall. Because the shelf does not actually touch the
opposing wall,
however, the fluid drops down to the next shelf 1102b and the flow then
reverses.
1o Meanwhile, ozone is introduced through ports 1104. Note that this
arrangement is unlike the
above cited patents because the flow reversal thoroughly mixes the material at
each step, with
the top layer becoming largely the bottom layer and vice versa.
The rotation continues until all of the fluid is emptied from the inlet
trough, which
occurs at about 90 degrees. The entire arrangement is then rotated back into
its original
position, and then on to -90 degrees to repeat the process from the opposite
direction. During
these movements, ozone is continuously fed into one side of the treatment
chamber, and spent
gas removed from the opposite side. Because the motion of the chamber is thus
essentially
two reversing half turns, the gas connections can be conventional flexible
hoses. This
arrangement thereby saves the costs and installation problems of the sealed
bearings, etc., that
2o are required for the continuous rotation units described earlier.
For all of the foregoing embodiments, the last step is to shut down the system
and
store the product. As noted earlier, small solenoids can be used to tilt the
components to
drain the quite valuable product as completely as possible. This concept can
also be extended
to drop the exit side of the "v" of the spray nozzle.
2s One approach is to collect the product after the last ozone injection pass.
This would
leave a substantial amount of ozone in the liquid. As it reacts and decays to
oxygen, this
residual ozone would provide a slight increase in decontamination
effectiveness. Also, the
resulting oxygen would be quite beneficial to red blood cells and platelets.
Conversely, if the product is to be frozen, the collection should be taken
after the
3o product is degassed as thoroughly as possible. This step reduces the
formation of gas bubbles
(commonly observed as small pockets and streaks in ice cubes) in the frozen
product, and
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thus leads to less product damage during the freezing process.
It is recognized that some variation of the exact conditions and/or parameters
of the
present methods may be needed to achieve optimum results for certain types of
pathogens
and infectious agents and fluids. In particular, it is recognized that some of
the conditions
and/or parameters of the present methods may need to be varied to achieve
optimum results
for certain types of pathogens and infectious agents, while minimizing plasma
protein
damage. Such conditions and/or parameters which might need to be varied
include the
precise intensity and/or frequency of the ultrasonic energy; the precise
intensity and/or
frequency of the UV, gamma, and/or x-ray radiation; the precise amount of
ozone to be
1 o mixed with the plasma; the pressure of the ozone; and the precise
temperature and/or time of
any step.
In recognition of the fact that the precise conditions and/or parameters of
the present
methods may need to be varied to achieve optimum results for certain types of
pathogens and
infectious agents, while minimizing damage to the plasma proteins, the
following discussion
of how to assess and optimize the precise conditions and parameters used in
the present
methods is provided. The efficacy of any of the present decontamination
methods for any
given pathogen or infectious agent may be determined by:
( 1 ) determining the concentration or activity of a selected pathogen in a
sample of
the fluid;
2o (2) carrying out one of the present decontamination methods on said sample
of
fluid, to obtain a sample of decontaminated fluid; and
(3) determining the concentration or activity of said selected pathogen in
said
sample of decontaminated fluid.
The parameters of any of the present decontamination methods may be optimized
by
assessing the efficacy of the method against the pathogen in a first test,
then again assessing
the efficacy of the method after varying one or more parameters and/or
conditions of the
method, and then comparing the results of the two tests. Of course, it may be
necessary to
compare the results of more than two tests to fully optimize any one condition
or parameter.
Accordingly, it may be preferable to carry out a battery of tests to construct
a hyper-
3o dimensional matrix of test results.
The amount of damage to a particular fluid (e.g., plasma) protein and the
optimization
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of any of the present methods in regard to the minimization of plasma protein
damage may be
carried out in the same way, with the exception of determining protein
concentration or
activity rather than pathogen concentration or activity.
Any such testing on plasma itself, of course, requires plasma. However,
unfortunately, the concentrations of plasma proteins vary widely from donor to
donor. This
is a significant problem because these variations are usually greater than the
fractional protein
damage caused by the decontamination methods themselves, thus making direct
comparisons
difficult. For example, the standard reference range for fibrinogen is 200 to
400 mg/dl, but
even a relatively poor (in terms of harshness to the plasma proteins)
decontamination
to technique would destroy less than 25% of this protein. As a result, the
variation in the
plasma proteins from individual unit to individual unit would thus mask the
entire range of
protein damage.
Accordingly, when assessing the efficacy or optimizing the present
decontamination
methods for a particular pathogen or infectious agent it is preferred to use a
reference plasma
t s obtained by pooling several donations and then extracting multiple units
of the same volume.
The use of such a reference plasma establishes a common basis for comparison.
Pooling also
eliminates much testing expense: with a pool, the protein levels can be tested
once to
establish starting conditions, but for individual units, each starting
condition must be tested
separately. This pooling technique has proven to be quite successful in
earlier work done by
2o the present inventor, in which it was found that the inherently large error
in blood testing
equipment could be better offset by multiple tests of a single pool, versus
repeated tests of
single units. It should be noted that this pooling is for assessment and
optimization purposes
only, i.e., it does not restrict in any way the ability of the present methods
to process
individual plasma units.
2s For the purposes of initial assessment and optimization for a particular
pathogen, it
may be preferred to lower costs by using non-human plasma. In this regard,
bovine plasma
provides a useful starting point without the cost or handling problems
associated with human
products. Once the certain parameters, such as flow rates, ultrasound
intensities, etc., have
thus been optimized for bovine plasma, human plasma may then be used.
3o Of course, it should be understood that the plasma as obtained from the
donors)
might not contain any detectable amounts of the pathogen or infectious agent
of interest. In
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such cases, a known amount of the pathogen or infectious agent may be added to
the plasma.
The next concern is what plasma protein to test. Given the multitude of
components
in plasma, there are many options, notably Factor VIII, fibrinogen, von
Willebrands factor
and various immunoglobulins. Of these proteins, fibrinogen is the most
appropriate: it is
clinically significant, it is commercially valuable, it is easy to test, it is
readily damaged by
existing decontamination techniques and its widespread use by other
investigators provides a
means for direct comparisons.
Obviously, handling live human pathogens is expensive and difficult. For this
reason,
model viruses, which simulate actual human viruses, may be used for
experimental
t o decontamination testing. Specifically, their low cost, low risk, and
direct applicability to
actual pathogens have led to complete industry and regulatory acceptance.
Because of these
benefits, many test viruses have been isolated and are now in common use.
Typical examples
of such model viruses include Sindbis and BVDV (Bovine Viral Diarrhea Virus)
for human
HCV, and duck HBV for human HBV (B. Horowitz, "Virus Inactivation by
Solvent/Detergent Treatment and the
Manufacture of SD-Plasma," Vox Sank vol. 74, Suppl. 1, pp. 203-206 (1998)).
While these are certainly significant viruses, however, much of the recent
interest in
the blood industry has focused on human parvovirus B 19. From a clinical
standpoint,
erythema infectiosum or "fifth disease" from parvovirus is mainly a concern
during
2o pregnancy, and thus poses much less risk to the general population than
hepatitis or AIDS. In
terms of the blood industry, parvovirus infection is actually so common that
Plas+SD is sold
with the claim that the pooled donors effectively contribute antibodies. On
the other hand,
parvovirus is extremely difficult to eradicate by conventional techniques. The
net result is
that there is some question within the blood industry about the cost-
effectiveness of attacking
this particular virus. Within the decontamination discipline, however,
parvovirus is of great
interest as a test standard because any technique that is effective against
such a robust virus
would also be extremely effective against lesser pathogens. Accordingly, it
may be preferred
to use parvovirus, in particular porcine parvovirus (PPV), as the standard
test virus for the
optimization of the conditions and/or parameters of any one of the present
methods.
3o Having thus selected the plasma and a test virus, the next concern is how
to measure
the effectiveness of the proposed technology. Fortunately, such measurements
are actually
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quite simple using standard procedures. Specifically, the plasma may be first
spiked with the
test virus and then split into two fractions. One fraction is then maintained
as a control, while
the other fraction is subjected to the decontamination methods being assessed
or optimized.
For statistical analysis, six samples may be taken at each test point. The
test results for a
given pathogen can be reported in terms of a logarithmic reduction factor.
It should be noted that the logarithmic reduction factor is a quantitative
measurement.
As such, it can be inserted directly into a standard test matrix. Because
ultrasonic effects are
nonlinear, this matrix is in turn nonlinear. This matrix can, however, be
reduced by standard
steepest descent techniques. The result is an optimized system, within the
limits of resolution
to of the test points. For example, given Beer's exponential law of optical
absorption, it is
anticipated that the logarithmic reduction factor will drop substantially
beyond a critical fluid
depth. Having thus established this critical value, the residence time can
then be optimized
by changing the pump rate and/or the dimensions of the treatment chamber.
Although the literature has some limited information on the ability of various
proteins
15 to withstand ultrasound, there is unfortunately a great deal of variation
in application
techniques, heat control, sonifier design, power measurements, etc. For
example, power can
be measured as the wattage applied to the transducer, or ultrasonic generator,
but if the
system is not tuned for resonance, the power actually applied to the sample
can be much less.
The net result is that transferring such results from one case to another is
thus quite
2o unreliable. Thus, it may be preferred to use the above-described
optimization method to
determine to original or factory settings for a particular apparatus design.
It may also be
preferred to use the above-described optimization method to perform routine
calibrations of
apparatus. In this regard, it may be preferred to use the amount of dissolved
oxygen in the
plasma before and after sonification as a measurement of the amount of sonic
energy being
25 applied to the plasma by a given apparatus.
It should be recognized that the present methods may not be effective by
themselves
for the complete removal of all contaminants. Accordingly, it may be desired
in certain
circumstance to utilize a small amount of quencher in conjunction with those
embodiments
which involve irradiation. The advantages in the context of the present
invention are lower
3o quench concentrations, reduced chemical cost, reduced removal cost, and
less bioburden.
It should also be recognized that in addition to using the present
combinations of
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ultrasound and vacuum prior to UV exposure, the present methods may also be
used to
prepare anaerobic synthesis systems for biotechnology applications. The
advantage here is
that the processed systems are then be ready for immediate UV and/or ozone
treatment.
Other features of the invention will become apparent in the course of the
following
descriptions of exemplary embodiments, which are given for illustration of the
invention and
are not intended to be limiting thereof.
EXAMPLES
The following examples are presented only as representative of the equipment
and
1 o techniques described above. These examples are not intended to limit the
scope of the
technology, or the materials that can be treated. The following examples
describe
increasingly more sophisticated experiments, beginning with a basic system,
and then
progressing towards more specialized devices. In each case, blood products are
used,
although different blood products are used for different experiments.
15 In each case, however, the same test virus is used: porcine parvovirus. As
described
above in embodiment VII, parvovirus is of particular interest because
destruction of this
small, robust, non-enveloped virus implies destruction of lesser viruses as
well. Also as
noted in embodiment VII, the porcine form of parvovirus is convenient to
handle because it
cannot infect humans.
20 As for sources, porcine parvovirus is quite widespread throughout the
agricultural
industry, and is thus commonly available. For detailed experimental work,
however, a well-
defined source is desired, such as the American Type Culture Collection,
Manassas, VA, Item
number VR-742.
Likewise, because parvovirus is so widespread, many veterinary facilities and
schools
25 can analyze it. A particularly well known group, however, is the American
BioResearch
Laboratories, located in Sevierville, Tennessee.
As for test solutions, plasma is particularly useful because it has both heat-
tolerant
components, notably fibrinogen, and heat-sensitive components, notably Factor
VIII. For
convenience, bovine plasma can be used instead of human plasma; in any case,
porcine
3o plasma should not be used due to the high likelihood of existing
antibodies. Although bovine
plasma is readily available from slaughterhouses, higher quality material is
available from
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facilities dedicated to the maintenance of healthy donor animals, such as Quad
Five, Ryegate,
MT. In addition to plasma, erythrocytes can also be obtained from bovine
sources;
alternatively, out of date human cells can also be used, following the
convention that scarce
transfusable materials should not be used for basic experimental purposes.
Likewise, human
platelets, which have a shelf life of only 5 days, are also available on an
out of date basis. All
blood banks have such materials, but the largest supplier is the American Red
Cross, which
has offices nationwide.
Having thus identified the materials and their sources, testing must then be
arranged.
Because all of the above blood products have significant clinical interest,
testing can be
1 o preformed readily in any modern hematology laboratory. For example, bovine
blood
products are routinely analyzed at the University of Georgia Veterinary School
in Athens,
GA, while human blood is tested at the Emory University Hospital Hematology
Laboratory in
Atlanta, GA.
With such suppliers and support services, a broad range of experiments can be
performed. The first such test is a heat-tolerant plasma protein, fibrinogen.
This protein is
particularly interesting because it is a crucial part of surgical glues, such
as Tisseel by Baxter
Healthcare, Deerfield, IL.
Example 1: Heat-tolerant plasma protein
2o This test is designed for a heat tolerant protein, fibrinogen. This test is
also designed
for a small quantity, on the order of several ml, because this is the volume
of material that can
be extracted from a single unit of donated plasma. The test procedure is
described below.
First, configure the equipment to handle a small, heat-tolerant material. The
required
system equipment includes a warming component, a small vacuum module, a UVC
exposure
module, and an ozone treatment module. The disposable is a small, single unit
device,
consisting of a liner for the heater, a Teflon~ bag to serve in both the
vacuum chamber and
UVC irradiator, a pair of bags for the ozone exposure unit, an ultrasonic
ozone contactor, two
virus tight filters, and sterile connecting tubing. Load the disposable items
and apply vacuum
to evacuate the system to 50 mbar.
3o Next, dilute I ml of porcine parvovirus by 10:1 in bovine plasma. For
statistical
purposes, prepare 6 sets of six samples each. Retain one set as a control.
Heat the remainder
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of the samples to 52 °C to aid in degassing. Remove 1 set at this point
to check for heat
effects. Next, immediately degas the remaining sets, and remove one set to
check for protein
damage. Monitor the degassing process with a hydrophone, and record the
elapsed process
time. Next, expose all but one of the remaining sets to UVC irradiation for 5
minutes at a
s distance of 10 cm from the lamps, record the total dosage, and remove one
set. Expose the
remaining two sets (one UVC treated, and one not) to ozone at 3 atmospheres
for 15 minutes.
After these experiments are complete, place all samples on ice and transport
to blood
and virus testing facilities. After testing, analyze samples to determine
confidence levels and
any statistically significant differences.
Results: Compared to the controls, the heating and degassing alone show no
significant differences in viral loading or protein damage. Conversely, the
UVC irradiation
alone shows a Log 6 reduction in active parvovirus loading; likewise the ozone
treatment
alone shows a Log 6 reduction in parvovirus. The combined UVC and ozone shows
an
apparent Log 12 reduction, although at such levels, detection is much more
difficult. In all
cases, the fibrinogen loss is less than 5%, which is essentially within the
limit of error of the
measuring equipment.
Example 2: Heat-sensitive plasma protein
This test is designed for a heat tolerant protein, Factor VIII, which is known
to be
2o quite labile. All of the test conditions are as above, except that no
heating is done on the
sample prior to degassing. To compensate for this difference, increase the
vacuum to 40
mbar, and extend the degassing time as indicated by the hydrophone monitor.
Results: As observed for fibrinogen, the degassing tests show no measurable
decontamination or protein damage. Also as observed for fibrinogen, both UVC
and ozone
show Log 6 reduction in parvovirus, and the combined process shows Log 12,
again with the
problem of low level detection. Finally, the protein levels are reduced by
less than 5%, which
is within the limits of the measuring equipment.
Example 3: Batch plasma
3o This test is designed to evaluate the system when processing sequential
batches of
material. The specific material is a single donor unit of plasma. As such, it
is larger than the
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volumes tested above, but smaller than the volumes that would be treated by
the continuous
flows discussed below in Example 4. To accommodate this volume, the equipment
described
in the previous example must be changed as follows.
Because the volume to be treated cannot be handled in the small disposables
described earlier, larger units, with provisions for transferring the flow
from module to
module, must be used. First, replace the single use vacuum chamber liner with
a multiple use
unit, which amounts to using a liner with an inlet and exit for the processed
fluid. Next,
replace the irradiation bag with a larger unit, and orient this unit upwards
as described earlier
to ensure proper residence times. Next, replace the ozone treatment bags with
larger units.
l0 Finally, as an additional test, degas and then ozone saturate the fluid
three times.
Results: The individual UVC and ozone cases each yield Log 6 virus reductions,
with
Log 12 for the combined process. For the repeated ozone exposure, the viral
reduction is Log
9. In all cases, the Factor VIII damage is less than 5 %.
Example 4: Continuous flows
This test is designed to evaluate the system when processing a continuous flow
of
material. The specific material is again plasma, but in this case the large
flow corresponds to
the treatment of a pool of material for subsequent fractionation. To
accommodate this
volume, the equipment described in the previous example must be changed as
follows.
2o The major modification is to change the batch flow equipment to continuous
flow
equipment. Specifically, the major changes are made to the degassing and ozone
treatment
modules, along with their associated modules; the batch flow UVC module can
also be used
for continuous flows with only the addition of a flow controller on the feed
pump.
For the degassing module, 4 separate steps are used for this experiment. The
degassed liquid is then returned to atmospheric pressure across the discharge
of a peristaltic
pump that is gravity fed from the degassing trays. UVC exposure follows
immediately, and
then the fluid is exposed to ozone. The residence time in the UVC and ozone
modules is
about 15 minutes each, with the actual treatment time being continuously
varied slightly
about this value according to the respective dosage monitors.
3o Results: The individual UVC and ozone cases each yield Log 6 virus
reductions, with
Log 12 for the combined process. The Factor VIII damage is less than 5 %.
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?xample 5: Erythrocytes
This test is designed to determine the effect of decontamination on a living
cellular
structure. The equipment and conditions are essentially as described for
Example 1, except
for three factors. The first concern is that a much larger illumination
chamber (30 cm by 10
cm) is used to treat a larger surface area than required for the relatively
transparent fluid used
in Example 1; the volumes are nevertheless similar because the erythrocyte
chamber is much
thinner at about 40 microns. The second difference is that the heating is done
only to 45 °C,
instead of 52 °C, because erythrocytes are known to withstand this
lower temperature well
during hyperthermia treatments, but the higher (52 °C) temperature
could compromise their
cell membranes. The third concern is that the erythrocytes are exposed to
oxygen, or
oxygen/ozone, immediately after irradiation because these cells require oxygen
to survive.
Note that nerve cells in the brain are known to suffer irreversible damage
after only 6 minutes
without oxygen, but erythrocytes are much more durable. Furthermore,
decreasing the
l5 temperature immediately after degassing also helps to maintain cellular
integrity.
After performing the previously described procedures, the viral test results
are Log 6
reduction for both UVC and ozone, while the combined process is about Log 12.
Cell
damage in such testing is commonly measured by hemolysis. Observing the number
of
damaged cells immediately after the process indicates gross mechanical damage,
while
?o measuring the cell damage 24 hours later indicates the viability of the
treated cells (see, G. F.
Doebbler, A. W. Rowe and A. P. Rinfret, "Freezing of Mammalian Blood," in
Cryobiology,
Harold T. Meryman (ed.), Academic Press Inc., London, 407, 1966). In this
experiment, the
treated materials could not be distinguished from the controls, thereby
indicating no
significant damage.
?5
Example 6: Platelets
This test is designed for the platelet ozone contactor. The equipment and
conditions
are essentially as described in Example 1 for heated, small volumes, except
for three factors.
The first difference is the use of a special ozone contactor designed solely
for platelets, and
.0 shown in Figure 11. The second difference is that the heating is done only
to 22 °C, instead
of 52 °C, because FDA regulations state that platelets must be
maintained at 22 ~ 2 °C to
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retain viability. The third factor is that the illumination must be done
immediately after
degassing, as reported above for erythrocytes, because platelets require
oxygen for long term
maintenance.
The results of this test are Log 6 reduction of parvovirus during UVC and
ozone, and
Log 12 for the combined processes. To determine platelet damage, the first
test is a simple
optical procedure widely used in the platelet industry. This test amounts to
simply observing
the flow of the platelets in their special oxygen-permeable bag. Normal
platelets should
sparkle, while damaged platelets often agglomerate, and thus do not sparkle
when
illuminated. In this experiment, the treated platelets exhibit the same
sparkle as shown by the
1o controls. The second test is to determine clotting effectiveness, which is
done by forming a
clot from the gel and then rupturing it, thereby indicating whether or not the
platelets function
as necessary. In this experiment, the clots formed from the control are
indistinguishable from
the clots formed from the treated material, thereby indicating no appreciable
damage to the
platelets during decontamination.
Summary: The above examples indicate that the new technology inactivates a
robust virus to
acceptable levels with minimum damage to the materials being treated.
Obviously, numerous modifications and variations of the present invention are
2o possible in light of the above teachings. It is therefore to be understood
that, within the scope
of the appended claims, the invention may be practiced otherwise than as
specifically
described herein.
All patents and other references mentioned above are incorporated in full
herein by
this reference, the same as if set forth at length.
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