Note: Descriptions are shown in the official language in which they were submitted.
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PURIFICATION OF RED BLOOD CELLS BY SEPARATION AND DIAFILTRATION
BACKGROUND OF THE INVENTION
The development of hemoglobin-based oxygen carriers has focused on oxygen
delivery
for use in medical therapies such as transfusions and the production of blood
products.
Hemoglobin-based oxygen carriers can be used to prevent or treat hypoxia
resulting from blood
loss (e.g, from acute hemorrhage or during surgical operations), from anemia
(e.g., pernicious
anemia or sickle cell anemia), or from shock (e.g, volume deficiency shock,
anaphylactic
shock, septic shock or allergic shock).
Existing hemoglobin-based oxygen carriers include perfluorochemicals,
synthesized
hemoglobin analogues, liposome-encapsulated hemoglobin, chemically-modified
hemoglobin,
and hemoglobin-based oxygen carriers in which the hemoglobin molecules are
cross-linked.
Preparation of hemoglobin-based oxygen carriers includes several purification
steps. In order
to remove plasma proteins from whole blood, a process of microfiltration is
used to wash the
cells of whole blood. The cell washing operation removes plasma proteins from
bovine whole
blood using diafiltration over a 0.2 m microfiltration membrane with isotonic
saline/citrate
solution. Diafiltration is a continuous filtration operation in which
saline/citrate solution is
added to the filter retentate to maintain a volume in the recirculation tank.
The blood solution
is recirculated across the filter and the filtrate, containing the plasma
proteins, is sent to waste.
Washing the blood solution using filtration results in highly variable
processing times
which adversely effect product throughput. Additionally, extended cell washing
process times
could lead to growth of unacceptable levels of bioburden and to cell lysis,
thereby further
reducing process yield.
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SUMMARY OF THE INVENTION
The invention generally is directed to a method of purifying red blood cells
for use in
the manufacture of a blood substitute. The method includes separating whole
blood, whereby a
red blood cell fraction and a liquid fraction are formed. The red blood cell
fraction is
diafiltered to thereby form purified red blood cells. The purified red blood
cells can then be
processed further to isolate the hemoglobin molecules.
In one embodiment, the present invention is drawn to forming a lysate of
purified red
blood cells for use in a hemoglobin blood substitute. The method comprises,
separating whole
blood, whereby a red blood cell fraction and a liquid fraction are formed. The
red blood cell
fraction is diafiltered to form purified red blood cells. The purified red
blood cells are lysed to
form a lysate of purified red blood cells.
In another embodiment, the method includes separating defibrinated whole
bovine
blood by centrifugation, whereby a red blood cell fraction and a liquid
fraction are formed.
The red blood cell fraction is diafiltered to thereby form purified red blood
cells. The purified
red blood cells then are mechanically lysed.
This invention has many advantages. For example, separating of whole blood
into a red
blood cell fraction and a liquid fraction removes many potential membrane
foulants from the
resultant red blood cell fraction, allowing more efficient processing of the
red blood cell
fraction. Specifically, separating whole blood to form a red blood cell
fraction and a liquid
fraction, and then diafiltering the red blood cell fraction can reduce the
period of time necessary
to obtain purified red blood cells by diafiltration or by centrifugation
alone. Generally, the
period of time necessary to diafilter a red blood cell fraction to thereby
form purified red blood
cells is normalized; the diafiltration period of a red blood cell fraction
will approximate more
closely the time necessary to diafilter a relatively pure sample of suspended
red blood cells.
The decrease in time necessary to obtain purified blood cells is obtained with
whole blood and
defibrinated whole blood. Reduced or normalized cell washing process times
reduces the
potential for growth of unacceptable levels of bioburden and cell lysis. In
turn, the reduction in
growth of unacceptable levels of bioburden and cell lysis increases process
yield.
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BRIEF DESCRIPTION OF THE DRAWINGS
The Figure is a schematic representation of apparatus suitable for conducting
the
method of the invention.
DETAILED DESCRIPTION OF THE INVENTION
The foregoing and other objects, features and advantages of the invention will
be
apparent from the following more particular description of preferred
embodiments of the
invention, as illustrated in the accompanying drawing. The drawing is not
necessarily to scale,
emphasis instead being placed upon illustrating the principles of the
invention.
The invention generally is directed to a method of purifying red blood cells
by
separating whole blood into a'red blood cell faction and a liquid fraction.
Potential membrane
foulants are believed to be partitioned from a resulting red blood cell
fraction. A liquid fraction
that includes a substantial portion of the foulants, and which is formed by
fractionating the
whole blood, is separated from the red blood cell-fraction and the red blood
cell fraction then is
diafiltered.
Referring to the Figure, shown therein is apparatus 10, which is one
embodiment of an
apparatus suitable for practicing the method of the invention. Whole blood is
collected in
vessel 12. Whole blood suitable for use in the invention can be freshly
collected or collected
from otherwise outdated sources, such as expired human blood from a blood
bank. Further, the
whole blood can have been maintained in a frozen and/or a liquid state,
although it is preferred
that the whole blood not be frozen prior to use in this method. Examples of
suitable sources of
whole blood include human, bovine, ovine, porcine, other vertebrates and
transgenically-
produced hemoglobin, such as the transgenic Hb described in BIQITECHNOLOGY,
12: 55-59
(1994). The whole blood can be collected from live or freshly slaughtered
animal donors.
One method for collecting bovine blood is described in U.S. Patent Nos.
5,084,558 and
5,296,465, issued to Rausch, et al.
In one embodiment, the whole blood is defibrinated in vessel 12 by a suitable
method.
Defibrination can be accomplished as described in U.S. Patent No. 6,518,010 by
Gawryl, et
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al., filed on February 28, 2001. Defibrinating the blood initiates the
clotting cascade, artificially removing the fibrin molecules involved in the
formation of blood clots. Defibrination can be induced by chemical or
mechanical means. Chemical coagulating agents are defined herein as substances
that induce
clotting. For example, collagen induces coagulation, so that when there is an
external wound, a
fibrin clot will stop blood from flowing. Artificially exposing blood to
collagen will cause the
formation of fibrin clots, which can be removed to produce defibrinated blood.
Examples of
other coagulating agents are tissue extract, tissue factor, tissue
thromboplastin, anionic
phospholipid, calcium, negatively charged materials (e.g., glass, kaolin, some
synthetic plastics,
fabrics). A preferred clotting agent is collagen. The liquid fraction obtained
by separating
blood cells from defibrinated whole blood typically is referred to as "serum."
The whole blood can be exposed to the clotting agent for a period of time
sufficient to
cause essentially all fibrin in the blood to be converted into a fibrin clot.
The appropriate time
is determined by the point at which fibrin molecules apparently stop
polymerizing. Chemical
defibrination, defined herein as defibrination that is induced by exposure to
a chemical
coagulating agent, is conducted at a suitable temperature, preferably a
temperature in a range of
between about 4 C and about 40 C.
In another embodiment, mechanical agitation, such as stirring, can also be
used to
initiate the clotting cascade. The whole blood can be stirred until fibrin
polymerization
apparently ceases. The accumulated fibrin is removed to complete
defibrination. Mechanical
defibrination, defined herein as defibrination induced by agitating the blood
solution, is
conducted at a suitable temperature, and preferably at a temperature in a
range of between
about 4 C and about 40 C.
In an alternative embodiment, the whole blood can be treated to prevent
coagulation.
For example, the whole blood can be treated with an anti-coagulation agent
such as sodium
citrate, heparin, ethylenediarnunetetraacetic acid (EDTA) and sodium oxylate
used at
concentrations sufficient to inhibit coagulation of the whole blood. In one
embodiment, 5L of
sodium citrate (34 g/1) is added to 15L of freshly collected whole blood to
yield a final
concentration of 8.5 g/1 citrate in the whole blood solution. In another
embodiment, EDTA is
added to the freshly isolated whole blood to yield a final concentration of
0.18%. The liquid
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fraction obtained by separating blood cells from whole blood treated with
anticoagulants
typically is referred to as "plasma."
It is also possible to defibrinate blood that already has been citrated by
saturating the
citrated blood with a divalent cation, and then defibrinating the solution,
similar to the means
by which noncitrated blood would be processed. The preferred divalent cation
is calcium.
Where the blood has been treated to induce coagulation, fibrin clots are
removed from
the whole blood by suitable means. An example of a suitable means is shown in
the Figure.
The whole blood, including the fibrin and fibrin clots, is directed from
vessel 12, through the
line 14 and strainer 16. A 60 mesh screen is an example of a suitable
strainer. Optionally, or
alternatively to the use of a strainer, cheesecloth or polypropylene filters
can be employed to
remove large debris, including fibrin clots. The fibrin clots are collected at
strainer 16 and the
remainder of the whole blood is directed to vessel 18.
Where the blood is treated with an anticoagulant, the treated blood can be
directed to
centrifuge 27, bypassing line 14, strainer 16, vessel 18, line 20, pump 22
filter 24 and filter 26.
However, the treated blood can also be directed through line 14, strainer 16,
vessel 18, line 20,
pump 22 filter 24 and filter 26. Directing the anticoagulant-treated whole
blood through
strainer 16 and filters 24 and 26 is useful to remove any large debris present
in the treated
whole blood.
As shown in the Figure, whole blood (either treated to induce coagulation or
not) is
directed from vessel 18 through line 20 by pump 22 and through first filter 24
and second filter
26 to centrifuge 27. In one embodiment, first filter 24 and second filter 26
are polypropylene
filters. In a particularly preferred embodiment, first filter 24 has a
permeability of about 800
m, and second filter 26 has a permeability of about 50 m. Where the whole
blood has been
treated to initiate the clotting cascade, removal of essentially all of the
fibrin by first filter 24
and second filter 26 completes the defibrination step.
After filter 26, the whole blood, either defibrinated or not, is subjected to
separation to
form a red blood cell fraction and a liquid fraction. "Separation," also
referred to herein as
"fractionation," includes partitioning of red blood cells from serum or plasma
to form a
separate liquid fraction which is preferably the serum or plasma,
respectively. The separation
techniques as used herein generally are based on separation by density, such
as by
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centrifugation, as distinguished from separation based on size, such as by
diafiltration.
As used herein, "liquid fraction" includes liquid resulting from separation of
whole
blood and separation of whole blood that has been defibrinated. In one
embodiment, the liquid
fraction is "serum," e.g., the liquid fraction resulting from the separation
of defibrinated whole
blood. In another embodiment, the liquid fraction is "plasma," e.g., the
liquid fraction resulting
from the separation of whole blood that has been treated to prevent
coagulation. In one
embodiment, the whole blood has been treated with anticoagulants such as
sodium citrate or
heparin.
In one embodiment, after filter 26, the whole blood, either defibrinated or
not, is
subjected to separation in centrifuge 27. Typically, during centrifugation,
the whole blood is
exposed to a G-force in a range of between about 1,000 and about 12,000 x G in
order to
separate the whole blood and thereby form the red blood cell fraction (blood
cell component)
and the liquid fraction. Typically, centrifugation is conducted over a period
of time in a range
of between about 30 seconds and about 4 minutes. Preferably, the
centrifugation is conducted
at about 8,000 - 10,000 x G for about 3 minutes. The temperature of the whole
blood during
the separation step generally is in a range of between about 4 and about 15
C.
In one embodiment, the red blood cell fraction includes red blood cells and
white blood
cells of the whole blood and the liquid fraction includes the platelets of the
whole blood. Also,
preferably, the red blood cell fraction includes most of the red blood cells
(RBCs) (e.g., at least
about 90%, or at least about 95%, or at least about 99%) of the whole blood.
The liquid fraction is substantially removed from the red blood cell fraction.
Typically,
the liquid fraction is removed from the red blood cell fraction simultaneously
with
centrifugation. In one embodiment, the liquid fraction is continuously removed
during
centrifugation using, for example, a tubular bowl centrifuge in continuous
feed mode. In
another embodiment, a tubular bowl centrifuge can be used in a batch mode. In
another
embodiment, the liquid fraction is removed from the red blood cell fraction
after separation of
the red blood cell fraction and the liquid fraction by decanting the liquid
fraction from the red
blood cell fraction.
The red blood cell fraction is directed from centrifuge 27 to vessel 28 of the
Figure.
The red blood cell fraction is suspended in a suitable solution (e.g.,
diafiltration buffer) in
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vessel 28. Acceptable isotonic solutions are known in the art and include
solutions, such as a
citrate/saline solution, having a pH and osmolarity which does not rupture the
cell membranes
of red blood cells and which displaces the liquid portion of the whole blood.
A preferred
isotonic solution has a neutral pH and an osmolarity between about 285-315
mOsm. An
example of a suitable solution is an isotonic citrate/saline buffer (sodium
citrate dehydrate - 6.0
g/L, sodium chloride - 8.0 g/L). In an alternative embodiment, the red blood
cell fraction can
be resuspended in any suitable isotonic solution, for example, 5% dextrose.
The red blood cell
fraction can be resuspended at a concentration of about 20 to about 200 g/l.
In one embodiment, the red blood cell fraction is suspended in a volume of
isotonic
solution such that the original volume of the whole blood from which the red
blood cell
fraction was obtained is restored. The resuspended blood cell fraction is
hereinafter referred to
as a "blood solution."
The blood solution is maintained at a suitable temperature in vessel 28.
Preferably, the
blood solution is maintained at a temperature in a range of between about 4 C
and about 15
C. The temperature of blood solution in vessel 28 is maintained by
recirculation of a suitable
medium, such as ethylene glycol, through jacket 30 at vessel 28. Recirculation
of medium
through jacket 30 is maintained by line 32, reservoir 34, pumps 36, 38 and a
chiller, or
refrigeration unit, 40.
Thereafter, the blood solution is filtered, thereby purifying the red blood
cells.
Preferably, the blood solution is filtered by diafiltration. In one
embodiment, diafiltration is
conducted by directing the blood solution from vessel 28 through line 42 and
pump 44 to
diafiltration module 46. Diafiltration module 46 includes inlet 48, retentate
outlet 50 and
permeate outlet 52. Membrane 54 partitions retentate portion 56 of
diafiltration module 46
from permeate portion 58 of diafiltration module 46. Preferably, membrane 54
has a
permeability limit in a range of between about 0.01 m and about 5 m. In one
embodiment,
the blood solution is diafiltered across a membrane having a permeability
limit in the range of
between 0.2 m and about 2.0 m. Alternate suitable diafilters include
microporous
membranes with pore sizes that will separate RBCs from substantially smaller
blood solution
components, such as a 0.1 m to 0.5 m filter (e.g., a 0.2 m hollow fiber
filter, Microgon
Krosflo II microfiltration cartridge, Laguna Hills, CA). In an especially
preferred embodiment,
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membrane 54 has a permeability limit in a range of between about 0.1 and about
2 m-
A portion of the liquid component of the blood solution in diafiltration
module 46
passes across membrane 54 from retentate portion 56 to permeate portion 58,
thereby purifying
red blood cells of retentate portion 56. Components of the blood solution,
such as plasma, or
components which are significantly smaller in diameter than RBCs pass through
the walls of
the diafilter forming a filtrate.
Purified red blood cells of the blood solution are directed through retentate
outlet 50
and line 60 back to vessel 28. Purified red blood cells can be collected from
vessel 28 through
valve 62 to line 64 for further processing. The membrane permeable liquid
component
contains any remaining liquid fraction of the whole blood (e.g., plasma or
serum) and
diafiltration buffer. The liquid component that permeates membrane 54 can be
directed from
permeate portion 58 of diafiltration module 46 through line 66 and collected
from vessel 68.
Blood solution recirculating through vessel 28 and diafiltration module 46 can
be sampled at
sampling ports (not shown) in line 42 or line 60.
Preferably, while diafiltering the blood solution to wash the red blood cells,
a liquid,
such as an isotonic solution, is directed from vessel 70 through line 72 to
the blood solution in
vessel 28 to dilute the concentration of the blood solution. In one
embodiment, the blood
solution is diluted to a concentration in a range of between about 25% and
about 75% of the
initial suspended concentration of red blood cells, by volume. Concentration
during
diafiltration then can reduce the volume back to the original concentration or
higher.
Generally, the process of adding a liquid to the suspended red blood cells and
then removing at
least a portion of the liquid is referred to as "cell washing." Preferably,
the isotonic solution
includes an ionic solute or is aqueous. Suitable istonic solutions are
described above. In an
alternate embodiment, the blood is washed through a series of sequential (or
reverse sequential)
dilution and concentration steps, wherein the blood solution is diluted by
adding at least one
isotonic solution, and is concentrated by flowing across a filter, thereby
forming a dialyzed
blood solution.
The red blood cells of the blood solution generally are washed described
above, to
separate red blood cells from residual extracellular plasma proteins, such as
serum albumins or
antibodies (e.g., immunoglobulins (IgG)) left in the red blood cell fraction.
The result is a
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reduction in the amount of microfiltration membrane-permeable species
(including membrane-
permeable plasma proteins) in the blood solution.
Cell washing generally is considered to be complete when the level of plasma
or serum
proteins contaminating the red blood cells has been substantially reduced
(typically by at least
about 90% of the plasma or serum proteins present in the red blood cell
fraction prior to
washing). Additional washing may further separate extracellular plasma
proteins from the
RBCs. For instance, diafiltration with six volumes of isotonic solution may be
sufficient to
remove at least about 99% of IgG from the blood solution.
The method of the present invention reduces the presence of potential membrane
foulants that can slow manufacturing runs. For example, small fibrin molecules
can be
problematic and may foul a membrane filter if they accumulate on the surface
of a membrane
with a permeability of 0.1 to 5 m and thus block the pores. A narrower range
in which the
foulants can be problematic is 0.2 to 0.4 m. However, as shown in Example 4,
fibrin alone
does not account for all of the potential membrane foulants. As shown in
Example 4, in 2 out
of 3 experiments, defibrinated whole blood had cell washing process times of
greater than 100
minutes. However, the red blood cell fraction generated from the same volume
of defibrinated
whole blood and diluted to the original volume in isotonic buffer had cell
washing times of 45
minutes or less.
Furthermore, defibrination can cause some red blood cell lysing. Red blood
cells, white
blood cells, or platelets that have broken open can stick to the filter. It is
believed that
fractionating whole blood and separating a red blood cell fraction from a
liquid fraction
removes a significant portion of such potential foulants, thereby normalizing
the time required
to diafilter, or "wash," red blood cells to purify them for use in the
manufacture of a blood
substitute.
To prepare a hemoglobin blood substitute from the purified red blood cells,
the purified
red blood cells of the washed blood solution can be processed further to
isolate the hemoglobin
molecules. The resulting washed blood solution can be exposed to means for
separating red
blood cells in the washed blood solution from white blood cells and platelets,
such as by
centrifugation. It is understood that other methods generally known in the art
for separating red
blood cells from other blood cell components can be employed. For example, one
embodiment
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of the invention separates red blood cells by sedimentation, wherein the
separation method
does not rupture the cell membranes of a significant amount of the RBCs, such
as less than
about 5% of the RBCs, prior to red blood cell separation from the other blood
components.
Following separation of the red blood cells from the other components of the
washed
red blood cell fraction, the RBCs are lysed, resulting in the production of a
hemoglobin (Hb)
solution. Methods of lysis include mechanical lysis, chemical lysis, hypotonic
or osmotic lysis
or other known lysis methods which release hemoglobin without significantly
damaging the
ability of the Hb to transport and release oxygen.
Following lysis, the lysed red blood cell phase is then ultrafiltered to
remove larger cell
debris, such as proteins with a molecular weight above about 100,000 Daltons.
The
hemoglobin is then separated from the non-Hb components of the filtrate.
Methods of ultrafiltration and methods of separating Hb from non-Hb components
by
pH gradients and chromatography are further described in U.S. Patent
5,691,452.
Preferably, the Jib eluate then is deoxygenated prior to polymerization to
form a
deoxygenated Jib solution (hereinafter deoxy-Hb) for further processing into a
hemoglobin-
based oxygen carrier. In a preferred embodiment, deoxygenation substantially
deoxygenates
the Hb without significantly reducing the ability of the Hb in the Hb eluate
to transport and
release oxygen, such as would occur from formation of oxidized hemoglobin
(metHb).
Alternatively, the hemoglobin solution may be deoxygenated by chemical
scavenging with a
reducing agent selected from the group consisting of N-acetyl-L-cysteine
(NAC), cysteine,
sodium dithionite or ascorbate. A suitable method of deoxygenation is
described in U.S. Patent
5,895,810, filed June 7, 1995.
The deoxygenated hemoglobin solution can be further processed into a
hemoglobin-
based oxygen carrier. As defined herein, a "hemoglobin-based oxygen carrier"
is a
hemoglobin-based composition suitable for use in humans, mammals, and other
vertebrates,
which is capable of transporting and transferring oxygen to vital organs and
tissues, at least,
and can maintain sufficient intravascular oncotic pressure, wherein the
hemoglobin has been
isolated from red blood cells. "Vertebrate" includes humans, or any other
vertebrate animals
which use blood in a circulatory system to transfer oxygen to tissue.
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"Stable polymerized hemoglobin," as defined herein, is a component of a
hemoglobin-
based oxygen carrier composition which does not substantially increase or
decrease in
molecular weight distribution and/or in methemoglobin content during storage
periods at
suitable storage temperatures for periods of about two years or more. Suitable
storage
temperatures for storage of one year or more are between about 0 C and about
40 C. The
preferred storage temperature range is between about 0 C and about 25 C.
A suitable low oxygen environment, or an environment that is substantially
oxygen-
free, is defined as the cumulative amount of oxygen in contact with the
hemoglobin-based
oxygen carrier, over a storage period of at least about two months, preferably
at least about one
year, or more preferably at least about two years, which will result in a
methemoglobin
concentration of less than about 15% by weight in the hemoglobin-based oxygen
carrier. The
cumulative amount of oxygen includes the original oxygen content of the
hemoglobin-based
oxygen carrier and packaging in addition to the oxygen resulting from oxygen-
leakage into the
hemoglobin-based oxygen carrier packaging.
Throughout this method, from RBC collection until hemoglobin polymerization,
blood
solution, RBCs and hemoglobin are maintained under conditions sufficient to
minimize
microbial growth, or bioburden, such as maintaining temperature at less than
about 20 C and
above 0 C. Preferably, temperature is maintained at a temperature of about 15
C or less.
More preferably, the temperature is maintained at 10 2 C.
In this method, portions of the components for the process of preparing a
stable
polymerized hemoglobin-based oxygen carrier are sufficiently sanitized to
produce a sterile
product. "Sterile" is as defined in the art, specifically, in the United
States Pharmacopeia
requirements for sterility provided in USP XXII, Section 71, pages 1483-1488.
Further,
portions of components that are exposed to the process stream, are usually
fabricated or clad
with a material that will not react with or contaminate the process stream.
Such materials can
include stainless steel and other steel alloys, such as Hasteloy.
In one embodiment, polymerization results from intramolecular cross-linking of
Hb.
The amount of a sulfhydryl compound mixed with the deoxy-Hb is high enough to
increase
intramolecular cross-linking of Hb during polymerization and low enough not to
significantly
decrease intermolecular cross-linking of Hb molecules, due to a high ionic
strength. Typically,
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about one mole of sulfhydryl functional groups (-SH) are needed to oxidation-
stabilize between
about 0.25 moles to about 5 moles of deoxy-Hb.
Optionally, prior to polymerizing the oxidation-stabilized deoxy-Hb, an
appropriate
amount of water is added to the polymerization reactor. In one embodiment, an
appropriate
amount of water is that amount which would result in a solution with a
concentration of about
to about 100 g/1 Hb when the oxidation-stabilized deoxy-Hb is added to the
polymerization
reactor. Preferably, the water is oxygen-depleted.
The temperature of the oxidation-stabilized deoxy-Hb solution in the
polymerization
reactor is raised to a temperature to optimize polymerization of the oxidation-
stabilized deoxy-
10 Hb when contacted with a cross-linking agent. Typically, the temperature of
the oxidation-
stabilized deoxy-Hb is about 25 to about 450 C, and preferably about 41 to
about 43 C
throughout polymerization. An example of an acceptable heat transfer means for
heating the
polymerization reactor is a jacketed heating system which is heated by
directing hot ethylene
glycol through the jacket.
The oxidation-stabilized deoxy-Hb is then exposed to a suitable cross-linking
agent at a
temperature sufficient to polymerize the oxidation-stabilized deoxy-Hb to form
a solution of
polymerized hemoglobin (poly(Hb)) over a period of about 2 hours to about 6
hours. A
suitable amount of a cross-linking agent is that amount which will permit
intramolecular cross-
linking to stabilize the Hb and also intermolecular cross-linking to form
polymers of Hb, to
thereby increase intravascular retention. Typically, a suitable amount of a
cross-linking agent
is that amount wherein the molar ratio of cross-linking agent to Hb is in
excess of about 2:1.
Preferably, the molar ratio of cross-linking agent to Hb is between about 20:1
to 40:1.
Examples of suitable cross-linking agents include polyfunctional agents that
will cross-
link Hb proteins, such as glutaraldehyde, succindialdehyde, activated forms of
polyoxyethylene
and dextran, a-hydroxy aldehydes, such as glycolaldehyde, N-maleimido-6-
aminocaproyl-(2'-
nitro,4'-sulfonic acid)-phenyl ester, m-maleimidobenzoic acid-N-
hydroxysucciniinide ester,
succinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxylate, sulfosuccinimidyl
4-(N-
maleimidomethyl)cyclohexane-1-carboxylate, m-maleimidobenzoyl-N-
hydroxysuccinimide
ester, m-inaleiinidobenzoyl-N-hydroxysulfosuccinimide ester, N-succinimidyl(4-
iodoacetyl)aminobenzoate, sulfosuccinimidyl(4-iodoacetyl)aminobenzoate,
succinimidyl 4-(p-
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maleimidophenyl)butyrate, sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate, 1-
ethyl-3-(3-
dimethylanninopropyl)carbodiimide hydrochloride, N,N'-phenylene dimaleimide,
and
compounds belonging to the bis-imidate class, the acyl diazide class or the
aryl dihalide class,
among others.
Poly(Hb) is defined as having significant intramolecular cross-linking if a
substantial
portion (e.g., at least about 50%) of the Hb molecules are chemically bound in
the poly(Hb).
In a preferred embodiment, glutaraldehyde is used as the cross-linking agent.
Typically,
about 10 to about 70 grams of glutaraldehyde are used per kilogram of
oxidation-stabilized
deoxy-Hb. More preferably, glutaraldehyde is added over a period of five hours
until
approximately 29-31 grams of glutaraldehyde are added for each kilogram of
oxidation-
stabilized deoxy-Hb.
Wherein the cross-linking agent used is-not an aldehyde; the poly(Hb)-formed
is
generally a stable poly(Hb). Wherein the cross-linking agent used is an
aldehyde, the poly(Hb)
formed is generally not stable until mixed with a suitable reducing agent to
reduce less stable
bonds in the poly(Hb) to form more stable bonds. Examples of suitable reducing
agents
include sodium borohydride, sodium cyanoborohydride, sodium dithionite,
trimethylamine, t-
butylamine, morpholine borane and pyridine borane. The poly(Hb) solution is
optionally
concentrated by ultrafiltration until the concentration of the poly(Hb)
solution is increased to
between about 75 and about 85 g/1. For example, a suitable ultrafilter is a
30,000 Dalton filter
(e.g., Millipore HeliconTM Cat # CDUF050LT; Amicon Cat # 540430, Bedford,
MA).
The pH of the poly(Hb) solution is then adjusted to the alkaline pH range to
preserve
the reducing agent and to prevent hydrogen gas formation, which can denature
Hb during the
subsequent reduction. The poly(Hb) is typically purified to remove non-
polymerized
hemoglobin. This can be accomplished by diafiltration or hydroxyapatite
chromatography (see,
e.g. U.S. Patent No. 5,691,453, filed June 7, 1995). Following pH adjustment,
at least one
reducing agent, preferably a sodium borohydride solution, is added to the
polymerization
step. The pH and electrolytes of the stable poly(Hb) can then be restored to
physiologic
levels to form a stable polymerized hemoglobin-based oxygen carrier, by
diafiltering the
stable poly(Hb) with a diafiltration solution having a suitable pH and
physiologic electrolyte
levels.
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Suitable methods of cross-linking hemoglobin and preserving the hemoglobin-
based
oxygen carrier are discussed in detail in U.S. Patent 5,691,452, issued
November 25, 1997.
Vertebrates that can receive the hemoglobin-based oxygen carrier, formed by
the
methods of the invention, include mammals, such as humans, non-human primates,
dogs, cats,
rats, horses, or sheep. Further, vertebrates, that can receive said hemoglobin-
based oxygen
carrier, include fetuses (prenatal vertebrate), post-natal vertebrates, or
vertebrates at time of
birth.
A hemoglobin-based oxygen carrier of the present invention can be administered
into
the circulatory system by injecting the hemoglobin-based oxygen carrier
directly and/or
indirectly into the circulatory system of the vertebrate, by one or more
injection methods.
Examples of direct injection methods include intravascular injections, such as
intravenous and
intra-arterial injections, and intracardiac injections. Examples of indirect
injection methods
include intraperitoneal injections, subcutaneous injections, such that the
hemoglobin-based
oxygen carrier will be transported by the lymph system into the circulatory
system or injections
into the bone marrow by means of a trocar or catheter. Preferably, the
hemoglobin-based
oxygen carrier is administered intravenously.
The vertebrate being treated can be normovolemic, hypervolemic or hypovolemic
prior
to, during, and/or after infusion of the hemoglobin-based oxygen carrier. The
hemoglobin-
based oxygen carrier can be directed into the circulatory system by methods
such as top loading
and by exchange methods.
A hemoglobin-based oxygen carrier can be administered therapeutically, to
treat
hypoxic tissue within a vertebrate resulting from many different causes
including anemia,
shock, and reduced RBC flow in a portion of, or throughout, the circulatory
system. Further,
the hemoglobin-based oxygen carrier can be administered prophylactically to
prevent oxygen-
depletion of tissue within a vertebrate, which could result from a possible or
expected reduction
in RBC flow to a tissue or throughout the circulatory system of the
vertebrate. Further
discussion of the administration of hemoglobin to therapeutically or
prophylactically treat
hypoxia, particularly from a partial arterial obstruction or from a partial
blockage in
microcirculation, and the dosages used therein, is provided in U.S. Patent
5,854,209, filed
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March 23, 1995.
Typically, a suitable dose, or combination of doses of hemoglobin-based oxygen
carrier,
is an amount which when contained within the blood plasma will result in a
total hemoglobin
concentration in the vertebrate's blood between about 0.1 to about 10 grams
Hb/dl, or more, if
required to nlalce up for large volume blood losses.
The invention will now be further and specifically described by the following
examples.
EXEMPLIFICATION
Example 1 - Bench Scale Experiment
Referring to the Figure, whole bovine blood was collected into a container
with
anticoagulant (EDTA) and was subjected to centrifugation at 2,600 rpm (1,200 X
G) for 30
minutes at 4 C in centrifuge 27 (Beckman J2-21 using a JA-10 rotor) which
separated the
whole blood into a heavy phase (red blood cell fraction, or cell component)
and a light phase
(liquid fraction). The starting volume of blood was 200 ml. The red blood cell
fraction and
liquid fraction were separated and each phase was processed in a bench-scale
cell washing
system. The liquid fraction was processed directly. The red blood cell
fraction was directed to
a recirculation vessel, 28, and diluted with isotonic citrate/saline buffer
(sodium citrate
dehydrate 6.0g/L, sodium chloride 8g/L) to its original volume. The
recirculation vessel was
kept at the appropriate temperature by recirculation of a suitable medium in a
recirculation
jacket, 30, surrounding the recirculation vessel. The diluted red blood cell
fraction, (e.g., blood
solution) was directed through a filter module, 46, (Microgon Minikros
sampler) to separate the
blood solution into a permeate and a retentate. The permeate was collected in
a graduated
cylinder. The retentate was directed back to recirculating vessel through line
60. Pressure was
monitored through a pressure input pressure gauge (0-30 PSI) in line 42 and
outlet pressure
gauge (0-15 PSI) in line 60. The cell component was washed with isotonic
citrate/saline buffer
in the bench-scale system until 400 ml of membrane permeate (2 retentate
volumes) were
obtained. The data is summarized in Table 1.
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Table 1
Sample Time to Collect 400 ml Permeate (min:sec)
Red Blood Cell Fraction 34:58
Liquid Fraction 52:20
As can be seen from Table 1, the liquid fraction contains a microfiltration
membrane
foulant, because the processing of the liquid fraction was slower than the
processing of the red
blood cell fraction.
Example 2 - Bench Scale Experiment II
Bovine blood was collected into sanitized stainless steel containers
containing sodium
citrate anti-coagulant and subjected to batch centrifugation in a second
centrifuge type, a CEPA
Tubular Bowl Centrifuge (New Brunswick Scientific Co, Edison, NJ) to generate
a red blood
cell fraction and a liquid fraction. The centrifuge was operated in two feed
configurations. In
one configuration, the feed was pumped in with a peristaltic pump and in the
second
configuration the blood was added using siphoning. The red blood cell fraction
obtained from
each configuration was than diluted to the original volume of the bovine
blood, to generate
blood solution with isotonic citrate/saline buffer. As a control, ctrated
whole blood without
centrifugation was included as a separate sample. The samples were washed in a
bench scale
washing apparatus until three retentate volumes of permeate was obtained
(about 600 ml). The
data is summarized in Table 2.
Table 2
Experiment Designation Blood Centrifuge Loading Time to Collect 600 ml
Method Permeate
Centrifuge #1 Peristaltic Pump 36:56
Centrifuge #2 Siphon 38:08
Citrated Control Not Centrifuged 1:20:53
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As can be seen in Table 2, centrifugation, which resulted in removal of the
liquid
fraction from the red blood cell faction increased the speed of cell washing.
Example 3 - Pilot Scale Experiment
Blood was pooled from three animals and treated with anticoagulant as
described in
TM
Example 2. The treated pooled blood was centrifuged in either a Westfalia
(Northvale, NJ)
SA-1 or SB-7 centrifuge, to generate a red blood cell fraction and a liquid
fraction, or washed
directly as a control. The red blood cell-fraction was washed using a pilot
scale washing
system.
The red blood cell f action was directed through a strainer using a pump
(Watson-
Marlow pump, Wilmington, MA). After passing through the strainer, the red
blood cell
fraction was directed into a recirculation vessel and diluted with isotonic
citrate/saline buffer.
The volume of the recirculation vessel was 9.6 liters. The recirculation
vessel-was kept at the
appropriate temperature by recirculation of ethylene glycol through h-a
recirculation jacket
surrounding the recirculation vessel. The red blood cell fraction/buffer
mixture was directed
through a filter (Microgon) using a pump (Waukesha, Delavan, WI) to separate
the red blood
cell fraction/buffer mixture into permeate and retentate. The permeate was
directed to a
permeate collection container on a floor scale. The retentate was directed
back to the
recirculating vessel. Pressure was monitored using a feed pressure gauge and a
retentate
pressure gauge. A total of three diafiltration volumes were passed over the
retained cells. The
data is summarized in Table 3.
Table 3
Sample Processing Time (min)
Citrated Whole Blood Control (1) 285
Citrated Whole Blood Control (2) >400
Red Blood Cell Fraction Westfalia SA-1 (1) 88
Red Blood Cell Fraction Westfalia SA-1 (2) 72
Red Blood Cell Fraction Westfalia SB-7 (1) 134
Red Blood Cell Fraction Westfalia SB-7 (2) 141
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As can be seen from Table 3, the red blood cell fraction from blood
centrifuged either
in the SA-1 or SB-7 centrifuge had greatly reduced processing time compared to
citrated whole
blood. While not wishing to be bound by theory, cell washing time was
decreased by a greater
amount when the whole blood was centrifuged in the SA-1 centrifuge as compared
to the SB-7
because the SB-7 caused significant lysis of the cells, the stroma of which
can cause microfilter
membrane fouling.
Example 4 - Effect of Defibrination on Process Time With and Without
Centrifugation
Approximately 4 liters of blood from two cows were pooled and defibrinated by
mechanical agitation. The blood contained approximately 400g of hemoglobin.
The resulting
defibrinated blood contained 12 gIL hemoglobin. The defibrinated blood was
either
centrifuged, resulting in a red blood cell fraction with approximately 5-10%
of the plasma
remaining, or not centrifuged. The defibrinated red blood cell fraction and
defibrinated whole
blood were washed as described in Example 3, except that five diafiltration
volumes were
collected. The starting volume for both samples was 7 liters.
Table 4
Sample Process Time (min)
Centrifuged Defibrinated Blood (1) 37
Defibrinated Whole Blood - Control (1) 28
Centrifuged Defibrinated Blood (2) 45
Defibrinated Whole Blood - Control (2) 150
Centrifuged Defibrinated Blood (3) 25
Defibrinated Whole Blood - Control (3) 135
In the first experiment shown in Table 4, the rate of cell washing of both
defibrinated
red blood cell fraction and defibrinated whole blood was very rapid and
centrifugation did not
improve the process time. However, in experiments 2 and 3, the rate of cell
washing was
improved for the defibrinated red blood cell fraction compared to the
defibrinated whole blood,
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demonstrating that the membrane foulant was not removed by defibrination.
Therefore,
centrifugation improves cell washing performance for both whole blood and
blood that has
been defibrinated.
EQUIVALENTS
Those skilled in the art will recognize, or be able to ascertain using no more
than
routine experimentation, many equivalents to the specific embodiments of the
invention
described herein. These and all other such equivalents are intended to be
encompassed by the
following claims.
