Note: Descriptions are shown in the official language in which they were submitted.
CA 02345230 2001-03-22
WO 00!18969 PCT/US99/21245
PATENT
ATTORNEY DOCKET NO: 50005/011 WO 1
ack ground of the Invention
This invention relates to methods and compositions for the selective
inactivation of animal viruses in biological compositions, such as blood.
Following traumatic injury {or during surgery), an organism may require
a blood transfusion to prevent death due to blood loss. In humans and certain
domesticated animals, blood transfusion has enabled the survival of injured
individuals who would otherwise have died from blood loss.
Whole blood is composed of many different types of proteins and cells.
Blood proteins include antibodies, complement proteins, and proteins involved
in the blood clotting cascade. In addition, each of the different types of
blood
cells plays a unique role in maintaining the health of the organism. Red blood
cells, for instance, are essential for the transport of oxygen and carbon
dioxide
gases to and from the cells of a multicellular organism. Another type of blood
cell, a platelet, is involved in initiating blood clotting; thrombocytopenia
patients have a platelet deficiency and are prone to bleeding disorders.
One caveat in using blood transfusions is the danger of transmitting
blood-borne viruses from donor blood to a recipient. The transmission of viral
diseases (e.g., hepatitis A, B, and C, acquired immunodeficiency syndrome, and
cytomegalovirus infections) by blood or blood products is a significant
problem
in medicine. Screening donor blood for viral markers can help reduce the
transmission of viruses to recipients, but many screening methods are directed
to only a few discrete viruses and are therefore incomplete or less than 100%
sensitive.
A number of agents have been developed that are capable of inactivating
viruses found in blood, as well as viruses found in other biological
CA 02345230 2001-03-22
WO 00/18969 PCT/US99/21245
-2-
compositions, such as mammalian and hybridoma cell lines, products of cell
lines, milk, colostrum, urine, and sperm. For example, ethyleneimine monomer
and ethyleneimine oligomers are very effective viral inactivating agents.
These
agents are themselves toxic, and must therefore be rendered non-toxic before a
product, such as blood or milk proteins, may be used clinically. Typically, a
viral inactivating compound, such as ethyleneimine dimer, is added to a
biological composition to inactivate infectious viruses that might be present
in
the composition. A quenching agent is then added to inactivate the
ethyleneimine dimer that remains after viral inactivation has taken place. The
end result is a biological composition that is relatively free of infectious
viruses, but that is contaminated with quenched inactivating agent and with
quenching agent.
In general, the present invention features a method for inactivating
viruses in purified mammalian a-nucleated cell preparations, and a method for
removing a virus inactivating agent from a treated biological composition by
washing with an inert solution.
Accordingly, in a first aspect, the invention features a method of
inactivating animal viruses in a preparation of purified mammalian a-nucleated
cells that includes contacting the preparation with a selective ethyleneimine
oligomer inactivating agent under viral inactivating conditions for a period
of
time sufficient to inactivate at least some of the animal viruses in the
preparation.
In various embodiments of the first aspect of the invention, the cells are
red blood cells or platelets, and the ethyleneimine oligomer is PEN 102. In
other embodiments, at least 90% of the animal viruses in the preparation are
inactivated, or preferably at least 98% of the animal viruses in the
preparation
CA 02345230 2001-03-22
WO 00/18969 PCT/US99/21245
-3-
are inactivated. In another embodiment, the mammalian cell is from a mammal
that is a human, a non-human primate, a domesticated mammal, or an
endangered mammal.
In another embodiment of the first aspect, the method further includes
washing the contacted preparation with a solution that does not quench the
ethyleneimine oligomer, where the washing reduces the concentration of the
ethyleneimine oligomer in the washed preparation. Preferably, the
concentration of the ethyleneimine oligomer in the washed preparation is at or
below a concentration of the ethyleneimine oligomer that is toxic. In other
embodiment, the solution is sterile unbuffered saline and the washing is
manual.
In another embodiment of the first aspect, the washing includes includes
the steps of (i) layering the contacted preparation on a mesh having pores
with
diameters smaller than the diameters of the a-nucleated cells; and (ii)
continuously flowing the solution that does not quench the ethyleneimine
oligomer over the contacted preparation. In yet another embodiment, the
washing includes the steps of (i) adding a volume of the solution that is at
least
three times the volume of the preparation; and (ii) removing the solution from
the preparation. Preferably, the washing is repeated at least two times.
In another embodiment of the first aspect, the washing is automated.
Preferably, in the washing process, a container containing the contacted
preparation is in a machine that performs the following steps under sterile
conditions: (i) pumping the preparation out of the container; (ii) diluting
the
preparation with the solution that does not quench the ethyleneimine oligomer;
(iii) removing the solution from the preparation, wherein the solution is
discarded; and (iv) pumping the preparation back into the container.
Preferably, the machine performs steps (ii) and (iii) at least two times.
In another embodiment of the first aspect of the invention, the method
CA 02345230 2001-03-22
WO 00/18969 PCT/US99/21245
-4-
further includes quenching the contacted preparation with a quenching agent.
The quenching agent may be soluble or may be immobilized on a solid-phase
support.
In a second aspect, the invention features a method for selectively
inactivating animal viruses in a biological composition that includes the
steps
of (a) contacting the composition with a selective ethyleneimine oligomer
inactivating agent under viral inactivating conditions for a period of time
sufficient to inactivate at least some of the animal viruses in the
composition;
and (b) washing the composition with a solution that does not quench an
ethyleneimine oligomer, wherein the washing reduces the amount of the
ethyleneimine oligomer in the composition. Preferably, the concentration of
the ethyleneimine oligomer in the washed composition is at or below a
concentration of the ethyleneimine oligomer that is toxic. In various
embodiments, the solution is sterile unbuffered saline and the washing step is
1 S automated.
In yet another embodiment of the second aspect of the invention, in the
washing step, a container containing the contacted composition is in a machine
that performs the following steps under sterile conditions: (i) pumping the
composition out of the container; (ii) diluting the composition with the
solution
that does not quench the ethyleneimine oligomer; (iii) removing the solution
from the composition, wherein the solution is discarded; and (iv) pumping the
composition back into the container. Preferably, the machine performs steps
(ii) and (iii) at least two times.
In yet another embodiment of the second aspect, the washing step
includes: (i) adding a volume of the solution that is at least three times the
volume of the composition; and (ii) removing the solution from the
composition. Preferably, the washing step is repeated at least two times.
In various other embodiments of the second aspect, the composition
CA 02345230 2001-03-22
WO 00/18969 PCTNS99/21245
-5-
includes a mammalian cell and the washing step includes: (i) layering the
contacted composition on a mesh having pores with diameters smaller than the
diameter of the mammalian cell; and (ii) continuously flowing the solution
that
does not quench the ethyleneimine oligomer over the contacted composition.
The mammalian cell may be an a-nucleated cell (e.g., a platelet or a red blood
cell). In other embodiments, the composition is a cell-free composition and
the
washing step is manual.
By "purified" is meant a preparation that contains, by volume, at least
50%, more preferably, at least 70%, more preferably at least 85%, even more
preferably at least 95%, and most preferably, at least 98% of the indicated
component. For example, a purified preparation of red blood cells contains at
least 50% by volume red blood cells.
By "ethyleneimine oligomer" is meant a compound having ( 1 ) an
aziridino moiety or a halo-hydrocarbon-amine moiety, and, preferably, (2) two
or more nitrogen atoms separated by hydrocarbon moieties. These compounds
are also referred to as "inactivating agents," or "selective inactivating
agents."
One preferred ethyleneimine oligomer of the invention is PEN 102, which has
the following formula:
CH2
~ N-CH2-CH2-NH2
CH2 /
A second preferred ethyleneimine oligomer of the invention is PEN103,
which has the following formula:
CH2
' N-CH2-CH2-NH-CH2-CH2-NHZ
CH~
An inactivating agent has "selectivity" for nucleic acids or "selectively"
CA 02345230 2001-03-22
WO 00/18969 PCT/US99/21245
-6-
reacts with nucleic acids if the comparative rate of reaction of the
inactivating
agent with nucleic acids is greater than the rate of reaction with other
biological
molecules, e.g., proteins, carbohydrates or lipids.
By "nucleic acid" is meant both DNA and RNA, both single and double
stranded.
"Inactivating," "inactivation," or "inactivate," when referring to nucleic
acids, means to substantially eliminate the template activity of DNA or RNA,
for example, by destroying the ability to replicate, transcribe or translate a
message. For example, the inhibition of translation of an RNA molecule can be
determined by measuring the amount of protein encoded by a definitive amount
of RNA produced in a suitable in vitro or in vivo translation system. When
referring to viruses, the term means diminishing or eliminating the number of
infectious viral particles measured as a decrease in the infectious titer or
number of infectious virus particles per ml. Such a decrease in infectious
virus
particles is determined by assays well known to a person of ordinary skill in
the
art.
"Viral inactivating conditions" refers to the conditions under which the
viral particles are incubated with the selective ethyleneimine oligomer
inactivating agents of this invention, including, for example, time of
treatment,
pH, temperature, salt composition and concentration of selective inactivating
agent so as to inactivate the viral genome to the desired extent. Viral
inactivating conditions are selected from the conditions for selective
modification of nucleic acids described in U.S. Patent Application Serial No.
08/855,378, hereby incorporated by reference.
By "inactivate at least some of the animal viruses" is meant that at least
50% of the viruses in the treated preparation are inactivated, preferably at
least
70% of the viruses are inactivated, more preferably at least 80%, still more
preferably at least 90%, still more preferably at least 95%, still more
preferably,
CA 02345230 2001-03-22
WO 00/18969 PCT/US99/21245
at least 99%, and most preferably, 100% of the viruses in the treated
preparation are inactivated. The number of viruses in a preparation may be
measured by the number or titer of infectious viral particles per ml of
preparation. Such a measurement may be accomplished by a variety of well
known virus titer assays.
By "animal virus" is meant a virus capable of infecting a cell from an
animal. Animal viruses may be DNA or RNA viruses, and may be enveloped
or non-enveloped viruses or viroids. Examples of animal viruses include,
without limitation, poxviruses, herpes viruses, adenoviruses, papovaviruses,
parvoviruses reoviruses, orbiviruses, picornaviruses, rotaviruses,
alphaviruses,
rubiviruses, influenza viruses, type A and B, flaviviruses, coronaviruses,
paramyxoviruses, morbilliviruses, pneumoviruses, rhabdoviruses, lyssaviruses,
orthmyxoviruses, bunyaviruses, phleboviruses, nairoviruses, hepadnaviruses,
arenaviruses, retroviruses, enteroviruses, rhioviruses and the filoviruses.
Specifically excluded from the definition of an animal virus are viruses which
infect non-animal cells (e.g., a bacteriophage which infects bacterial cells).
By "biological composition" is meant a composition containing cells or
a composition containing one or more biological molecules, or a composition
containing both cells and one or more biological molecules. Cell-containing
compositions include, for example, mammalian blood, red cell concentrates,
platelet concentrates, leukocyte concentrates, blood plasma, platelet-rich
plasma, semen, placental extracts, mammalian cell culture or culture medium,
products of fermentation, and ascites fluid. Biological compositions may also
be cell-free, and contain at least one biological molecule. By "biological
molecule" is meant any class of organic molecule normally found in living
organisms including, for example, nucleic acids, polypeptides, post-
translationally modified proteins (e.g., glycoproteins), polysaccharides, and
lipids. Biological molecule-containing biological compositions include, for
CA 02345230 2001-03-22
WO 00/18969 PCT/US99/21245
-g_
example, serum, blood cell proteins, blood plasma concentrate, blood plasma
protein fractions, purified or partially purified blood proteins or other
components, a supernatant or a precipitate from any fractionation of the
plasma,
purified or partially purified blood components (e.g., proteins or lipids),
mammalian colostrum, milk, urine, saliva, a cell lysate, cryoprecipitate,
cryosupernatant, or portion or derivative thereof, compositions containing
proteins induced in blood cells, and compositions containing products produced
in cell culture by normal or transformed cells (e.g., via recombinant DNA or
monoclonal antibody technology).
By an "a-nucleated cell" is meant a cell which, when mature, lacks a
nucleus. Preferred examples of a-nucleated cells are platelets and red blood
cells.
By a "solution that does not quench an ethyleneimine oligomer" is
meant a solution that does not contain a quenching agent (e.g., a
thiophosphate
or a thiosulfate). A quenching agent, when contacted with an ethyleneimine
oligomer, renders the contacted ethyleneimine oligomer non-toxic. Preferred
solutions that are incapable of reacting with an ethyleneimine oligomer are
unbuffered saline and water.
By a "quenching agent" is meant a thiophosphate or a thiosulfate, or a
compound containing a thiophosphate or a thiosulfate that, when contacted with
an ethyleneimine oligomer, is capable of rendering the contacted ethyleneimine
oligomer non-toxic.
By "domesticated mammal" is meant any non-human mammal that a
human maintains. For example, cows, donkeys, mules, elephants, horses,
llamas, camels, goats, sheep, reindeer, dogs, cats, pigs, ferrets, rabbits,
mice,
rats, hamsters, and guinea pigs are domesticated animals. In addition, any
mammal that a human keeps as a pet, such as a pet skunk or a pet wolf, is also
a
domesticated mammal.
CA 02345230 2001-03-22
WO 00/18969 PCT/US99/21245
-9-
By "an endangered mammal" is meant a mammal that is included on the
Endangered Species List compiled by the U.S. Fish and Wildlife Service as of
September 25, 1998.
Currently, red blood cell preparations are used to transfuse a recipient,
S without prior inactivation of viruses. Hence, the methods and compositions
of
the present invention allow the inactivation of viruses in red blood cell
preparations prior to their use in transfusions. As the inactivating agents
described herein are selective for the nucleic acids that are a major
component
of viruses, viral nucleic acid can be selectively inactivated over the other
molecules (e.g., proteins and lipids) present in the red blood cell
preparation.
In addition, the invention features an in vitro method for removing an
ethyleneimine oligomer from a biological composition following virus
inactivation without using a quenching agent. This method results in a
biological composition that is relatively free not only of contaminating
viruses,
but also relatively free of quenched (i.e., non-toxic) ethyleneimine oligomer
and unreacted quenching agent.
Other features and advantages of the invention will be apparent from the
following description and from the claims.
Fig. 1 is a schematic diagram showing the chemical reactions that take
place during the post-column derivatization of PEN102 or quenched PEN102.
Fig. 2 is a schematic flow diagram showing the post-column reaction
hardware used in the HPLC analysis of ethyleneimine oligomers.
Fig. 3 is the HPLC elution profile of PEN102.
Fig. 4 is a bar graph showing the distribution of PEN102 between the
red blood cell component and the plasma component of fresh baboon whole
blood (spun hematocrit (HCT) of 38%) following treatment with 12 mM (1000
CA 02345230 2001-03-22
WO 00/18969 PCTlUS99/21245
-10-
~,g/ml; 0.1% v/v) PEN102 for 6 hours at 20°C.
Fig. 5 is a bar graph showing the effectiveness of manual washing of
PEN102 from the red blood cell fraction of whole baboon blood (spun HCT
38%) following treatment with 12 mM PEN102 for 6 hours at 20°C. The
number of manual washes of the RBC fraction with nonbuffered saline ( 1:10
v/v) for 10 min. at 20°C is indicated, and residual PEN102 in the RBC
fraction
is shown as a percentage.
Fig. 6 is a bar graph showing the effectiveness of manual washing of
PEN102 from the red blood cell fraction of whole baboon blood (SPUN HCT
38%) following treatment with 1000 ~,g/ml PEN102 for 6 hours at 20°C.
The
number of manual washes of the RBC fraction with nonbuffered saline (1:10
v/v) for 10 min. at 20°C is indicated, and residual PEN102 in the RBC
fraction
is shown as ~,g of PEN102 per ml of RBC.
Fig. 7 is a bar graph showing the quenching of the ethyleneimine dimer
PEN102 (6mM) from human blood (plasma (gray bar) and red blood cells
(black bar)) following incubation for 2 hours at 23°C with either 50 mM
Na-
thiosulfate or an equimolar amount of AgroPore-Thiophosphate solid-phase (a
solid-phase quencher containing thiophosphomonoester groups).
We have discovered a method to selectively inactivate viruses in a-
nucleated cell preparations by treating the preparations with ethyleneimine
oligomer inactivating agents. For example, most mature mammalian red blood
cells, unlike those of other vertebrate animals, lack nuclei and, hence, lack
nucleic acid. Thus, treatment of the cells with an ethyleneimine oligomer
inactivating agent that inactivates nucleic acids allows for the selective
inactivation of the nucleic acid of any viruses contaminating the red blood
cell
preparation, while leaving the red blood cells unaffected. Accordingly, as we
demonstrate below, ethyleneimine oligomer-mediated inactivation of nucleic
CA 02345230 2001-03-22
WO 00/18969 PCT/US99/21245
-11-
acids in red blood cell preparations does not affect the in vivo longevity of
the
cells. Likewise, since mature platelet cells (also known as platelets} lack
nuclei, they are similarly unaffected by treatment with virus-inactivating
ethyleneimine oligomers.
The invention also provides a method for removing the ethyleneimine
oligomer from the treated biological composition (e.g., blood), prior to use
of
the composition by repeatedly washing the composition with a solution that
does not quench the ethyleneimine oligomer (e.g., sterile unbuffered saline).
Where the biological composition is a composition containing cells (e.g.,
sperm), the treated cells may be washed by repeated steps of resuspension in a
solution that does not quench an ethyleneimine oligomer and isolating the
cells
by centrifugation. Where the biological composition is a cell-free composition
(e.g., rnilk), the treated milk proteins may be, for example, diluted with a
solution that does not quench an ethyleneimine oligomer, and then dialyzed to
remove the ethyleneimine oligomer.
Thus, unlike the current methods which inactivate ethyleneimine
oligomer in a treated biological composition with a quenching agent, leaving
the biological composition contaminated with the quenched ethyleneimine
oligomer and extraneous quenching agent, the method of the present invention
allows the generation of a biological composition free of both virus and
quenching agent.
F;lh~Cl~n~i aS
The ethyleneimine oligomer inactivating agents of the present invention
are compounds having (1) an aziridino moiety or a halo-hydrocarbon-amine
moiety, and, preferably, (2) two or more nitrogen atoms separated by
hydrocarbon moieties. For example, an ethyleneimine oligomer may have one
of the following five formulas:
CA 02345230 2001-03-22
WO 00/18969 PCTNS99/21245
-12-
(1) CH2~
NH
CH ~
2
(2) CH2
~ N-CHZ-CHa-~2
CH2
(3) CHZ
~ N-CH2-CH2-NH-CH2-CHZ-NH2
CH~
(4) CH2
~ N-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH2
CH2
CH2 ~ CH2-CH2-~2
~N-CH2-CH2-N~
CH2 ~ CH2-CHz-NH2
Ethyleneimine oligomers can also be substituted so long as this does not
eliminate the essential property of the ethyleneimine (i.e., the inactivation
of
nucleic acids). In one variation, the ethyleneimine oligomers are substituted
with halogens and have the general formula (3-Hal-(CH2-CH2-NH)nH.
Preferably, the "n" is an integer between 2-5, inclusive.
The ethyleneimine oligomer inactivating agents of the present invention
also include both aziridino compounds and halo-hydrocarbon-amine
compounds.
The aziridino compounds have the formula:
(R~,R2)C
~NURS-N+(~~R7)-~nRs~ x n
(R3,Ra)C
CA 02345230 2001-03-22
WO 00/18969 PCTNS99/21245
-13-
where each of R1, R2, R3, R4, R6, R~, and R8 is, independently, H or a
monovalent hydrocarbon moiety containing between 1 and 4 carbon atoms,
inclusive, provided that R~, R2, R3, R4, R6, R~, and Rg cannot all be H; RS is
a
divalent hydrocarbon moiety containing between 2 and 4 carbon atoms,
S inclusive; X is a pharmaceutically acceptable counter-ion; and n is an
integer
between 2 and 10, inclusive. These compounds can be prepared by the
aziridine-initiated oligomerization of a halo-hydrocarbon-amino compound.
The halo-hydrocarbon-amine compounds can have the formula co-Xl-
[Rl-N+(RZ, R3)-]"R4~ (X2 )n , where X1 is CI or Br; R1 is a divalent
hydrocarbon
moiety containing between 2 and 4 carbon atoms, inclusive; each of R2, R3, and
R4 is, independently, H or a monovalent hydrocarbon moiety containing
between 1 and 4 carbon atoms, inclusive, provided that R2, R3, and R4 cannot
all be H when Rl contains 2 carbon atoms; X2 is a pharmaceutically acceptable
counter-ion; and n is an integer between 2 and 10, inclusive. These compounds
can be prepared by the oligomerization of the corresponding halo-hydrocarbon-
amino compounds.
Alternatively, these compounds can have the formula (3-X~-CH2CH2
N+H(Rt)-[R2-N+(R3, R4)-~"RS' (X2 )n+~, where X~ is Cl or Br; each of R~, R3,
R4,
and RS is, independently, H or a monovalent hydrocarbon moiety containing
between 1 and 4 carbon atoms, inclusive; R2 is a divalent hydrocarbon moiety
having 3 or 4 carbon atoms; X2 is a pharmaceutically acceptable counter-ion;
and n is an integer between 2 and 10, inclusive. These compounds can be
prepared by the aziridine-initiated oligomerization of an halo-hydrocarbon-
amino compound, followed by conversion of the aziridino group to the
corresponding halide compound.
Methods for producing and using ethyleneimine oligomers for
inactivating viruses in biological compositions are generally described in
U.S.
Application Serial Nos. 08/835,446 (filed April 8, 1997), 08/521,245 (filed
CA 02345230 2001-03-22
WO UO/18969 PCTNS99/21245
-14-
August 29, 1995), 08/855,378 (filed May 13, 1997), 09/005,606 (filed January
12, 1998), and 09/005,719 (filed January 12, 1998), hereby incorporated by
reference.
Since the goal of a blood transfusion is often the transfer of red blood
cells, it may be desirable to separate these cells from the other blood
components, such as white blood cells (e.g., lymphocytes, neutrophils, and
platelets) and biological molecules (e.g., clotting factors and complement).
In
one example, prior to transfusion, whole blood may be separated into the
following components: (1) the red blood cell (RBC) portion (which includes a
small portion of the white blood cells) and (2) the plasma (which also
includes
a small portion of the white blood cells).
Standard methods exist for the separation of red blood cells from other
blood components. For example, a Ficoll or Percoll gradient may be used to
separate the different components of whole blood based on their differences in
density. Such gradients may be generated using reagents commercially
available from, for example, Pharmacia Biotech (Uppsala, Sweden).
In addition, commercially available systems, such as the MCS~+
Apheresis System (commercially available form Haemonetics Corp., Braintree,
MA) may be used to isolate red blood cells from whole blood. It should be
noted that this system may also be used to separate other a-nucleated cells
(e.g.,
platelet cells) from whole blood.
Although ethyleneimine oligomers are useful compounds for the
selective inactivation of viral nucleic acids, their inherent alkylating
abilities
CA 02345230 2001-03-22
WO 00/18969 PCT/US99/21245
-1 S-
may render them toxic to most nucleated cells. Thus, prior to introduction of
the treated red blood cells into the recipient animal, it is desirable to
remove the
ethyleneimine oligomer from the cells, or at least reduce the concentration of
the ethyleneimine oligomer to a level that is non-toxic. Of course, if the
cells
or biological molecules are to be used in vivo, the washing step, in addition
to
the ethyleneimine oligomer treatment step, must be conducted under sterile
conditions.
We have employed a murine lymphoma mutagenesis assay to detect
toxic (i.e., mutagenic) levels of the ethyleneimine oligomers and have found
that the toxic level for the ethyleneimine oligomer used in the studies
described
below, PEN102, is greater than 1 pg/ml (i.e., a concentration of 1 p.g/ml
PEN102 or less is non-toxic). The toxic levels of other ethyleneimine
oligomers may be readily assessed using the murine lymphoma mutagenesis
assay. Hence, the goal of the washing step is to reduce the concentration of
the
ethyleneimine oligomer in the desired treated biological composition to a
level
at or below that determined to be non-toxic.
One method to remove ethyleneimine oligomer compounds from a
treated red blood cell preparation is to subject the cells to repeated
washings
with nonbuffered sterile saline (i.e., sterile 0.9% NaCI). Following each
washing step, a sample of the biological composition being treated (and
washed) may be tested for the presence of the ethyleneimine oligomer at a
concentration higher than that previously determined to be toxic. If the
concentration is found to be at a toxic level, at the very minimum, at least
one
additional washing step should be performed prior to the in vivo use of the
treated composition. As a safety measure, once a non-toxic level of an
ethyleneimine oligomer is achieved, an additional washing step is preferably
performed prior to the in vivo use of the treated composition.
In one example of washing red blood cells (RBCs) following treatment
CA 02345230 2001-03-22
WO 00/18969 PCT/US99/Z1245
-16-
of whole blood, the treated whole blood is diluted with approximately 3X
volume of sterile 0.9% NaCI (i.e., 15 ml saline is added to 5 ml blood).
Following centrifugation to isolate the RBC component, the packed RBC
volume is resuspended in approximately 9X volume of sterile 0.9% NaCI, and
allowed to mix (under gentle mechanical agitation) for 10 minutes at
22°C. The
RBC component is then isolated by centrifugation, and the washing step with
9X volume of sterile saline is repeated until the concentration of the
ethyleneimine oligomer in the RBC component is at or below the concentration
determined to be non-toxic (as determined, for example, using the mouse
lymphoma mutagenicity assay described below).
In a second example, isolated platelets (isolated using, for example,
plateletpheresis at a standard blood bank facility), are treated with a
concentration of ethyleneimine oligomer for an amount of time and at an
incubation temperature sufficient to inactivate at least some of the viruses
in the
platelet preparation. The platelets are next repeatedly washed in at least 4X
volume of sterile saline solution until the concentration of the ethyleneimine
oligomer is at or below the concentration determined to be non-toxic.
In another example, where the composition contains cells having a
known diameter, the cells may be collected in mesh having pores of a diameter
smaller than the cells' diameter, and then washed under a continuous flow of a
solution that does not quench an ethyleneimine oligomer for a period of time
sufficient to lower the concentration of the ethyleneimine oligomer in the
cells
to a non-toxic level. Of course, the fewer cells layered on the mesh, the
lower
the period of time necessary to wash the cells under a continuous flow. In a
variation of this washing method, the ethyleneimine oligomer-treated cells may
collected in a mesh bag having pores of a diameter smaller than the cells'
diameter. The bag may then be repeatedly dipped in a solution that does not
quench the ethyleneimine oligomer until the concentration of the ethyleneimine
CA 02345230 2001-03-22
WO 00/18969 PCT/US99/21245
-17-
oligomer in the cells is reduced to a non-toxic level.
Although the washing steps described in the following examples are
manual washings under sterile conditions, it will be understood that automated
washing may be employed to free a biological composition from an
ethyleneimine oligomer. For example, a machine may be designed to wash
ethyleneimine oligomer treated cells under sterile conditions.
In one example of such a machine, purified platelets may be treated with .
ethyleneimine oligomer under viral inactivating conditions for a period of
time
sufficient to inactivate at least some of the viruses in the platelet
preparation.
This treatment step may be performed by combining the platelets with the
ethyleneimine oligomer in a sterile container, such as a sterile plastic bag.
The
bag may then be attached to the machine such that machine can, under sterile
conditions, pump the cells out of the bag (and, additionally rinse the bag
with
sterile 0.9% NaCI). Under completely sterile conditions, the machine may then
dilute the platelets with sterile saline, gently mix the platelets for a
desired time
at a desired temperature, collect the platelets by centrifugation, discard the
"used" sterile 0.9% NaCI, and add "fresh" 0.9% NaCl, and repeat the mixing-
centrifugation-discarding process for a desired number of times. After the
final
collection of the platelets by centrifugation, the platelets may be
resuspended in
"fresh" 0.9% NaCI, or in another desired solution (e.g., blood), and returned
to
the original container. Platelets thus virally inactivated and washed may be
used immediately, stored, or frozen as desired.
Toxi i r screening.of an ,yleneimine olig~o; mer
The same alkylating abilities of ethyleneimine oligomers that render
them able to inactivate viral nucleic acids also enable them to damage and/or
induce mutations in the genomic DNA of mammalian cells. Hence, before a
ethyleneimine oligomer-treated biological composition may be used in vivo (or
CA 02345230 2001-03-22
WO 00/18969 PCTNS99/21245
-18-
in vitro, in, for example, the case of treated sperm for in vitro
fertilization), it is
desirable to reduce the concentration of the ethyleneimine oligomer in the
composition to a level that is non-toxic to most mammalian cells.
The mouse lymphoma mutagenicity assay is one method to determine
the toxicity concentration level of a particular ethyleneimine oligomer. This
assay uses a murine lymphoma cell which is heterozygous at the thymidine
kinase (TK) locus (i.e., TK +/-) grown in the presence of the toxin, 5-
trifluorothymidine (TFT), to screen different concentrations of the particular
ethyleneimine oligomer. Both TK +/- cells and TK -/- cells are viable in
normal culture media; however, in the presence of TFT, only the TK -/- cells
will grow because the TK +/- cells will incorporate the toxic TFT into their
DNA. If the TK+/- murine lymphoma cells are exposed to a toxic
concentration of an ethyleneimine oligomer, they may undergo a single-step
forward mutation to a TK-/- genotype, enabling them to grow in the presence of
TFT. Thus, a concentration of an ethyleneimine oligomer which does not result
in the growth of TK +/- murine lymphoma cells in the presence of 5-
trifluorothymidine (TFT) is non-toxic.
The mutagenicity test we used below to screen the toxicity level of the
ethyleneimine oligomer PEN102 was performed by Convance Laboratories Inc.
(Vienna, VA). L5178Y murine lymphoma cells (which are heterozygous at the
TK locus) grown in the presence of 5-trifluorothymidine (TFT) were exposed
to different concentrations of PEN102. The results demonstrated that a
concentration of 1 ~g/ml PEN102 did not allow the growth (i.e., the forward
mutation) of LS 178Y TK +/- cells in the presence of 5-trifluorothymidine
(TFT). Hence, a concentration of less than or equal to i ~,g/ml PEN102 is non-
toxic, and a PEN102-treated biological composition (e.g., a treated RBC
preparation) containing up to 1 ~,g/ml PEN102 is safe for infusion into a
recipient.
CA 02345230 2001-03-22
WO 00/18969 PCTNS99/21245
-19-
High nerforma-n~~uid chromatog~phy~HPLC, ana ~r i of yjeneimine
Ethyleneimine oligomers are routinely analyzed by cation exchange
HPLC. Because ethyleneimine oligomers do not contain a chromophore, the
following method uses post-column derivatization which reacts only with
primary amino groups, thereby eliminating complications of multiple reaction
sites (ethyleneimine oligomers contain only one primary amino group) or
interfering compounds. A schematic of the reactions that take place during
HPLC of PEN102 and quenched PEN102 is shown on Fig. 1. The method has
been determined to be linear within the range of 10-230 ng of PEN102.
a. S a in ~ materials for HP .C,~ analysis
A schematic diagram of the HPLC post column reaction hardware is
shown on Fig. 2.
1. Potassium phosphate eluent, Picketing laboratories Catalog No.1700-
1101; pH 6.00; 0.1 N. (Picketing Laboratories Inc., Mountain View,
CA)
2. Potassium chloride eluent, Picketing Laboratories Catalog No. 1700-
1102; pH 6.00; O.I N.
3. o-Phthalaldehyde (OPA) diluent, Picketing Laboratories Catalog No.
OD 104.
4. o-Phthalaldehyde (OPA), Picketing Laboratories Catalog No. 0120.
5. Thiofluor, Picketing Laboratories Catalog No. 3700-2000.
6. Nitrogen, grade 4.8
7. Equipment and conditions
Beckman 126 solvent module (or equivalent); (Beckman Instruments
Inc., Fullerton, CA)
Beckman Gold Nouveau software (or equivalent)
Jasco FP-920 intelligent Fluorescent detector (Jansco Inc., Easton, MD)
CA 02345230 2001-03-22
WO 00/18969 PCT/US99/21245
-20-
Pickering Laboratories PCX 3100 post-column reaction module
Alkion cation-exchange column 4 x 150 mm; Pickering Laboratories
Catalog No. 9410917
Excitation Wavelength. 330nm
Emission Wavelength: 465nm
Column temperature: 40°C
Reactor temperature: 45°C
Reactor pump flow rate: 0.3 ml/min.
1. Degas 450 ml OPA diluent for approximately 10 min. by bubbling
nitrogen gas through the diluent.
2. Dissolve approximately 0.05 g o-pthalaldehyde (OPA) in a minimum
amount of methanol, add to OPA diluent.
3. Dissolve approximately 1 g thiofluor in a small amount of OPA diluent,
add to OPA solution from Step 2.
This method uses cationic exchange chromatography with post column
derivatization of ethyleneimine oligomer primary amino groups and fluorescent
detection. The HPLC elution profile of PEN102 is shown on Fig. 3.
1. Prepare samples in potassium phosphate eluent, pH 6. Dilute 1 ~l
PEN i 02 at a final concentration of 1: 8 x 104 with the eluent.
2. Use potassium chloride eluent pH 6.
3. Inject 10 ~,1 of the sample for analysis. Flow rate is set at 0.8 ml/min.
4. 0-4 min., 100% flow potassium phosphate eluent; 4-8 min., 0-100%
flow potassium chloride eluent; 9-10 min., 100-0% flow potassium
chloride eluent.
5. Reequilibrate the column with 100% potassium phosphate eluent for an
additional 10 min. between analyses.
CA 02345230 2001-03-22
WO 00/18969 PCT/US99/21245
-21-
Since this method is very sensitive to contamination, gloves should be
worn at all times when preparing buffers or samples.
The following specific examples are to be construed merely as
illustrative, and not limitative of the remainder of the disclosure in any way
whatsoever. Furthermore, although some of the examples describe the addition
of the ethyleneimine oligomer to whole blood prior to the isolation of blood
components, preferably the desired blood component (e.g., red blood cells) is
isolated prior to addition of the ethyleneimine oligomer. This not only
reduces
the amount of the ethyleneimine oligomer required to inactivate the viruses
contained in the desired blood component, but also allows the retainment of
other, untreated blood components (e.g., plasma), which may be subsequently
(or simultaneously) virally inactivated with an ethyleneimine oligomer. For
example, virally inactivated plasma may be used to purify virus-free plasma
proteins, such as blood clotting factors or albumin.
Exavm~l~I
Biochemistry of baboon RRCs treated with PEN102 or PF,N10~
Fifteen milliliters (ml) of fresh baboon blood was collected from baboon
205 (Naval Blood Research Laboratory) in CPD-ADSOL {a standard blood
storage solution that prevents the blood from clotting). The fresh whole blood
was treated with 0.79 ml of 240 mM PEN102 in 0.25 M NaH2P04 (20X stock
solution of PEN102 was prepared immediately before the treatment). The final
concentration of PEN102 in the blood was 12 mM (1000 ~,g/ml; 0.1% v/v). In
a control experiment, 0.79 ml of 0.25 M NaH2P04 was added to 1 S ml of fresh
baboon blood. Both control and PEN102-treated samples were incubated, with
rocking, for 6 hours at room temperature. After the end of the incubation
period, the red blood cells were isolated by centrifugation, and a series of
CA 02345230 2001-03-22
WO 00/189b9 PCT/US99/21245
-22-
biochemical parameters were immediately determined without removal of
PEN102.
In addition, a parallel experiment was performed with a second
ethyleneimine oligomer, PEN 103. As above, fifteen milliliters {ml) of fresh
baboon blood was collected from baboon 205 (Naval Blood Research
Laboratory) in CPD-ADSOL. The fresh whole blood was treated with 0.79 ml
of 310 mM PEN103 in 0.5 M NaH2P04 (20X stock solution of PEN103 was
prepared immediately before the treatment). The final concentration of
PEN103 in the blood was 15.5 mM (2000 ~,g/ml; 0.2% v/v). In a control
experiment, 0.79 ml of 0.5 M NaH2P04 was added to 15 mI of fresh baboon
blood. Both control and PEN103-treated samples were incubated, with
rocking, for 6 hours at room temperature (i.e., 22°C). After the end of
the
incubation period, the red blood cells were isolated by centrifugation, and a
series of biochemical parameters were immediately determined without
removal of PEN 103.
Table I shows the results of the RBC biochemistry tests.
CA 02345230 2001-03-22
WO 00/18969 PCT/US99/2i245
-23-
v
h
a
..,
O N -~O~
N N N
cd
zN
V
0 0
tj ~Q
U
.d a ~ N et
N ~ ~ i i
a ~ r r
f~ N
O N
U x O
yo
." .d a,
U
M
O '..~
O O
+
x C"
CA W h
~ ~
I-~1 ~...~ M M N
N x ~ U
O ~ M M M
O
U N
i
~x
0
os
p ~n ~Cv1
.'.,
N N N
CO
N
M ~ V
U CA ~o ~o~o
.. ~ ...
N ~
R
~ t~ t'~l~
r.
N M
O O
U fs,Cs,
CA 02345230 2001-03-22
WO 00/18969 PCT/US99/21245
-24-
As can be seen from Table I, no significance difference was found
between the control (i.e., untreated), the PEN102-treated, or the PEN103-
treated blood. Hence, treatment with PEN102 or PEN103 does not appear to
affect the biochemical functioning of red blood cells.
E~s.ampl~.ii
IIa. Treatment of fresh CPD baboon blood with PEN102
Eighty ml of fresh baboon blood collected from baboon 214 (Naval
Blood Research Laboratory) in CPD (resulting in a spun HCT of 38%) was
treated with 4.2 ml of 240 mM PEN102 in 0.25 M NaH2P04 (20X stock
solution of PEN102 was prepared immediately before treatment). The final
calculated total concentration of PEN102 in the blood was 12 mM (1000 ~g/ml;
0.1 % v/v).
IIb. Distribution of PEN102 between RBC and plasma in fresh whole CPD
baboon blood.
Following 6 hours of incubation of the blood at room temperature, the
blood was separated into a plasma fraction and a red blood cell (RBC)
fraction.
The concentration of PEN102 in the plasma and RBC fractions was determined
using the HPLC analysis described above.
The concentration of PEN102 in the plasma fraction was found to be
10.7 mM (893 ~g/ml), while the concentration of PEN102 in the RBC fraction
was only 5.6 mM (469 ~,g/ml). Therefore, after 6 hours of incubation, the RBC
and plasma fractions of blood contained 24% and 76% of the total PEN 102
respectively (Fig. 4).
CA 02345230 2001-03-22
WO 00/18969 PCTNS99/21245
-25-
IIc. Manual washing of baboon RBC after PEN102 treatment.
Eighty ml of treated baboon blood was divided in 6 portions (about 13
ml each) in 50 ml sterile tubes. The average volume of RBC in each portion
was about 5 ml. Unbuffered sterile saline (i.e., 0.9% NaCI in water) was added
to fill each tube to about 50 ml. The RBC fraction was separated by
centrifugation at 2000 rpm ( 1248 x g) for 5 min. at room temperature, and the
diluted plasma fraction removed.
The RBC fraction was next subjected to a washing cycle: To the
remaining RBC (about 5 ml in each tube), a new portion of unbuffered sterile
saline (about 4.5 ml) was added. The tubes were incubated with gentle
agitation at room temperature for 10 min. At the end of the incubation, RBC
fraction was separated by centrifugation. After each cycle, a small aliquot of
the RBC suspension was removed to determine the concentration of PEN102
by HPLC (see method above).
The washing cycle was repeated four times. After the fourth cycle, the
six tubes of RBC were combined together and unbuffered sterile saline added
to give a final hematocrit (HCT) of 46%. The effectiveness of the washing
procedure is shown on Fig. 5, which demonstrates a rapid reduction in the
percentage of PEN102 in the RBC fraction. The concentration of PEN102
remaining in the RBC fraction (in ~g/ml of RBC) after each washing cycle is
shown on Fig. 6. The data in Fig. 6 demonstrates that the residual
concentration of PEN102 in the RBC after 4 washings was lower than the
safety level for PEN102, 1 ~g/ml, which was determined by mutagenicity
testing on mouse lymphoma cells (performed by Covance, Vienna, VA).
Iid. Biochemistry of baboon RBC after PEN102 treatment, washing, and
storage for 18 hours.
Washed, treated RBC (see section IIc above) were resuspended in 0.9%
CA 02345230 2001-03-22
WO 00/18969 PCT/US99121245
-26-
saline-0.2% glucose to a hematocrit {HCT) of about 46%, and stored overnight
( 18 hours) at 4°C. Following storage, a series of biochemical
parameters were
determined. The results of biochemistry tests performed on these treated,
washed, and stored ItBC are presented in Table II.
S Table II
Biochemistry of Control (Untreated) and PEN102-treated,
Washed, and Stored Baboon RBC
MOS SUP HB spun MCV SUP Red P50 RBC MCHC
HB K+
mOs/kg(mg/dL)(g/dL)HCT (spun(mEq/L)Cell (mmHg pH (spun
HCT) + ) HCT)
K
control*394 3 12.6 38 80.9 3.5 7.9 31.8 6.93929.0
PEN102 NA 90 13.3 46 91.1 2.2 8.0 30.6 6.64932.0
**
* fresh. ed boon
untreatCPD blood
ba
** baboon CPD blood, treated with PEN102, washed, and stored for 18 hours at
4°C
As shown in Figs. 5 and fi, four manual washing cycles of treated RBC
effectively reduced the concentration of PEN102 below the level of safety
1 S determined in a companion mutagenicity study. Table II demonstrates that
treatment of RBC with PEN102, followed by washing and 18 hours of storage
at 4°C, did not affect the major biochemical characteristics of the
RBC.
IIe. Post-treatment in vivo survival of RBC.
Five ml portions of PEN102 treated ItBC from baboon 214 were labeled
with either SICr or biotin using standard labeling protocols (see, e.g.,
Valeri et
al., Transfusion 24: 105-108, 1984). For example, to label RBCs with 5'Cr,
approximately 20-30 ml of RBCs were incubated for 30 min. at 37°C with
0.5
pCi StCr (disodium chromate) per ml of blood (commercially available from,
SUBSTITUTE SHEET (RULE 26)
CA 02345230 2001-03-22
WO 00/18969 PCT/US99/21245
-27-
for example, Dupont/NEN, Boston, MA), and then washed to remove the
unincorporated 5'Cr-label. The RBCs were biotin-labeled using the Biotin-X-
NHS kit, commercially available from Calbiochem (San Diego, CA). The 5'Cr-
labeled ltBCs and biotin-labeled RBCs were then combined and infused back
S into baboon 214. Table II shows the in vivo survival time of the PENi02
treated, washed, and stored RBC compared to the in vivo survival time of
normal RBCs that had been removed from baboon 214, labeled, and re-infused.
The half life (T50) of the RBCs is shown in days.
Table III
In vivo Survival of RBC Post-PEN102 Treatment
T50 S~Cr T50 biotin
Experimental 12.2 days 33 days
Historical for Baboon13.8 days +/- 1.1 38 days +/- 5 days
214 days
As Table III demonstrates, the treatment of baboon RBC with 12 mM
PEN102, followed by manual washing and storage of the RBC for 18 hours at
4°C, did not affect either the biochemistry or the lifespan of the RBC.
CA 02345230 2001-03-22
WO 00/18969 PCT/US99/21245
-28-
Exampl~I
~uenchin~.e~hyleneimine oligomer i ..~;~ted human blood with Na-thiosulfate
or a solid- hp ase quencher
In another method to remove the ethyleneimine oligomer from treated
red blood cells, a quenching agent, either soluble {i.e., Na-thiosulfate) or
solid-
phase, was used. The reagents and methods described in this example are
described in more detail in Purmal et al., Solid ~hace ~uenching~~rc emc,
(U.S.
Patent Application Serial No. 09/161,078, filed September 25, 1998).
In this experiment, 50 ,ul of 120 mM PEN102 in 0.25 M NaH2P04 was
added to 0.9 ml of whole human CPD blood (final concentration of PEN102
was 6 mM, 6~cmole total), and incubated at 23 ° C for 4 hours. At the
end of the
4-hour incubation period, 68 mg (50 ~cmole-equivalents of
phosphothiomonoester groups) of ArgoPore-Thiophosphate support was added.
In the parallel experiment, 50 ,ul of 1 M NazSz03 (final concentration 50 mM)
was added to same amount of PEN 102 treated blood. Both samples were
allowed to incubate for 2 hours at 23 °C. The red blood cell (RBC) and
plasma
fraction of the blood were separated by centrifugation ( 10,000 rpm, 5 min.),
and the RBCs were opened by adding 9 volumes of water. The concentration
of PEN102 was determined in RBC and in the plasma fraction of the blood by
HPLC {Fig. 7).
As shown in Fig. 7, both sodium thiosulfate and solid phase-bound
thiophosphate groups were capable of quenching PEN102. After 2 hours, the
plasma quenched with sodium thiosulfate contained only 6.8 ~,g/ml PEN102,
and the red blood cells quenched with sodium thiosulfate contained 2.2 ~g/ml
PEN102. The solid phase quencher containing thiophosphate groups was even
more effective. The plasma quenched with this system contained only 1.5
~,g/ml PEN102, and the red blood cells contained only 0.9 gg/ml PEN102 after
2 hours. Thus, incubation of PEN102 treated red blood cells with the solid-
CA 02345230 2001-03-22
WO 00/18969 PCT/US99/21245
-29-
phase quencher for two hours lowered the concentration of PEN102 in the red
blood cells to a non-toxic level.
S BBC
Blood is collect from a dog and divided into two portions. The first
portion is incubated with an amount of ethyleneimine oligomer sufficient to
inactivate at least some of the animal viruses in the portion. Following
incubation, the red blood cells from this treated portion are isolated, washed
four times with sterile unbuffered saline, as described above, and then frozen
in
glycerol for two weeks at -70°C.
At the same time, the second portion is incubated for 6 hours at
22°C,
but without any addition of ethyleneimine oligomer. Following incubation, the
red blood cells are isolated and washed four times with sterile saline, and
then
frozen in glycerol for two weeks at -70°C.
Following the two weeks of frozen incubation time, the cells are thawed.
and cells in the first portion (i.e., the ethyleneimine oligomer treated
portion)
are labeled with 5'Cr while the cells in the second portion (i.e., the
untreated
portion) are labeled with biotin. The cells are then combined and re-infused
to
the donor dog. The survival time of the 5'Cr labeled red blood cells is
compared to the survival time of the biotin-labeled cells, and no significant
difference is predicted to be found between the two. Hence, ethyleneimine
oligomer treatment does not affect the survival time of canine red blood cells
in
vivo.
CA 02345230 2001-03-22
WO 00/18969 PCT/US99/21245
-30-
Example VV
jn vivo SLrvivai of PENIO~~t.~_d, washed,, and frozen baboon platelets
Eighty ml of fresh baboon blood is collected from a baboon and the
platelets are immediately separated from the remaining blood components
(which are discarded). The purified platelets are divided into two equal
portions, one of which (i.e., the PEN102 treated portion) is treated for 6
hours
at room temperature with 2.1 ml of 240 mM PEN102 in 0.25 M NaH2P04 {20X
stock solution of PEN 102 is prepared immediately before treatment) with the
final calculated total concentration of PEN 102 in the platelet-containing
solution being 12 mM (1000 pg/ml; 0.1% v/v). The second portion (i.e., the
untreated portion) is treated for 6 hours at room temperature with 2.1 ml of a
0.25 M NaH2P04.
The two portions of platelets are next separated by centrifugation, and
the supernatant removed. The pelleted cells are next resuspended in sterile
unbuffered saline, incubated at room temperature on a rocker for 10 min., and
re-pelleted by centrifugation. After four cycles of this resuspension/re-
pelleting, the platelets are frozen in glycerol for two weeks at -70°C.
After two weeks, both the treated and the untreated portions of platelets
are thawed and labeled with 5'Cr, as described above. In a blind study, the
5'Cr
labeled PEN102 treated platelets are transfused into one recipient baboon and
the 5'Cr labeled untreated platelets are transfused into a second recipient
baboon. Both of the recipient baboons are the same gender and roughly the
same age. The T50 of the PEN102 treated platelets is predicted to be
approximately equal to that of the untreated platelets.
CA 02345230 2001-03-22
WO 00/18969 PCT/US99/21245
-31-
Example VI
BiochemiStr~r and in vivo sLrvival of eth~rleneimine oligomer-treated, washed.
and stored human RRC
Blood donated by a human blood donor is incubated with an amount of
ethyleneimine oligomer sufficient to inactivate at least some of the animal
viruses in the blood. Following incubation and using the MCS~+ Apheresis
System (commercially available form Haemonetics Corp., Braintree, MA), the
ethyleneimine oligomer treated blood is separated into three components: (1)
the red blood cells (RBCs), (2) the platelets, and {3) the plasma. The plasma
and platelets are frozen in glycerol and stored at -70°C.
The isolated RBCs are spread onto a mesh having pores with diameters
that are smaller than the diameter of a human RBC, and rinsed with a
continuous flow of sterile unbuffered saline until the concentration of
ethyleneimine oligomer in the cells is at or below the concentration
determined
to be non-toxic in the mouse lymphoma forward mutation assay described
above. The washed cells are next stored for 18 hours at 4°C. The sample
of
cells is then divided into two. The first of the two portions of washed,
treated,
and stored RBCs are subjected to testing for a series of biochemical
parameters.
No significant changes in the biochemical characteristics of the washed,
treated
RBC are predicted to be seen as compared to a sample of washed, untreated
RBC.
The second of the two portions of washed, treated, and stored RBCs are
labeled with biotin, and re-infused back into the same human donor. Tracking
of the biotin-labelled cells demonstrates that the ethyleneimine oligomer
treated
cells survive in vivo for a length of time comparable that of untreated biotin-
labeled red blood cells that are similarly isolated (i.e., using the MCS~+
Apheresis System), washed, and stored.
CA 02345230 2001-03-22
WO 00/18969 PCT/US99/21245
-32-
Exam In a VII
Pro in pLrification from ethylcneimine oli~~omer treated and washed bovine
uliri~
A f rst sample of bovine urine is collected and incubated with an amount
of an ethyleneimine oligomer sufficient to inactivate at least some of the
viruses in the urine. Contemporaneously, a second sample of bovine urine is
collected and subjected to the same incubation conditions as the first sample,
but in the absence of any ethyleneimine oligomer. Both urine samples are next
greatly diluted with sterile water until the concentration of the
ethyleneimine
oligomer is below that determined to be toxic in a mouse lymphoma
mutagenicity assay (such as that described above and commercially performed
by Covance, Vienna, VA).
The urine from both samples is then subjected to protein purification
techniques to isolate the desired urine protein (e.g., the Tamm-Horsfall
glycoprotein). Standard protein purification techniques include HPLC, and
described in general technique laboratory manuals (see, e.g., Scopes, R. K.,
Protein Eurification: Principles and Practice, ed. C. R. Cantor, Spring-Verlag
Inc., New York, NY, 1982; Coligan, J.E., C''nrrent Protocols in Protein
Science,
John Wiley & Sons, New York, NY, 1996). A comparable of amount of the
desired urine protein is predicted to be purified from both the ethyleneimine
oligomer treated urine and the untreated urine.
From the above description, one skilled in the art can easily ascertain the
essential characteristics of the present invention, and without departing from
the spirit and scope thereof, can make various changes and modifications of
the
invention to adapt it to various usages and conditions. Thus, other
embodiments are also within the claims.
CA 02345230 2001-03-22
WO 00/18969 PCT/US99/21245
-33-
Without further elaboration, it is believed that one skilled in the art can,
based on the description herein, utilize the present invention to its fullest
extent.
All publications and patent applications mentioned in this specification are
herein incorporated by reference to the same extent as if each independent
publication or patent application was specifically and individually indicated
to
be incorporated by reference.
What is claimed is:
