Title: Use of defibrinated blood for manufacture of a hemoglobin-based oxygen carrier
Abstract: Red blood cells are purified by defibrinating whole blood and then filtering the defibrinated whole blood, whereby at least a portion of a plasma component is separated from the red blood cells to form a suspension of red blood cells, thereby purifying the red blood cells. Whole blood is defibrinated by, for example, using a chemical coagulating agent or mechanical agitation. Separation of the plasma component from red blood cells can be completed by, for example, diafiltration. The suspension of red blood cells can then be employed to produce a hemoglobin-based oxygen carrier.
Patent Number: 6,986,984 Issued on 01/17/2006 to Gawryl, et al.
| Inventors:
|
Gawryl; Maria S. (Charlestown, MA);
Houtchens; Robert A. (Milford, MA);
Light; William R. (Natick, MA)
|
| Assignee:
|
Biopure Corporation (Cambridge, MA)
|
| Appl. No.:
|
306819 |
| Filed:
|
November 26, 2002 |
| Current U.S. Class: |
435/2; 424/529; 530/354; 530/363; 530/380; 530/385; 530/829 |
| Current Intern'l Class: |
A01N 1/02 (20060101); A61K 38/17 (20060101); C07K 1/00 (20060101) |
| Field of Search: |
424/529
514/6
530/354,363,380,385,829
|
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|
Primary Examiner: Gitomer; Ralph
Assistant Examiner: Srivastava; Kailash C.
Attorney, Agent or Firm: Hamilton, Brook, Smith & Reynolds, P.C.
Parent Case Text
RELATED APPLICATIONS
This application is a Divisional of U.S. application Ser. No. 09/795,821, filed
Feb. 28, 2001, now U.S. Pat. No. 6,518,010. The entire teaching of the above application
is incorporated herein by reference.
Claims
What is claimed is:
1. A method for preparing a hemoglobin solution, comprising the steps of:
a) defibrinating whole blood, wherein the whole blood comprises red blood cells
and a plasma component;
b) filtering the defibrinated whole blood by diafiltration across a membrane
having a permeability limit in a range of between about 0.01 μm and about
5 μm, whereby at least a portion of the plasma component is separated from
the red blood cells to form a red blood cell suspension; and thereafter
c) releasing hemoglobin molecules from the red blood cells of the red blood cell
suspension by lysing the red blood cells of the red blood cell suspension and isolating
the hemoglobin molecules by centrifuging or filtering the lysed red blood cell
suspension;
whereby a hemoglobin solution is formed.
2. The method of claim 1, wherein centrifuging the red blood cell suspension
causes at least a portion of the red blood cells to lyse, thereby releasing hemoglobin molecules.
3. The method of claim 2, wherein the released hemoglobin is isolated by centrifugation.
4. The method of claim 2, wherein the released hemoglobin is isolated by filtration.
5. The method of claim 1, wherein the red blood cells are lysed by centrifuging
the red blood cell suspension cells that have been separated in step b).
6. The method of claim 1, wherein the red blood cells are lysed hypotonically.
7. The method of claim 1, further comprising the step of deoxygenating the hemoglobin solution.
8. The method of claim 7, wherein the content of an oxyhemoglobin component of
the hemoglobin solution is reduced to less than about 20%.
9. The method of claim 7, wherein the oxyhemoglobin component of the hemoglobin
solution is reduced to less than about 10%.
10. The method of claim 7, wherein the hemoglobin solution is deoxygenated by
chemical scavenging with a reducing agent.
11. The method of claim 10, wherein the reducing agent is selected from the group
consisting of N-acetyl-L-cysteine (NAC), cysteine, sodium dithionite or ascorbate.
Description
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 crosslinked.
Preparation of hemoglobin-based oxygen carriers includes several purification steps.
Among the components that must be removed from collected blood is fibrinogen, which
is a soluble protein that is converted into fibrin by the action of thrombin during
clotting. Current techniques for processing blood often include addition of chemical
agents, such as sodium citrate, to prevent coagulation. However, additional techniques
which might, for example, reduce the expense of processing, without diminishing
other qualities, such as ultimate product purity, are sought.
SUMMARY OF THE INVENTION
The present invention relates to the use of defibrinated blood for purifying
red blood cells, preparing a hemoglobin solution, and preparing a hemoglobin-based
oxygen carrier. Chemical clotting agents (such as collagen) and mechanical agitation
(such as stirring) are methods used to defibrinate blood. Subsequent cell washing
removes plasma proteins that may lead to incompatibility between donor and recipient blood.
In one embodiment, the method for purifying red blood cells includes defibrinating
whole blood, the whole blood including red blood cells and a plasma component.
Subsequently, the whole blood is filtered to purify the red blood cells and thereby
form a red blood cell suspension.
In an embodiment of the method for preparing a hemoglobin solution, whole blood
is defibrinated. Red blood cells are separated from the whole blood, and hemoglobin
molecules are isolated from the red blood cells to form thereby a hemoglobin solution.
In one embodiment of the method to prepare a hemoglobin-based oxygen carrier,
whole blood is defibrinated. Red blood cells are separated from the whole blood.
Hemoglobin molecules are isolated and stabilized to form the hemoglobin-based oxygen carrier.
The advantages of this invention are numerous. One advantage is that the invention
obviates the need for an anticoagulant solution to be mixed with whole blood (human,
bovine, mammalian). Adding an anticoagulant involves manpower and capital for the
processes of preparation of high purity water mixing solutions, preparation of
citrated collection containers, collection, mixing, and purification. In addition,
when shipping blood, generally it is easier to defibrinate blood than it is to
build facilities for addition of an anticoagulant at the shipper's location.
BRIEF DESCRIPTION OF THE DRAWINGS
The FIGURE is a schematic of an embodiment 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 made more 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.
Generally, the invention is a method for purifying blood to form a red
blood cell (RBC) suspension, to isolate a hemoglobin solution, and to manufacture
a hemoglobin-based oxygen carrier. The method includes defibrinating whole blood.
For the purpose of describing the invention, whole blood is considered to be comprised
of red blood cells and plasma components.
Referring to the FIGURE, shown therein is one embodiment of apparatus
10
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 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 has not been frozen
prior to use in this method. Examples of suitable whole blood sources include human,
bovine, ovine, porcine, other vertebrates and transgenically-produced hemoglobin,
such as the transgenic Hb described in
BIO/TECHNOLOGY, 12: 55-59 (1994),
the teachings of which are incorporated herein by reference in their entirety.
The blood can be collected from live or freshly slaughtered animal donors. One
method for collecting bovine whole blood is described in U.S. Pat. Nos. 5,084,558
and 5,296,465, issued to Rausch et al., the teachings of which are incorporated
by reference in their entirety.
The whole blood is defibrinated in vessel
12 by a suitable method. Defibrinating
the blood sets off the clotting cascade to remove artificially 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.
In one embodiment, the blood is defibrinated by exposure to a coagulating agent.
Examples of coagulating agents are collagen, 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 whole blood is 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, also has the effect
of initiating the clotting cascade. After stirring until fibrin polymerization
apparently ceases, it is possible to remove the accumulated fibrin 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.
Fibrin is then removed from the whole blood by a suitable means. An example
of a suitable means is by directing the whole blood, including the fibrin, from
vessel
12, through line
14 and strainer
16. A 60 mesh screen
is an example of a suitable strainer. Fibrin is collected at strainer
16
and the remainder of the whole blood is directed to vessel
18. Optionally,
or alternatively to the use of a strainer, cheesecloth or polypropylene filters
can be employed to remove large debris, including fibrin.
As shown in the FIGURE, whole blood is directed from vessel
18 through
line
20 by pump
22 and through first filter
24 and second
filter
26 to vessel
28. 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 μm, and
second filter
26 has a permeability of about 50 μm. Removal of essentially
all of the fibrin by first filter
24 and second filter
26 completes
the defibrination step.
The whole blood is maintained at a suitable temperature in vessel
28.
Preferably, the whole blood is maintained at a temperature in a range of between
about 4° C. and about 15° C. The temperature of whole blood 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 chiller, or refrigeration unit,
40.
Thereafter, the whole blood is filtered, whereby at least a portion of
the plasma component is separated from the red blood cells to form a red blood
cell suspension, thereby purifying the red blood cells. Preferably, the whole blood
is filtered by diafiltration.
In one embodiment, diafiltration is conducted by diverting whole blood 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 diafiltrate module
46 from permeate portion
58 of diafiltrate
module
46. Preferably, membrane
54 has a permeability limit in a
range of between about 0.01 μm and about 5 μm.
A portion of the plasma component of whole blood in diafiltrate module
46
passes across membrane
54 from retentate portion
56 to permeate portion
58, thereby purifying red blood cells at retentate portion
56. Purified
red blood cells are directed through retentate outlet
50 and line
60
back to vessel
28. Purified blood can be collected from vessel
28
through valve
62 to line
64 for further processing. Plasma that permeates
membrane
54 can be directed from permeate portion
58 of diafiltration
module
46 through line
66 and collected from vessel
68. Blood
recirculating through vessel
28 and diafiltrate module
46 can be
sampled at sampling ports (not shown) in line
42 or line
60.
Preferably, prior to filtering whole blood to remove at least a portion
of the plasma component, a liquid is added to the whole blood in vessel
28
from vessel
70 and line
72 to dilute its concentration. In one embodiment,
the whole blood is diluted to a concentration in a range of between about 25% and
about 75% of its initial concentration (before dilution), by volume. Concentration
then can reduce the volume back to the original concentration or more. Generally,
the process of adding a liquid to the whole blood and then removing at least a
portion of the liquid, is referred to as "cell washing."
In one embodiment, cell washing includes the processes of dilution and diafiltration
in a continuous filtration operation; a saline/citrate solution is added to the
filter retentate to maintain a constant volume in the recirculation tank. The result
is a reduction in the concentration of microfiltration membrane-permeable species
(including membrane-permeable plasma proteins). Subsequent reconcentration of the
diluted blood solution back to the original volume produces a purified blood solution.
In a preferred embodiment, the blood solution is washed by diafiltration or by
a combination of discrete dilution and concentration steps with at least one solution,
such as an isotonic solution, to separate red blood cells from extracellular plasma
proteins, such as serum albumins or antibodies (e.g., immunoglobulins (IgG)). Preferably,
the isotonic solution includes an ionic solute or is aqueous. It is understood
that the red blood cells can be washed in a batch or continuous feed mode.
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 plasma portion of
the whole blood. The blood may be diluted to a concentration in the range between
about 25% and 75% of the original concentration. A preferred isotonic solution
has a neutral pH and an osmolarity-between about 285-315 mOsm. In a preferred embodiment,
the isotonic solution is composed of an aqueous solution of sodium citrate dihydrate
(6.0 g/l) and of sodium chloride (8.0 g/l).
In one method, the whole blood is diafiltered across a membrane having a permeability
limit in the range of between 0.2 μm and about 2.0 μm. Alternate suitable
diafilters include microporous membranes with pore sizes that will separate RBCs
from substantially smaller blood solution components, such as a 0.1 μm to
0.5 μm filter (e.g., a 0.2 μm hollow fiber filter, Microgon Krosflo
II microfiltration cartridge). During cell washing, fluid 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 in the filtrate. Erythrocytes,
platelets and larger bodies of the blood solution, such as white blood cells, are
retained and mixed with isotonic solution, which is added continuously or batch-wise
to form a dialyzed blood solution.
Concurrently, a filtered isotonic solution is added continuously (or
in batches) as makeup to maintain volume of filtrate to compensate for the portion
of the solution lost across the diafilter. In a more preferred embodiment, the
volume of blood solution in the diafiltration tank is initially diluted by the
addition of a volume of a filtered isotonic solution to the diafiltration tank.
Preferably, the volume of isotonic solution added is about equal to the initial
volume of the blood solution.
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.
Cell washing generally is considered to be complete when the level of plasma
proteins contaminating the red blood cells has been substantially reduced (typically
at least about 90%). 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.
Potential foulants of the membrane could cause problems with washing, such
as slow manufacturing runs, which may be minimized by using new membranes for each
run of washing. However, it is still possible to make an effective hemoglobin-based
oxygen carrier, despite potential membrane foulants. Small fibrin molecules can
be problematic and may foul the filter if they accumulate on the surface of a membrane
with a permeability of 0.1 to 5 μm and thus block the pores. A narrower range
in which the foulants can be problematic is 0.2 to 0.4 μm. Defibrinating
(mechanical, chemical, any kind) could cause red blood cell lysing. Red blood cells,
white blood cells, or platelets that have broken open might stick to the filter.
In another embodiment of the invention, it is possible to defibrinate blood that
has already been citrated by saturating the citrated blood with a divalent cation,
and then defibrinating the solution, similar to the means by which uncitrated blood
would be processed. The preferred divalent cation is calcium.
To prepare a hemoglobin blood solution, the purified blood sample can be further
processed to isolate the hemoglobin molecules. The resulting dialyzed blood solution
is exposed to means for separating red blood cells in the dialyzed 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 components can be employed. For example, one embodiment 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 30% of the RBCs, prior to red blood cell separation from the other blood components.
Following purification of the red blood cells, the RBCs are lysed, resulting
in the production of a hemoglobin (Hb) solution. Methods of lysis include mechanical
lysis, chemical lysis, hypotonic 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. Pat. No. 5,691,452,
filed Jun. 7, 1995, which is incorporated by reference in its entirety.
The Hb eluate is then preferably deoxygenated prior to polymerization to form
a deoxygenated Hb 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.
The method of deoxygenation is further described in U.S. Pat. No. 5,895,810,
filed Jun. 7, 1995, which is incorporated herein by reference in its entirety.
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. A vertebrate is as classically
defined, including humans, or any other vertebrate animals which uses blood in
a circulatory system to transfer oxygen to tissue. Additionally, the definition
of circulatory system is as classically defined, consisting of the heart, arteries,
veins and microcirculation including smaller vascular structures such as capillaries.
"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, 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 10 to about 100 g/l 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-Hb when contacted with a cross-linking agent. Typically, the temperature
of the oxidation-stabilized deoxy-Hb is about 25 to about 45° 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, α-hydroxy aldehydes, such as glycolaldehyde,
N-maleimido-6-aminocaproyl-(2′-nitro,4′-sulfonic acid)-phenyl ester,
m-maleimidobenzoic acid-N-hydroxysuccinimide ester, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate,
sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, m-maleimidobenzoyl-N-hydroxysuccinimide
ester, m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester, N-succinimidyl(4-iodoacetyl)aminobenzoate,
sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, succinimidyl 4-(p-maleimidophenyl)butyrate,
sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate, 1-ethyl-3-(3-dimethylaminopropyl)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), and only a small amount, such as less than about 15%, are
contained within high molecular weight poly(Hb) chains. High molecular weight poly(Hb)
molecules have a molecule weight, for example, above about 500,000 Daltons.
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/l. For example, a suitable ultrafilter is a 30,000 Dalton filter (e.g.,
Millipore Helicon Cat # CDUF050LT; Amicon Cat # 540430).
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 dialfiltration or hydroxyapatite
chromatography (see, e.g. U.S. Pat. No. 5,691,453, filed Jun. 7, 1995, which is
incorporated herein by reference in its entirety). Following pH adjustment, at
least one reducing agent, preferably a sodium borohydride solution, is added to
the polymerization step typically through the deoxygenation loop. 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.
Suitable methods of cross-linking hemoglobin and preserving the hemoglobin-based
oxygen carrier are discussed in detail in U.S. Pat. No. 5,691,452, issued Nov.
25, 1997, which is incorporated herein by reference in its entirety.
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. Pat. No. 5,854,209, filed Mar. 23, 1995, which
is incorporated herein by reference in its entirety.
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 make 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
The bench-scale experiments were performed in the apparatus shown in the FIGURE.
The defibrinated blood sample used in the bench scale experiment was defibrinated
by exposure to collagen. Initially, whole blood is diluted approximately 1:1 with
isotonic citrate saline buffer. The diluted blood was then concentrated back to
produce a Hb level of 10.5 g/dl (approximately a two-fold concentration). The process
volume for the diafiltration was 200 ml, therefore approximately 200 ml buffer
was added to approximately 200 ml whole blood followed by concentration back to
its original volume. This produced approximately 200 ml of membrane permeate. The
200 ml whole blood at a Hb concentration of 10.5 g/dL was then diafiltered against
citrate/saline buffer. The time to collect 400 mls permeate volume (2 retentate
volumes) was used as a point of comparison for the citrated blood and the defibrinated
blood. The time included the time to concentrate the diluted blood back to its
original volume (200 ml) and the time to perform the first diafiltration volume
(200 ml). The longer the time, the slower the process. Table 1 summarizes the results.
| TABLE 1 |
|
| |
|
Time to Collect 400 ml |
| Animal Number |
Whole Blood Sample |
Permeate (Hr: Min: Sec) |
|
| 1 |
Citrated |
0:25:08 |
| 1 |
Defibrinated |
0:51:06 |
| 2 |
Citrated |
0:24:47 |
| 2 |
Defibrinated |
0:25:15 |
| 3 |
Citrated |
0:25:08 |
| 3 |
Defibrinated |
0:24:28 |
| 4 |
Citrated |
0:30:18 |
| 4 |
Defibrinated |
0:15:57 |
|
As can be seen from Table 1, the time required to collect 400 ml of permeate
was
between about fifteen minutes and an hour.
EXAMPLE 2
Pilot Scale Experiment
The pilot-scale experiments were performed in the apparatus shown in the FIGURE.
The defibrinated blood sample used in the pilot-scale experiment was defibrinated
by mechanical agitation. Again, the whole blood is diluted with isotonic citrate
saline solution and concentrated, but because of the large volume required for
processing in the pilot scale system, the initial whole blood was diluted with
a greater than 1:1 ratio of citrate/saline buffer to whole blood. The Hb concentration
of the blood during diafiltration is less than 10.5 g/dl (approximately a two-fold
concentration). After concentration back to the minimum process volume of the system
(approximately 4.5 L), the blood was diafiltered for 5 diafiltration volumes. As
in the bench scale experiment, a longer time indicates a slower process. Table
2 summarizes the results.
| TABLE 2 |
|
| Experiment |
|
Processing Time to Collect 5 |
| Number |
Whole Blood Sample |
Diafiltration Volumes (minutes) |
|
| |
| 1 |
Citrated (Control) |
91 |
| |
Defibrinated |
28 |
| 2 |
Citrated (Control) |
83 |
| |
Defibrinated |
150 |
|
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.
While this invention has been particularly shown and described with references
to preferred embodiments thereof, it will be understood by those skilled in the
art that various changes in form and details may be made therein without departing
from the scope of the invention encompassed by the appended claims.
*
