U.S. patent application number 12/426566 was filed with the patent office on 2010-08-05 for method and apparatus for preparing an acellular red blood cell substitute.
This patent application is currently assigned to Northfield Laboratories, Inc.. Invention is credited to Richard E. DeWoskin, Marc D. Doubleday.
Application Number | 20100197566 12/426566 |
Document ID | / |
Family ID | 21765191 |
Filed Date | 2010-08-05 |
United States Patent
Application |
20100197566 |
Kind Code |
A1 |
DeWoskin; Richard E. ; et
al. |
August 5, 2010 |
Method and Apparatus for Preparing an Acellular Red Blood Cell
Substitute
Abstract
A process is disclosed for the preparation of an essentially
tetramer-free, substantially stroma-free, polymerized,
pyridoxylated hemoglobin. Also disclosed is an essentially
tetramer-free, substantially stroma-free, polymerized,
pyridoxylated hemoglobin product capable of being infused into
human patients in an amount of up to about 5 liters.
Inventors: |
DeWoskin; Richard E.; (St.
Charles, IL) ; Doubleday; Marc D.; (Cary,
IL) |
Correspondence
Address: |
MCDONNELL BOEHNEN HULBERT & BERGHOFF LLP
300 S. WACKER DRIVE, 32ND FLOOR
CHICAGO
IL
60606
US
|
Assignee: |
Northfield Laboratories,
Inc.
|
Family ID: |
21765191 |
Appl. No.: |
12/426566 |
Filed: |
April 20, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11972322 |
Jan 10, 2008 |
7521417 |
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12426566 |
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10993228 |
Nov 19, 2004 |
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11972322 |
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10274099 |
Oct 17, 2002 |
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10993228 |
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09155419 |
May 10, 1999 |
6498141 |
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PCT/US1997/005088 |
Mar 27, 1997 |
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10274099 |
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60014389 |
Mar 28, 1996 |
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Current U.S.
Class: |
514/1.1 |
Current CPC
Class: |
A61K 38/42 20130101;
A61P 7/00 20180101; Y10S 514/832 20130101; A61P 7/06 20180101; A61P
41/00 20180101; C08H 1/00 20130101; A61P 7/08 20180101; Y10S
530/829 20130101; C07K 14/805 20130101 |
Class at
Publication: |
514/6 |
International
Class: |
A61K 38/42 20060101
A61K038/42; A61P 41/00 20060101 A61P041/00 |
Claims
1. An aqueous solution of pyridoxylated, polymerized hemoglobin,
where the hemoglobin solution is capable of being infused into a
human patient in an amount of up to about 1.5 liters without
causing a decrease in kidney performance.
2. A solution of hemoglobin according to claim 1, wherein the
solution has a half-life in a human patient of about 15 hours.
3. A solution of hemoglobin according to claim 1, wherein the
solution has a half-life in a human patient of about 24 hours.
4. A solution according to claim 1, wherein the solution may be
infused in an amount of up to about 5 liters.
5. A solution of hemoglobin according to claim 1, wherein the
solution is capable of being infused into a human patient in an
amount of up to about 3 liters without causing a decrease in kidney
performance, and the solution has a half-life in a human patient of
about 24 hours.
6. A process for preparing a solution of pyridoxylated, polymerized
hemoglobin capable of being infused into a human patient in an
amount of up to about 3 liters, comprising: (a) removing leucocytes
by filtering a mixture containing red blood cells through a filter
having a minimum average pore size sufficient prevent the passage
of leucocytes; (b) lysing the red blood cells; (c) adding carbon
monoxide to and heating the product of (b) to a temperature of
about 60-62.degree. C. for about 10 hours to yield a heat-treated
hemoglobin solution; (d) filtering the heat treated hemoglobin
solution to remove stroma and stromal contaminants precipitated by
the heating; (e) degassing the heat-treated hemoglobin solution by
sparging oxygen and then nitrogen through the heat-treated solution
at a temperature of about 10.degree. C. to produce a foam to yield
a degassed, heat-treated hemoglobin solution; (e) pyridoxylating
the degassed solution to yield a solution of pyridoxylated
hemoglobin; (f) polymerizing the solution of pyridoxylated
hemoglobin to produce a solution of pyridoxylated, polymerized
hemoglobin; (g) oxygenating the solution; (g) purifying the
solution to remove tetrameric hemoglobin and collecting purified
pyridoxylated, polymerized hemoglobin, where the solution contains
less than 0.8% based on the total weight of hemoglobin of tetramer;
(h) deoxygenating the purified pyridoxylated, polymerized
hemoglobin; and (i) adjusting the pH and electrolyte levels in the
solution of purified pyridoxylated, polymerized hemoglobin to
physiological levels.
7. A process according to claim 6, wherein the degassed solution is
pyridoxylated by contacting the degassed solution with
pyridoxal-5-phosphate at a molar ratio of pyridoxal-5-phosphate to
hemoglobin of from about 1:1 to 3:1.
8. A process according to claim 6, wherein the degassed solution is
pyridoxylated by contacting the degassed solution with
pyridoxal-5-phosphate at a molar ratio of pyridoxal-5-phosphate to
hemoglobin of about 2:1.
9. A process according to claim 7, wherein the
pyridoxal-5-phosphate is contacted with the hemoglobin and
borohydride for about one hour.
10. A process according to claim 9, wherein the hemoglobin in the
pyridoxylated solution is polymerized by contacting the
pyridoxylated solution with glutataldehyde at a glutaraldehyde to
hemoglobin molar ratio of from about 24:1.
11. A process according to claim 10, wherein the pyridoxylated
solution is contacted with glutaraldehyde for about 18 hours and
then quenched.
12. A method of transfusing a human patient in need of a
transfustion, comprising administering to the patient up to about
1.5 liters of a pyridoxylated, polymerized hemoglobin solution.
13. A method of transfusing a human patient in need of a
transfustion, comprising administering to the patient up to about
3.0 liters of a pyridoxylated, polymerized hemoglobin solution.
14. A method of transfusing a human patient in need of a
transfustion, comprising administering to the patient up to about
5.0 liters of a pyridoxylated, polymerized hemoglobin solution.
15. A method according to claim 14, wherein the hemoglobin is a
glutaraldehyde-polymerized hemoglobin.
16. A method according to claim 15, wherein the hemoglobin has the
molecular weight distribution shown in FIG. 4.
17. A solution according to claim 1, wherein the hemoglobin is a
glutaraldehyde-polymerized hemoglobin having the molecular weight
distribution shown in FIG. 4.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] This invention relates to methods and apparatus for
preparing red blood cell substitute products, i.e., hemoglobin
products. It further relates to an acellular red blood cell
substitute comprising an essentially tetramer-free, cross linked,
polymerized, pyridoxylated hemoglobin solution which is free of
stromal contaminants.
[0003] 2. Description of Related Art
[0004] For a number of years, blood banks have provided whole blood
for replacement during surgery, because of trauma, or for other
situations. However, whole blood obtained from human donors is not
suitable for a variety of uses. In particular, the use of whole
blood is problematic because of the requirement for donor-typing,
stability and shelf-life problems and toxicity caused by viruses
and other contaminants. These problems are especially pertinent to
emergency situations, such as the use of blood by the military.
Consequently, much effort has been devoted to the development of
substitutes for whole blood obtained from human donors. This
development has resulted in various modifications to blood from
human or other mammalian sources. Stroma-free hemoglobin is known
in the art to have oxygen transport and reversible oxygen (or
ligand) binding capacities. Since toxicity problems have precluded
use as a blood substitute, stroma-free hemoglobin has required
further modifications to provide a nontoxic, useful pharmaceutical
product.
[0005] These modifications include (1) rendering hemoglobin free or
substantially free of stroma and stromal contaminants; (2)
pyridoxylation; (3) polymerization or cross-linking; (4) removal of
tetramer; and (5) modification with carbon monoxide or other
ligands.
[0006] However, hemoglobin solutions prepared by these techniques,
while capable of carrying sufficient quantities of oxygen to
support life, have been plagued with many undesirable side effects
and properties. For example, a major troubling side effect is a
decrease in kidney performance. These changes were thought to be
due to the presence of unwanted contaminants such as bacterial
endotoxin or fragments of red cell membranes (stroma). While
contaminants such as these can indeed produce renal alterations,
hemoglobin solutions essentially free of the above contaminants
still produce substantial renal dysfunction. The cause for the
renal dysfunction has been ascribed to physiologically unacceptable
amounts of unpolymerized hemoglobin tetramer. Other undesirable
side effects of the infusion of tetrameric hemoglobin are
vasoconstriction, hemoglobinuria, depression of heart rate,
elevation of mean arterial blood pressure and extravasation of
infusate especially into the peritoneal cavity.
[0007] In practice, no known hemoglobin-derived blood substitute
has been successful in totally avoiding toxicity problems. These
products also have unacceptably low half-lives after administration
to human patients. Such half-lives require replacement of blood
volume repeatedly over short periods of time. Consequently, there
is a substantial need for hemoglobin products that are non-toxic to
patients and have substantial half-lives after administration. Of
course, these products must be capable of reversibly transporting
oxygen to tissues in a manner similar to that achieved by whole
blood.
SUMMARY OF THE INVENTION
[0008] The present invention provides hemoglobin substitutes that
are non-toxic to humans and have substantial half-lives of at least
15 hours when administered to humans. The hemoglobin products of
the invention are stroma-free, pyridoxylated and polymerized as
well as being free of viral and other toxic contaminants. Further,
these products are substantially free of leukocytes (white blood
cells) and platelets.
[0009] The present invention also encompasses processes for
preparing the inventive hemoglobin substitutes. The processes
include removing leukocytes and platelets from blood; washing and
lysing the red blood cells; removing stromal contaminants and
stroma by filtration and heat-treating; preparing the deoxy form of
the hemoglobin; pyridoxylation and polymerization; further
purification and concentration; and deoxygenation. The resulting
hemoglobin product may be then formulated to provide a hemoglobin
product having levels of various electrolytes within normal
physiological ranges.
[0010] The invention also provides an aqueous formulation of
pyridoxylated, polymerized hemoglobin, where the hemoglobin is a
glutaraldehyde-polymerized hemoglobin containing tetrameric
material, having the molecular weight profile of FIG. 3. This
formulation may be used to prepare an acellular red blood cell
substitute. In this aspect, the formulation is first purified to
remove tetramer and then combined with appropriate amounts of
electrolytes to produce a physiologically acceptable, acellular,
red blood cell substitute that may subsequently be used to treat a
human patient requiring an infusion of an oxygen carrier.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 is a schematic diagram depicting the portion of the
process and equipment used to result in a deoxygenated hemoglobin
solution prepared for pyridoxylation and polymerization.
[0012] FIG. 2 is a schematic diagram depicting the portion of the
process and apparatus beginning with pyridoxylation and
polymerization and resulting in a deoxygenated, purified,
pyridoxylated, polymerized hemoglobin product and the portion of
the process and apparatus used to formulate the final hemoglobin
product having physiological levels of electrolytes.
[0013] FIG. 3 is an HPLC tracing of polymerized material after
glycine treatment prior to purification. Polymerized product is
indicated by peaks at retention times (RT) 15.57, 16.08, 17.00, and
18.19. Tetramic material is indicated by peaks at RT 19.88 and
20.51. Polymer is 76.2% of this material.
[0014] FIG. 4 is an HPLC tracing of the hemoglobin product of the
invention. Polymerized hemoglobin is indicated by the peaks at RT
15.7, 16.33, 17.32, and 18.56. Tetramer is indicated by the peak at
RT 21.18.
[0015] FIG. 5 is a schematic diagram depicting a column
chromatography purification process employed in the invention.
[0016] FIG. 6 is a schematic diagram depicting a membrane
filtration purification process employed in the invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0017] The present invention concerns an acellular red blood cell
substitute comprising an essentially tetramer-free, cross-linked,
polymerized, pyridoxylated hemoglobin which is substantially free
of stroma, stromal contaminants and other contaminants.
[0018] As used herein, the term "cross-linked" means the chemical
emplacement of molecular "bridges" onto or into a molecule, or
between molecules with the purpose of altering the shape, size,
function or physical characteristics of the molecule. Cross-linked
molecules may be polymerized or non-polymerized, i.e., cross-linked
molecules may be tetrameric.
[0019] As used herein, the term "tetramer" refers to hemoglobin
molecules having a molecular weight of about 64 Kd; that is, the
term refers to both native and intramolecularly crosslinked
hemoglobin molecules.
[0020] As used herein, the term "essentially tetramer free" denotes
the level of purity with respect to tetramer contamination at which
certain biological responses to tetramer administered into a mammal
are no longer present. A main criterion is the absence of
alterations in renal function when pharmaceutically effective
amounts are infused, that is, at a level of purity of about 99% or
better (less than about 1% of tetramer is present). The preferred
product produced by the inventive process contains no more than
about 0.8% tetramer based on the weight of total hemoglobin (THb).
In other words, an essentially tetramer-free product according to
the invention contains no more then physiologically acceptable
amounts of unpolymerized hemoglobin tetramer. Particularly
preferred products of the invention contain less than about 0.5%
tetramer; the most particularly preferred products of the invention
contain about 0.3-0.4% tetramer. Such amounts of tetramer have been
found to be physiologically acceptable.
[0021] As used herein, the terms "ultrapurified product" or
"purified product" have the same meaning as the term "essentially
tetramer-free."
[0022] As used herein, % total hemoglobin (THb) is defined as grams
of hemoglobin/100 mL of solution.
[0023] As used herein, the term "polymerizing solution" means a
solution containing a "cross-linking" or polymerizing agent, such
as glutaraldehyde, imido esters, diaspirin or others, in a
biochemically suitable carrier.
[0024] As used herein, the term polymerized means the placement of
molecular bridges between molecules or tetrameric submits where the
size and weight of the resulting polymerized molecule is increased
with respect to native or tetrameric hemoglobin. Polymerized
hemoglobin is not tetrameric hemoglobin.
[0025] By a solution of hemoglobin as used herein is meant a
solution of tetrameric hemoglobin or polymerized hemoglobin
molecules where the molecules are not contained within a red blood
cell. Such a solution need not be free of or substantially free of
red blood cell stroma or stromal contaminants. However, preferred
polymerized hemoglobin solutions are free of red blood cell stroma
and stromal contaminants.
[0026] By the term "semipermeable membrane" is meant a membrane
permeable to some molecular species and not to others and, i.e., a
membrane which acts as a selective filter excluding certain
molecular weights.
[0027] The product of the process according to the present
invention, a polymerized, pyridoxylated, hemoglobin solution
essentially free of tetrameric (native or intramolecularly
crosslinked) hemoglobin, stromal and various other contaminants,
produced from heat treated, virally inactivated tetrameric
hemoglobin, is physiologically acceptable as well as
therapeutically and clinically useful. The product has reversible
oxygen binding capacity which is necessary for oxygen transport
properties. Most notably, the product demonstrates good loading and
unloading characteristics in usage which correlates to having an
oxygen-hemoglobin dissociation curve (P.sub.50) similar to whole
blood. The product binds oxygen with high affinity in the
capillaries through the lungs and then adequately releases oxygen
to the tissues in the body. The product also does not require
compatibility studies with the recipient.
[0028] The product also has a half-life when administered to humans
of about at least 15 hours and more preferably of about 24 hours.
This hemoglobin product may be infused into patients in amounts of
up to about 3.0 L and even up to about 5.0 L. In other words, the
inventive hemoglobin product can be used to replenish essentially
all of a human patient's blood volume without causing
vasoconstriction, renal toxicity, hemoglobinuria or other problems
associated with intravenous administration of synthetic or
semisynthetic oxygen carriers and blood substitutes. Thus, the
invention includes a method of transfusing a patient, preferably a
human patient, with an amount of a stroma-free, tetramer-free,
polymerized, pyridoxylated hemoglobin product that is non-toxic to
the patient, where the amount is up to at least about 5.0 L. Such a
method includes attaching the patient or subject to an infusion
device or other such equipment for infusing or transfusing the
patient.
[0029] The process of this invention is unique in that it yields a
product having a level of tetramer of no more than about 1% and,
more preferably, no more than about 0.8% by weight based on the
weight of total hemoglobin in the solution. The process of this
invention provides a further advantage in that it can render the
final product substantially free of microbial and viral antigens
and pathogens. Such microbial and viral antigens and pathogens are
reduced to nondetectable levels i.e. The product is sterile as
determined by the analysis set forth in the United States
Pharmacopoeia, XXIII Chapter <71>. Examples of such antigens
and pathogens include, for example, bacterial, rickettsial, fungal,
protozoan, viral and other organisms. Most importantly, the process
provides a biological product free of viruses that cause hepatitis
and acquired immune deficiency syndrome (AIDS).
[0030] Insofar as the physiological properties are concerned, the
biological product of this invention, when infused in amounts of up
to at least about 5.0 L, does not cause vasoconstriction, renal
toxicity, hemoglobinuria and other problems implicated with
intravenous administration of known hemoglobin solutions containing
physiologically undesirable amounts of tetrameric hemoglobin.
Intravenous administration of the product produced by the process
described herein results in no appreciable decrease in urine
production, no appreciable decrease in glomerular filtration rate,
no appreciable extravasation into the peritoneal cavity and no
appreciable change in the color of urine produced.
[0031] Therefore, the process of the invention provides an
acellular red blood cell substitute useful in the treatment of
trauma, myocardial infarction, stroke, acute anemia and oxygen
deficiency disorders such as hypoxemia, hypoxia or end stage
hypoxia due to impairment or failure of the lung to fully oxygenate
blood. The product also is useful in the treatment of any disease
or medical condition requiring a resuscitative fluid (e.g., trauma,
specifically hemorrhagic shock), intravascular volume expander or
exchange transfusion. In addition to medical treatment, the product
can be useful in preserving organs for transplants.
[0032] The inventive process comprises the following
procedures:
[0033] 1. red cell aspiration and filtration
[0034] 2. cell wash/lyse
[0035] 3. heat treatment
[0036] 4. ultrafiltration concentration
[0037] 5. degassification
[0038] 6. chemical modification
[0039] 7. purification
[0040] 8. UP poly concentration
[0041] 9. deoxygenation
[0042] 10. formulation
[0043] The preferred starting material in the process of the
present invention is outdated whole human blood or packed red blood
cells. In addition, non-outdated blood (indated) may also be used.
Preferably, whole blood is not used in this process if it has been
in storage for more than 2 weeks past the expiration date indicated
on the bag. The use of whole blood outdated by more than 2 weeks
provides additional difficulty in extracting the hemoglobin and
removing cellular remnants such as stromal proteins and
contaminants.
[0044] All processes described herein are applicable to other
mammalian blood with possible minor modifications within the skill
of the art. Most of the process may be carried out at about
2.degree. C. to about 8.degree. C., preferably about 4.degree.
C.
[0045] During red cell aspiration and filtration, the red blood
cells (RBC) are aseptically extracted from donor bags without
introducing air into the blood and passed across a series of
filters to result in a RBC suspension having reduced amounts of
leukocytes and platelets. The resulting suspension is then
subjected to cell washing/lysing.
[0046] The suspension is washed under carbon monoxide atmosphere
with an about 1% NaCl solution to remove residual plasma proteins.
The washed RBC are then treated with water for injection (WFI) to
lyse the cells and the resulting mixture clarified using a cross
flow filtration unit. The clarified product is then heat-treated to
precipitate additional stromal material which is removed by
filtration. The product of this procedure is a stroma-free
hemoglobin (SFH) solution with a THb of about 3% (w/v).
[0047] The heat treated and stroma-free hemoglobin solution
containing carboxyhemoglobin is concentrated and degassed to yield
a SFH solution containing deoxyhemoglobin. Degassification involves
first saturating the carboxyhemoglobin solution with oxygen for
about 16 hours to yield a solution of oxygenated hemoglobin and
about 7% by weight, based on the total weight of hemoglobin, of
carboxyhemoglobin. Subsequently, the oxygen is driven off with
nitrogen, argon or helium to form a solution containing free
hemoglobin, i.e., uncomplexed hemoglobin, and about 7% by weight,
based on the total weight of hemoglobin, of oxyhemoglobin. The
resulting degassed solution is filtered and transferred into a
vessel for chemical modification.
[0048] Subsequent to degassification, the stroma-free hemoglobin
solution is pyridoxylated using pyridoxal-5'-phosphate (P5P) at a
molar ratio of pyridoxal-5'-phosphate to hemoglobin of about 1:1 to
3:1. Alternatively, the stroma-free hemoglobin may be pyridoxylated
using 2-Nor-2 formyl pyridoxal-5'-phosphate. A reducing agent such
as sodium cyanoborohydride or preferably sodium borohydride is
added to the pyridoxylation mixture. Excess reagents and salts are
removed by dialysis against pyrogen free water or, preferably,
diafiltration with WFI. The pyridoxylated hemoglobin is then
polymerized with a glutaraldehyde solution.
[0049] The stroma-free, pyridoxylated hemoglobin solution is
polymerized using an aqueous glutaraldehyde solution. The duration
of polymerization and the amount of glutaraldehyde added is
dependent on volume of the hemoglobin solution, the desired yield
of polymers and the desired molecular weight distribution. In
general, longer polymerization times increase the yield and the
molecular weight distribution of the polymers. A yield of
approximately 75% by weight of polymers, based on the total weight
of hemoglobin, is obtained in about 16-18 hours. The preferred end
point of the polymerization is defined as that point where the
solution contains about 75% by weight of polymers, based on the
total hemoglobin weight, as monitored by size-exclusion HPLC.
Alternatively, the endpoint is defined as the point at which the
solution contains about 65% of polymers based on the total weight
of hemoglobin, i.e., about 2.5 hours.
[0050] The polymerization reaction is quenched by the addition of
aqueous glycine. The buffer must be added as quickly as possible.
The cross-links are then stabilized by adding, again as quickly as
possible, a solution of aqueous sodium borohydride. This
polymerized solution is subsequently concentrated and then
diafiltered under an atmosphere of oxygen to oxygenate the
solution. Water is finally added to the solution until the solution
contains about 4% by weight hemoglobin.
[0051] Polymerization according to the invention results in a high
yield of polymers having a narrow molecular weight range as shown
in FIG. 3 and Example 1 below.
[0052] The polymerized, pyridoxylated hemoglobin solution is then
purified by column chromatography, filtration, e.g., membrane
filtration, or both, to remove residual unpolymerized (tetrameric)
hemoglobin from the solution. The purified polymerized hemoglobin
solution is then concentrated to about 6% using an ultrafiltration
apparatus in preparation for gas exchange.
[0053] The concentrated solution is then deoxygenated with
nitrogen. The deoxygenation takes place at about 10-12.degree. C.
until the amount of oxyhemoglobin in the solution is less than
about 16% by weight of the total hemoglobin.
[0054] The resulting deoxygenated, purified, and polymerized
hemoglobin solution is then concentrated by ultrafiltration under a
nitrogen atmosphere in a cooled vessel. The pH is adjusted to about
8.8-9.0, and the amounts of electrolytes may be adjusted as
necessary to levels representing that of normal plasma. In
addition, conventional antioxidants such as glutathione, ascorbate
or glucose may also be optionally added. After the solution is
concentrated to the desired level, preferably about 10% by weight
polymerized, pyridoxylated, purified, tetramer-free, stroma-free
hemoglobin, the solution is sterilized by filtration and
transferred via a sterile transfer apparatus into suitable
pharmaceutically acceptable containers.
[0055] The characteristics of the resulting hemoglobin solution are
shown below:
TABLE-US-00001 Polymerized Hemoglobin Total Hemoglobin (g/dl).sup.1
9.5-12.0 Methemoglobin (% of total Hb.sup.)1 <8.0
Carboxyhemoglobin (% of total Hb).sup.1 <5.0 P.sub.50
(torr).sup.1 23-32 Osmolality (mmol/Kg).sup.2 280-360 Sodium
(mmol/L).sup.3 135-155 Potassium (mmol/L).sup.3 3.5-4.5 Chloride
(mmol/L).sup.3 85-110 Free Iron (ppm).sup.4 <2.0 Molecular Wt.
Dist. - 128 Kd peak (%).sup.5 10-24 Molecular Wt. Dist. - 192 Kd
peak (%).sup.5 18-30 Molecular Wt. Dist. - 256 Kd peak (%).sup.5
45-70 Tetramer (64K)(%).sup.5 <0.8 Endotoxin (EU/mL).sup.6
<0.03 Phospholipids ng/Hb.sup.7 <50 Glycolipids (ng/Hb).sup.7
<2 .sup.1Level in polymerized hemoglobin determined
spectrophotometrically. .sup.2Level in polymerized hemoglobin
determined by osmometry. .sup.3Level in polymerized hemoglobin
determined by ion specific electrode. .sup.4Level in polymerized
hemoglobin determined by atomic aborption. .sup.5Determined by size
exclusion-HPLC. .sup.6Determined by LAL using an assay commercially
available from Associates of Cape Cod, assay components have
catalog nos. 100-5, 800-1, and 3100-5. .sup.7Determined by
HPTLC
[0056] The following examples demonstrate certain aspects of the
present invention. However, it is to be understood that these
examples are for illustrative purposes only and do not purport to
be wholly definitive as to conditions and scope of this invention.
All temperatures are expressed in degrees Celsius unless otherwise
specified. Unless otherwise noted, all percentages, e.g., of total
hemoglobin (THb), are expressed as weight/volume (w/v). It also
should be appreciated that when typical reaction conditions (e.g.,
temperature, reaction times) have been given, the conditions which
are both above and below these specific ranges can also be used,
though generally less conveniently.
[0057] Unless noted to the contrary, all vessels and tanks used in
the inventive process are made of 316-L Stainless Steel, preferably
a pharmaceutical grade of such stainless steel that has been highly
polished and therefore easily and rapidly cleaned. The various
connecting pipes and tubes are made of the same stainless steel or
of a pharmaceutical grade Teflon or silicone tubing. The filters
and membranes used in the process may be purchased from Millipore
Inc., Pall-Filtron, or Cuno Inc.
[0058] The half-life of the resulting product of the invention is
determined in vivo in mammals, e.g., humans. Typically, a blood
sample is removed from the mammal a period of time after the mammal
has been infused with the product. The amount of the product is
then determined by centrifuging the blood sample, expressing the
plasma portion, determining plasma hemoglobin levels
spectrophotometrically, and then correlating the amount of product
remaining in the mammal to the half-life of the product.
[0059] Size Exclusion Chromatography HPLC according to the
invention is carried out follows:
[0060] The sample is diluted with 0.2 M pH 6.9 potassium phosphate
buffer to 0.2 g/dl, filtered through a 0.2.mu. filter and injected
into an HPLC system consisting of the following components (in
order of system flow):
[0061] 1. Pharmacia model 2248 pump [0062] mobile phase is 0.2 M pH
6.9 potassium phosphate [0063] flow rate is 1.0 mL/minute
[0064] 2. 45 cm PEEK or titanium tubing, 0.010 in. I.D.
[0065] 3. Rheodyne model 7725i injector with 200 .mu.L PEEK sample
loop
[0066] 4. 18 cm PEEK or titanium tubing, 0.010 in. I.D.
[0067] 5. Upchurch model A431 0.5.mu. filter
[0068] 6. 9 cm PEEK or titanium tubing, 0.010 in. I.D.
[0069] 7. Phenomenex Biosep SEC S-3000 75.times.7.8 mm Guard
column
[0070] 8. 24 cm PEEK or titanium tubing, 0.010 in. I.D.
[0071] 9. Phenomenex Biosep SEC S-3000 600.times.7.8 mm Analytical
column
[0072] 10. 23 cm PEEK or titanium tubing, 0.010 in. I.D.
[0073] 11. Pharmacia Uvicord SD UV detector
TABLE-US-00002 wavelength: 280 nm flow cell: 8 .mu.L vol., 2.5 mm
pathlength range: 2 AUFS time constant: 10 seconds
[0074] The peak absorbance at 280 nm is recorded by a LKB 2221
Integrator, which integrates the individual peak areas and
calculates the total Hemoglobin area for each polymeric
species.
[0075] A further understanding of the invention may be obtained
from the following nonlimiting examples.
Example 1
[0076] Referring now to FIG. 1, donor bags 20 of outdated blood
(whole blood or packed red blood cells) are situated in a suitable
aseptic aspiration apparatus 22. As an example of a suitable
aspiration apparatus is a system having two aspiration stations.
Once a donor bag is placed in the aspiration station, a needle in
the aspiration apparatus punctures the donor bag, introduces about
150 ml of a 1% (w/v) aqueous sodium chloride solution and aspirates
the outdated blood from the donor bag under reduced pressure or
vacuum. The aspirated blood is passed through a 100.mu. depth
filter 24 and subsequently through two 5.mu. depth 26 filters in
series. As the blood passes through the 5.mu. depth filters,
leukocytes are removed from the blood. Typically, about 170 units
of outdated whole blood are aspirated, filtered and subsequently
transferred to Tank 1 as shown in FIG. 1. The filters are then
rinsed with about 75 liters of a 1% (w/v) agueous sodium chloride
solution. Prior to the introduction of the blood into Tank 1, Tank
1 is charged with about 70 L of a 1% aqueous sodium chloride
solution. After all 170 units of outdated whole blood have been
aspirated, filtered and transferred, and the filters have been
rinsed, the tank contains about 250 liters of a 4% total hemoglobin
solution. During the aspiration and filtering steps, Tank 1 is
maintained at a reduced pressure, i.e., a vacuum of 20-28'' Hg.
Once all the outdated blood has been transferred to Tank 1, the
vacuum is switched off and carbon monoxide is introduced into the
tank so that the tank contains an atmosphere of carbon
monoxide.
[0077] Tank 1 is coupled to a 0.65.mu. tangential flow filter 28 as
shown in FIG. 1. The initial charge of 250 liters of 4% total
hemoglobin solution is concentrated to approximately 125 L of an 8%
total hemoglobin solution by microfiltration through the tangential
flow filter. The pH of the hemoglobin solution at this point is
about 6 to 6.5. Subsequent to concentrating to 8% total hemoglobin,
the solution is washed by adding a 1% (w/v) sodium chloride
solution, diafiltering and removing the filtrate at the same rate
sodium chloride solution is added. The 125 L of hemoglobin solution
is typically washed with about 8 volumes of the 1% sodium chloride
solution (about 1,000 L). Subsequent to washing, the solution is
concentrated to about 70 L, i.e., about 14% total hemoglobin, and
"water for injection" (WFI) is added to bring the volume of the
solution up to about 180 L. With the addition of the WFI, the cells
swell and rupture releasing hemoglobin into solution. The
concentration of the resulting hemoglobin solution is about 5%
total hemoglobin (THb).
[0078] The resulting solution is clarified while still in Tank 1.
The solution is first concentrated to about 50 L and the filtrate
transferred to Tank 2. As the solution is pumped across the filter,
red blood cells stroma contaminants and cell wall material is
retained and removed by the filter. The remaining 50 L of solution
in Tank 1 is washed (diafiltered) with about 2.5 volumes of WFI.
This 2.5 volumes of wash is added to Tank 2. The material remaining
in Tank 1 is then concentrated to about 20 L and the filtrate added
to Tank 2. The volume resulting in Tank 2 is about 280 L of a 3.3%
total hemoglobin solution.
[0079] The resulting solution of stroma-free hemoglobin is then
heat treated in Tank 2 at a temperature of about 60-62.degree. C.
over a period of about 10 hours. During this time, the solution is
moderately agitated. As the solution is heated and passes a
temperature of about 55.degree. C., a precipitate forms.
[0080] The resulting 3.3% THb (w/v) stroma-free, heat treated
hemoglobin solution is then filtered through a 0.2.mu. filter 30
followed by a 0.1.mu. 32 filter and transferred to Tank 3. The
filtered hemoglobin solution is then concentrated to about 18% THb
and subsequently washed and diafiltered with 4 volumes of WFI (180
L). The concentration and diafiltration is accomplished using a 10
kilodalton (kd) molecular weight ultrafilter 34. Drain 35
associated with ultrafilter 34 collects filtrate. At this point,
the 45 L of 18% total hemoglobin solution contains less than 50 ng
of phospholipid per gram of hemoglobin, less than 2 ng of
glycolipid per gram of hemoglobin, less than 1% methemoglobin, less
than about 0.03 endotoxin units of endotoxin per milliliter at a pH
of about 6 to 6.5. This hemoglobin in the solution is
carboxyhemoglobin.
[0081] The resulting carboxy hemoglobin solution is then
transferred to Tank 4 where the carboxyhemoglobin is first
oxygenated and then deoxygenated. Tank 4 is fitted with a gas
sparge ring coupled to oxygen and nitrogen gas lines, a feed from
the tank bottom to a metered spray apparatus positioned at the top
of Tank 4, and a foam overflow collector connected to Foam Can 1
such that foam generated in Tank 4 is fed into Foam Can 36 where
the foam condenses into liquid and is fed back into Tank 4. Tank 4
further includes a set of Pall Rings filling approximately
one-third of the tank volume. Foam Can 36 includes a gas vent for
removal of gas. The solution in Tank 4 is a 13% total hemoglobin
solution.
[0082] During a first oxygenation step, oxygen is sparged through
the solution at a rate sufficient to have uniform dispersion of gas
in the vessel. The vessel, 200 L in volume, is sparged at a rate of
about 25 L/min. with gas. Oxygenation of the carboxyhemoglobin is
conducted for a period of about 16 hours such that the resulting
solution contains less than 5% carboxyhemoglobin based on the
weight of total hemoglobin. Oxygenation is conducted at a
temperature of about 10.degree. C. The foam generated in Tank 4 is
collected in Foam Can 36 and after settling, the resulting solution
is transferred back into Tank 4.
[0083] After oxygenation, the solution is sparged with a similar
flow of nitrogen for about 6 hours or until less than 10%
oxyhemoglobin based on the weight of total hemoglobin remains in
the solution. The nitrogen sparge is conducted at a temperature of
about 10.degree. C. and a pH of about 6 to 6.5. Alternatively,
carboxyhemoglobin could be converted to deoxyhemoglobin using a
membrane exchanger. It is noted that there is substantially no
denaturing of the hemoglobin as would normally be expected from the
foaming step. The resulting deoxygenated solution is now prepared
for chemical modification.
[0084] Referring now to FIG. 2, the deoxyhemoglobin solution is
transferred to Tank 5 for chemical modification. To Tank 5
containing the deoxyhemoglobin at about 4.degree. C. solution is
then added an aqueous solution of pyridoxyl-5-phosphate (P5P)
(93.75 g/L) at a 1:1 to 3:1 P5P to hemoglobin molar ratio. A 2:1
molar ratio of P5P to hemoglobin is preferred. The pyridoxylation
is conducted at a temperature of about 4.degree. C. The P5P
solution is typically added over about 1 minute and mixed for
approximately 15 minutes, after which a sodium borohydride/sodium
hydroxide solution is added to the hemoglobin solution at a molar
ratio of sodium borohydride to hemoglobin of about 20:1. A suitable
agueous sodium borohydride/sodium hydroxide solution contains 0.8 g
of sodium hydroxide per 2 liters and 90.8 g of sodium borohydride
per 2 liters. The borohydride solution is added as rapidly as
possible over a period of about 1 minute and then stirred for one
hour. The resulting 50 L solution of pyridoxylated hemoglobin is
subsequently diafiltered using 10K Dalton ultrafilter 38 to remove
excess reactants with 4 volumes of WFI. Drain 40 associated with
ultrafilter 38 collects the filtrate from filter 38.
[0085] After diafiltration with 4 volumes, i.e., 200 L, of WFI, the
hemoglobin is polymerized. To Tank 5 containing the pyridoxylated
hemoglobin is added sufficient WFI to prepare a 4.5% total
hemoglobin solution (about 175 L of hemoglobin solution). A
glutaraldehyde solution is added to the pyridoxylated hemoglobin
solution at a molar ratio of glutaraldehyde to hemoglobin of about
24:1. The glutaraldehyde solution is typically added over a period
of about 2.5 hours by metering pump to the hemoglobin solution. The
polymerization reaction is allowed to proceed for about 18 hours.
The target molecular weight distribution is about 75% polymer and
25% tetramer. The target polymers have molecular weights of less
than about 600,000 with a predominant fraction of the molecular
weights residing in the 100,000-350,000 range.
[0086] When the polymerization reaction reaches the target
molecular weight distribution (after about 18 hours), aqueous
glycine (about 166 g/L) is added (as a quench) to the hemoglobin
solution at a 140:1 molar ratio of glycine to hemoglobin. See FIG.
3 which is an HPLC tracing of the resulting polymerized,
glycine-quenched hemoglobin product. The resulting solution is then
mixed for about 10 minutes after which a sodium borohydride
sodium/hydroxide solution (having the concentration identified
above) is added to the hemoglobin solution at a 28:1 molar ratio of
sodium borohydride to hemoglobin. This resulting mixture is stirred
for about 1 hour. The solution is then concentrated to about 50 L
(ultrafilter 38) and washed with 4 volumes (200 L) of WFI. An
additional aliquot of sodium borohydride at the same molar ratio as
indicated above is added to the concentrated solution and again
mixed for 1 hour. The resulting solution is washed with 4 volumes
of WFI (200 L) resulting in polymerized, pyridoxylated, stroma-free
hemoglobin that has been heat treated.
[0087] The resulting solution is oxygenated by allowing the
solution to stand under an oxygen atmosphere and is subsequently
transferred to a purification system 42. The purification may be
achieved by column chromatography, filtration, preferably membrane
filtration (diafiltration), or a combination of filtration and
column chromatography.
[0088] In one embodiment, the solution is transferred to
chromatography feed vessel, Tank 6, as shown in FIG. 5. In this
embodiment, the resulting solution of oxyhemoglobin is then diluted
to about 200 L (4% total hemoglobin) in Tank 6 and the
concentration of chloride is adjusted to 22 mM with sodium chloride
solution. No adjustment of sodium concentration is necessary.
[0089] Five 40 L aliquots of the resulting hemoglobin solution are
then chromatographed using Column 44. Column 44 contains an
affinity gel which is an agarose gel modified with a yellow dye
(commercially available from Affinity Chromatography, Ltd., as
Mimetic Yellow No. 1) having greater affinity for polymer than
tetramer.
[0090] The chromatography is accomplished as follows. 40 L of
oxygenated, polymerized, pyridoxylated, stroma-free hemoglobin
solution is loaded onto Column 44. The column is washed with 15
column volumes (about 750 L) of 30 mM aqueous NaCl buffer to remove
tetramer. The column is then washed with about 250 L of a 300 mM
sodium chloride buffer to wash the polymer off. Polymer fractions
are collected in Tank 7. Unwanted fractions are sent to drain 46.
After each aliquot is removed, the column is regenerated with 15 mM
HCL solution (150 L), reequilibrated with 30 mM equeous NaCl (250
L) and another aliquot of feed solution (40 L) is loaded to the
column. The column is again washed with 30 mM NaCl followed by 300
mM NaCl. 40 L aliquots of hemoglobin solution are added to the
column and chromatographed until Tank 6 is empty.
[0091] The collected fractions in Tank 7 are ultrafiltered
(concentrated) using filter 48 associated with drain 50 to a volume
of about 40 L (6% total hemoglobin). The concentrated hemoglobin
solution is then transferred to gas exchange Tank 8 for
deoxygenation.
[0092] Alternatively, the solution is transferred to a filtration
recycle vessel 10, as shown in FIG. 6. The hemoglobin is then
diluted to about 4% THb in Tank 10. The 4% THb solution is then
diafiltered using 10 mM NaCl and a 300,000 molecular weight filter
52 commercially available from Pall-Filtron. The filtration is
continued until about 97% of the hemoglobin material passes through
the filter and into Tank 11. (About 3% of the material, i.e., high
molecular weight polymers is retained in Tank 10). The amount of
hemoglobin is determined spectrophotometrically using a
cooximeter.
[0093] The resulting material in Tank 11 is about 2-4% TH6 and
contains about 7-10% tetramer based on THb. The 2-4% THb is then
diafiltered using 10 mM NaCl and a 100,000 molecular weight filter
54 commercially available from Pall-Filtron associated with drain
or trap 56. The filtration is continued until the level of
tetramer, as determined by size exclusion chromatography using a
BioSep SEC-S3000 600.times.7.8 mm column is less than 0.8% of the
hemoglobin mass by weight. The resulting purified hemoglobin
solution remains initially in Tank 11 and is subsequently
transferred to gas exchange Tank 8 for deoxygenation.
[0094] Gas exchange Tank 8 may be the same tank as Tank 4 or,
preferably, a different tank. Gas exchange Tank 8 is equipped in
essentially the same fashion as gas exchange Tank 4 in FIG. 1 and
is attached to foam can 58 in a fashion identical to that of Tank 4
and foam can 36. Deoxygenation is accomplished in about 2.5 hours
with a nitrogen sparge at about 10.degree. C. and a solution pH of
about 7.5. Nitrogen sparging is continued until less than about 16%
oxyhemoglobin, based on the weight of total hemoglobin, remains in
the solution. The resulting deoxyhemoglobin solution is
subsequently transferred to Tank 9 for formulation.
[0095] In formulation Tank 9, the solution is first concentrated to
about 7% total hemoglobin, and the pH adjusted to about 8.8 to 9.0
at 4.degree. C. The pH is adjusted using 0.2 M NaOH. Electrolyte
solutions are then added to the pH 8.8 to 9.0 hemoglobin solution.
Glucose and glycine are added to achieve final concentrations of
about 5 g/L and 1.75 g/L respectively. Potassium chloride is added
to the solution to obtain a potassium concentration of about 3.5 to
4.5 mM. 3 M sodium chloride is then added to obtain a 85-110 mM
chloride concentration. Sodium lactate is subsequently added to
obtain a 135-155 mM concentration of sodium ion. Finally, a 0.45
molar ascorbic acid solution is added to raise the ascorbic acid
concentration up to about 1000 mg/L. Ascorbic acid is added as a
preservative/antioxidant for storage. The resulting hemoglobin
solution has a final osmolality of about 280-360 mmole per kg.
[0096] The formulated hemoglobin solution is then concentrated to
about 10% total hemoglobin using filter 60 associated with trap 62
and the 10% hemoglobin solution is then sterilized by filtration by
filter 64 and aseptically filled into presterilized bags.
[0097] The characteristics of the product prepared in this example,
Batch A, are shown in Table 1. In addition, the characteristics of
Batches B and C, both prepared according to the procedure set forth
above for Batch A, are shown in Table 1.
TABLE-US-00003 TABLE 1 Batch TEST A B C Hemoglobin, g/dL 10.4 10.2
10.2 Methemoglobin, % 4.6 6.0 5.6 Carboxyhemoglobin, % 0.2 1.4 1.5
P50 (Torr, pH 7.35-7.45, 28.5 26.8 27.0 pCO.sub.2 35-40 torr)
Osmolality, mmol/KG 318 320 317 Sodium, mmol/L 142 144 142
Potassium, mmol/L 4.0 4.0 4.0 Chloride, mmol/L 98 99 94 Free iron,
ppm 0.7 1.2 1.0 Molecular Weight 128:16 128:11 128:16 distribution,
% at each 192:26 192:23 192:26 MW (Kd) 256.sup.8:58.sup.
256.sup.8:66.sup. 256.sup.8:58.sup. Tetramer, % 0.4 0.3 0.4
Endotoxin, EU/mL <0.03 <0.03 <0.03 .sup.8Material also
includes a small amount of higher molecular weight material.
[0098] In the foregoing, there has been provided a detailed
description of preferred embodiments of the present invention for
the purpose of illustration and not limitation. It is to be
understood that all other modifications, ramifications and
equivalents obvious to those having skill in the art based on this
disclosure are intended to be within the scope of the invention as
claimed.
* * * * *