U.S. patent application number 13/225797 was filed with the patent office on 2013-02-28 for method for the preparation of a heat stable oxygen carrier-containing composition facilating beta-beta cross-linking.
The applicant listed for this patent is Kwok Chu BUTT, Sui Yi KWOK, Bing Lou WONG. Invention is credited to Kwok Chu BUTT, Sui Yi KWOK, Bing Lou WONG.
Application Number | 20130052232 13/225797 |
Document ID | / |
Family ID | 45508128 |
Filed Date | 2013-02-28 |
United States Patent
Application |
20130052232 |
Kind Code |
A1 |
WONG; Bing Lou ; et
al. |
February 28, 2013 |
METHOD FOR THE PREPARATION OF A HEAT STABLE OXYGEN
CARRIER-CONTAINING COMPOSITION FACILATING BETA-BETA
CROSS-LINKING
Abstract
Methods for preparation of a heat stable hemoglobin based
oxygen-carrier-containing pharmaceutical composition such that
beta-beta cross-linking is favored are provided. Using the methods
of the present invention, the oxygen affinity of the resultant
molecule can be controlled so that hemoglobin based oxygen carriers
tailored for specific applications can be produced. Lower oxygen
affinity crosslinked hemoglobin is useful for applications
requiring rapid tissue oxygenation (e.g. hemorrhagic shock) while
higher oxygen affinity cross-linked hemoglobin is useful for
applications requiring a slower rate of oxygenation (e.g. cancer
adjunct therapy). A highly purified and heat stable crosslinked
non-polymeric tetrameric hemoglobin having beta-beta cross-linking
of greater than 40-60% and suitable for use in mammals without
causing renal injury and vasoconstriction is produced. A high
temperature and short time (HTST) heat processing step is performed
to effectively remove any undesired dimeric hemoglobin,
non-crosslinked tetrameric hemoglobin, and plasma protein
impurities.
Inventors: |
WONG; Bing Lou; (Irvine,
CA) ; KWOK; Sui Yi; (Hong Kong, HK) ; BUTT;
Kwok Chu; (Hong Kong, HK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
WONG; Bing Lou
KWOK; Sui Yi
BUTT; Kwok Chu |
Irvine
Hong Kong
Hong Kong |
CA |
US
HK
HK |
|
|
Family ID: |
45508128 |
Appl. No.: |
13/225797 |
Filed: |
September 6, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61529279 |
Aug 31, 2011 |
|
|
|
Current U.S.
Class: |
424/400 ;
435/325; 514/13.5 |
Current CPC
Class: |
A61P 7/08 20180101; A61P
9/00 20180101; A61P 17/00 20180101; A61K 38/42 20130101; A61P 7/00
20180101; A61P 43/00 20180101 |
Class at
Publication: |
424/400 ;
514/13.5; 435/325 |
International
Class: |
A61K 9/00 20060101
A61K009/00; C12N 5/071 20100101 C12N005/071; A61P 9/00 20060101
A61P009/00; A61K 38/42 20060101 A61K038/42 |
Claims
1. A method for the preparation of a highly purified and heat
stable oxygen carrier-containing pharmaceutical composition, the
oxygen carrier-containing pharmaceutical composition including
hemoglobin, the hemoglobin consisting essentially of non-polymeric
crosslinked tetrameric hemoglobin having a beta-beta cross-linking
of greater than 40%, the method comprising: a) providing mammalian
whole blood including at least red blood cells and plasma; b)
separating the red blood cells from the plasma in the mammalian
whole blood; c) filtering the red blood cells that were separated
from the plasma to obtain a filtered red blood cell fraction; d)
washing the filtered red blood cell fraction to remove plasma
protein impurities, resulting in washed red blood cells; e)
disrupting the washed red blood cells to create a solution
comprising a lysate of disrupted red blood cells; f) performing
filtration to remove at least a portion of the waste retentate from
the lysate; g) extracting a first hemoglobin solution from the
lysate; h) performing at least one purification process to remove
one or more of viruses, waste retentate, or protein impurities; i)
cross-linking the hemoglobin by bis-3,5-dibromosalicyl fumarate to
form crosslinked hemoglobin in an oxygenated environment wherein
the crosslinked hemoglobin is non-polymeric crosslinked tetrameric
hemoglobin having at least 40% beta-beta cross-linking; j) heat
treating the crosslinked hemoglobin in a deoxygenated environment
to denature and precipitate any residual
non-stabilized/non-crosslinked hemoglobin, any dimeric hemoglobin
and any other protein impurities such that the resulting heat
stable crosslinked tetrameric hemoglobin has an undetectable
concentration of dimer and consists essentially of non-polymeric
crosslinked tetrameric hemoglobin with a beta-beta cross-linking of
at least 40% and an oxygen affinity greater than the oxygen
affinity of native hemoglobin of the same species measured under
substantially similar conditions; k) removing precipitate by a
centrifugation or a filtration to form a clear solution; and l)
adding the purified and heat stable crosslinked tetrameric
hemoglobin to a pharmaceutically acceptable carrier.
2. The method for the preparation of a highly purified and heat
stable oxygen carrier-containing pharmaceutical composition
according to claim 1 wherein the heat processing step is a high
temperature short time (HTST) process conducted at approximately
70.degree. C. to 95.degree. C. for 30 seconds to 3 hours followed
immediately by cooling and adding N-acetyl cysteine in an amount of
0.2 to 0.4% immediately following the cooling.
3. The method for the preparation of a highly purified and heat
stable oxygen carrier-containing pharmaceutical composition
according to claim 1 wherein the whole blood is bovine whole blood
and the beta-beta cross-linking is greater than 50% and the p50
value is less than approximately 23 mm Hg.
4. The method for the preparation of a highly purified and heat
stable oxygen carrier-containing pharmaceutical composition
according to claim 1 wherein the whole blood is bovine whole blood
and the beta-beta cross-linking is greater than 60% and the p50
value is less than approximately 23 mm Hg.
5. (canceled)
6. (canceled)
7. The method for the preparation of a highly purified and heat
stable oxygen carrier-containing pharmaceutical composition
according to claim 1 wherein the whole blood is human blood.
8. The method for the preparation of a highly purified and heat
stable oxygen carrier-containing pharmaceutical composition
according to claim 1 wherein the whole blood is porcine blood,
equine blood, or canine blood.
9. A method for the preparation of a highly purified and heat
stable oxygen carrier containing pharmaceutical composition
according to claim 1 further comprising packaging the hemoglobin
solution in a low oxygen permeability package such that hemoglobin
solution has a shelf life on the order of two years.
10. A highly purified and heat stable oxygen carrier-containing
pharmaceutical composition comprising hemoglobin, the hemoglobin
consisting essentially of non-polymeric crosslinked tetrameric
hemoglobin having beta-beta cross-linking of greater than 40% is
formed by a process comprising a) providing mammalian whole blood
including at least red blood cells and plasma; b) separating the
red blood cells from the plasma in the mammalian whole blood; c)
filtering the red blood cells that were separated from the plasma
to obtain a filtered red blood cell fraction; d) washing the
filtered red blood cell fraction to remove plasma protein
impurities, resulting in washed red blood cells; e) disrupting the
washed red blood cells to create a solution comprising a lysate of
disrupted red blood cells; f) performing filtration to remove at
least a portion of the waste retentate from the lysate; g)
extracting a first hemoglobin solution from the lysate; h)
performing at least one purification process to remove one or more
of viruses, waste retentate, or protein impurities; i)
cross-linking the hemoglobin by bis-3,5-dibromosalicyl fumarate to
form crosslinked hemoglobin in an oxygenated environment wherein
the crosslinked hemoglobin is non-polymeric crosslinked tetrameric
hemoglobin having at least 40% beta-beta cross-linking; j) heat
treating the crosslinked hemoglobin in a deoxygenated environment
to denature and precipitate any residual
non-stabilized/non-crosslinked hemoglobin, any dimeric hemoglobin
and any other protein impurities such that the resulting heat
stable crosslinked tetrameric hemoglobin has an undetectable
concentration of dimer and consists essentially of non-polymeric
crosslinked tetrameric hemoglobin with a beta-beta cross-linking of
at least 40% and an oxygen affinity greater than the oxygen
affinity of native hemoglobin of the same species measured under
substantially similar conditions; k) removing precipitate by a
centrifugation or a filtration to form a clear solution; and l)
adding the purified and heat stable crosslinked tetrameric
hemoglobin to a pharmaceutically acceptable carrier.
11. The highly purified and heat stable oxygen carrier-containing
pharmaceutical composition of claim 10, wherein the hemoglobin
consists essentially of non-polymeric crosslinked tetrameric
hemoglobin having beta-beta cross-linking of greater than 60% and a
p50 value of less than approximately 23 mm Hg
12. A method of oxygenating tissue comprising providing the
composition of claim 10 to the tissue either in vivo or ex
vivo.
13. A method of oxygenating tissue comprising providing the
composition of claim 11 to the tissue either in vivo or ex
vivo.
14. (canceled)
15. The method for the preparation of a highly purified and heat
stable oxygen carrier-containing pharmaceutical composition
according to claim 14 wherein the whole blood is bovine whole blood
and the oxygen affinity of the crosslinked tetrameric bovine
hemoglobin has a p50 value on the order of approximately 38 to 50
mm Hg.
16. (canceled)
17. A highly purified and heat stable oxygen carrier-containing
pharmaceutical composition comprising hemoglobin, the hemoglobin
consisting essentially of non-polymeric crosslinked tetrameric
hemoglobin having beta-beta cross-linking of greater than 50% is
formed by a process comprising: a) providing mammalian whole blood
including at least red blood cells and plasma; b) separating the
red blood cells from the plasma in the mammalian whole blood; c)
filtering the red blood cells that were separated from the plasma
to obtain a filtered red blood cell fraction; d) washing the
filtered red blood cell fraction to remove plasma protein
impurities, resulting in washed red blood cells; e) disrupting the
washed red blood cells to create a solution comprising a lysate of
disrupted red blood cells; f) performing filtration to remove at
least a portion of the waste retentate from the lysate; g)
extracting a first hemoglobin solution from the lysate; h)
performing at least one purification process to remove one or more
of viruses, waste retentate, or protein impurities; i)
cross-linking the hemoglobin by bis-3,5-dibromosalicyl fumarate to
form crosslinked hemoglobin in a deoxygenated environment wherein
the crosslinked hemoglobin is non-polymeric crosslinked tetrameric
hemoglobin having at least 50% beta-beta cross-linking; j) heat
treating the crosslinked hemoglobin in a deoxygenated environment
to denature and precipitate any residual
non-stabilized/non-crosslinked hemoglobin, any dimeric hemoglobin
and any other protein impurities such that the resulting heat
stable crosslinked tetrameric hemoglobin has an undetectable
concentration of dimer and consists essentially of non-polymeric
crosslinked tetrameric hemoglobin with a beta-beta cross-linking of
at least 50% having an oxygen affinity less than the oxygen
affinity of native hemoglobin of the same species measured under
substantially similar conditions); k) removing precipitate by a
centrifugation or a filtration to form a clear solution; and l)
adding the purified and heat stable crosslinked tetrameric
hemoglobin to a pharmaceutically acceptable carrier.
18. The highly purified and heat stable oxygen carrier-containing
pharmaceutical composition of claim 17, wherein the hemoglobin
consists essentially of non-polymeric crosslinked tetrameric
hemoglobin having beta-beta cross-linking of greater than 60% and
the oxygen affinity of the crosslinked tetrameric hemoglobin has a
p50 value on the order of approximately 38-50 mm Hg.
19. A method of oxygenating tissue comprising providing the
composition of claim 17 to the tissue either in vivo or ex
vivo.
20. A method of oxygenating tissue comprising providing the
composition of claim 18 to the tissue either in vivo or ex
vivo.
21. The highly purified and heat stable oxygen carrier-containing
pharmaceutical composition of claim 10, wherein the hemoglobin
consists essentially of non-polymeric crosslinked tetrameric
hemoglobin having beta-beta cross-linking of greater than 50% and a
p50 value of less than approximately 23 mm Hg.
22. The highly purified and heat stable oxygen carrier-containing
pharmaceutical composition of claim 10, wherein the mammalian whole
blood is selected from bovine blood, human blood, porcine blood,
equine blood, or canine blood.
23. The highly purified and heat stable oxygen carrier-containing
pharmaceutical composition of claim 17, wherein the mammalian whole
blood is selected from bovine blood, human blood, porcine blood,
equine blood, or canine blood.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority to provisional U.S.
Patent Application No. 61/529,279 filed Aug. 31, 2011, the
disclosure of which is incorporated by reference herein.
COPYRIGHT NOTICE/PERMISSION
[0002] A portion of the disclosure of this patent document contains
material which is subject to copyright protection. The copyright
owner has no objection to the facsimile reproduction by anyone of
the patent document or the patent disclosure as it appears in the
Patent and Trademark Office patent file or records, but otherwise
reserves all copyright rights whatsoever. The following notice
applies to the processes, experiments, and data as described below
and in the drawings attached hereto: Copyright .COPYRGT. 2011,
Billion King International Limited, All Rights Reserved.
TECHNICAL FIELD
[0003] The present invention relates to methods for the preparation
of a heat stable oxygen-carrier-containing pharmaceutical
composition such that beta-beta cross-linking is favored. Using the
methods of the present invention, the oxygen affinity of the
resulting molecule can be controlled so that hemoglobin based
oxygen carriers tailored for specific applications can be produced.
Lower oxygen affinity crosslinked hemoglobin is useful for
applications requiring rapid tissue oxygenation (e.g. hemorrhagic
shock) while higher oxygen affinity crosslinked hemoglobin is
useful for applications requiring a slower rate of oxygenation
(e.g. cancer adjunct therapy).
BACKGROUND OF INVENTION
[0004] Hemoglobin plays an important role in most vertebrates for
gaseous exchange between the vascular system and tissue. It is
responsible for carrying oxygen from the respiratory system to the
body cells via blood circulation and also carrying the metabolic
waste product carbon dioxide away from body cells to the
respiratory system, where the carbon dioxide is exhaled. Since
hemoglobin has this oxygen transport feature, it can be used as a
potent oxygen supplier if it can be stabilized ex vivo and used in
vivo.
[0005] Naturally-occurring hemoglobin is a tetramer which is
generally stable when present within red blood cells. However, when
naturally-occurring hemoglobin is removed from red blood cells, it
becomes unstable in plasma and splits into two .alpha.-.beta.
dimers. Each of these dimers is approximately 32 kDa in molecular
weight. These dimers may cause substantial renal injury when
filtered through the kidneys and excreted. The breakdown of the
tetramer linkage also negatively impacts the sustainability of the
functional hemoglobin in circulation.
[0006] In order to solve the problem, recent developments in
hemoglobin processing have incorporated various cross-linking
techniques to create intramolecular bonds within the tetramer as
well as intermolecular bonds between the tetramers to form
polymeric hemoglobin. The prior art teaches that polymeric
hemoglobin is the preferred form in order to increase circulatory
half-life of the hemoglobin. However, as determined by the present
inventors, polymeric hemoglobin more readily converts to
met-hemoglobin in blood circulation. Met-hemoglobin cannot bind
oxygen and therefore cannot oxygenate tissue. Therefore, the
cross-linking taught by the prior art that causes the formation of
polymeric hemoglobin is a problem. There is a need in the art for a
technique that permits intramolecular cross-linking to create
stable tetramers without the simultaneous formation of polymeric
hemoglobin.
[0007] Further problems with the prior art attempts to stabilize
hemoglobin include production of tetrameric hemoglobin that
includes an unacceptably high percentage of dimer units (or
non-crosslinked tetrameric hemoglobin that quickly dissociates to
dimeric hemoglobin if administered to a patient); the presence of
dimers makes the hemoglobin composition unsatisfactory for
administration to mammals. The dimeric form of the hemoglobin can
cause severe renal injury in a mammalian body; this renal injury
can be severe enough to cause death. Therefore, there is a need in
the art to create stable tetrameric hemoglobin with undetectable
dimeric form in the final product.
[0008] Another problem with prior art hemoglobin products is a
sudden increase in blood pressure following administration. In the
past, vasoconstriction events have been recorded from older
generation of hemoglobin based oxygen carriers. Thus there is a
need in the art for a process to prepare hemoglobin which will not
cause vasoconstriction and high blood pressure when applied to a
mammal.
[0009] Further problems with prior art attempts to create stable
hemoglobin include the presence of protein impurities such as
immunoglobin G that can cause allergic effects in mammals.
Therefore, there is a need in the art for a process which can
produce stable tetrameric hemoglobin without protein
impurities.
[0010] In addition to the above problems, there is a need in the
art for stabilized tetrameric hemoglobin that is dimer free,
phospholipid free and capable of production on an industrial
scale.
[0011] Hemoglobin-based oxygen carriers can be used in a wide
variety of medical applications; depending upon the medical
application, different levels of oxygen affinity are desirable. For
example, a hemoglobin molecule with low oxygen affinity can
transfer oxygen more easily to a target tissue than a hemoglobin
molecule with higher oxygen affinity. Therefore it would be
desirable to control the oxygen affinity of the crosslinked
tetrameric hemoglobin. Thus, there is a need in the art to control
the type of cross-linking and cross-linking conditions to produce
crosslinked tetrameric hemoglobin with precise levels of oxygen
binding. Related, commonly owned patents and applications directed
to formation of purified, tetrameric hemoglobin are U.S. Pat. Nos.
7,989,593 and 7,932,356 and U.S. patent application Ser. No.
12/821,214, the disclosures of which are incorporated by reference
herein.
SUMMARY OF INVENTION
[0012] The present invention provides a method for processing a
non-polymeric, heat stable purified crosslinked tetrameric
hemoglobin suitable for use in mammals without causing severe renal
injury, vascular detrimental effects and severe adverse events
including death. The present invention removes the dimeric form of
hemoglobin, non-crosslinked tetrameric hemoglobin, phospholipids
and protein impurities. The present invention also provides a
technique for controlling the oxygen affinity of the resultant
crosslinked tetramer by controlling the type of cross-linking in
the tetramer (e.g., the amount of beta-beta cross-linking,
alpha-alpha cross-linking, alpha-beta cross-linking in the
tetramer), the quaternary structure of tetramer, and the
cross-linking conditions. Lower oxygen affinity crosslinked
hemoglobin is useful for applications requiring rapid tissue
oxygenation (e.g. hemorrhagic shock) while higher oxygen affinity
crosslinked hemoglobin is useful for applications requiring a
slower rate of oxygenation (e.g. cancer adjunct therapy).
[0013] The method includes a starting material of mammalian whole
blood including at least red blood cells and plasma. Red blood
cells are separated from the plasma in the mammalian whole blood
followed by filtering to obtain a filtered red blood cell fraction.
The filtered red blood cell fraction is washed to remove plasma
protein impurities. The washed red blood cells are disrupted to
form a lysate. Filtration is performed to remove at least a portion
of the waste retentate from the lysate. A first hemoglobin solution
is extracted from the lysate.
[0014] One or more purification processes are performed on the
hemoglobin solution such as ultrafiltration and/or
chromatography.
[0015] The purified hemoglobin is crosslinked by
bis-3,5-dibromosalicyl fumarate (DBSF) to form heat stable
crosslinked hemoglobin without the formation of polymeric
hemoglobin. The expression "non-polymeric" as used herein, refers
to tetrameric hemoglobin that is not intermolecularly crosslinked
with other hemoglobin molecules or any other non-hemoglobin
molecules such as PEG. Depending upon the hemoglobin source, the
quaternary structure of the hemoglobin and on the cross-linking
conditions, a tetrameric product with a high percentage of
beta-beta cross-linking can be produced. Further, the oxygen
affinity of the resultant molecule can be controlled so that
hemoglobin based oxygen carriers tailored for specific applications
can be produced.
[0016] Following this procedure, the crosslinked hemoglobin is
heat-treated to remove any residual non-crosslinked tetrameric
hemoglobin and any non-stabilized hemoglobin, for example the
dimeric form of hemoglobin, and any other protein impurities. Prior
to the heat treatment N-acetyl cysteine is optionally added at a
concentration of approximately 0.2% to the crosslinked tetrameric
hemoglobin to prevent formation of met-hemoglobin. Immediately
following heat treatment and cooling, N-acetyl cysteine is
optionally added at a concentration of approximately 0.2% to 0.4%
to further prevent formation of met-hemoglobin. The heat treatment
is preferably a high temperature short time treatment conducted at
approximately 70.degree. C. to 95.degree. C. for 30 seconds to 3
hours with subsequent cooling to 25.degree. C. Any precipitates
formed during the heat treatment are removed by centrifugation or
filtration.
[0017] The dimer-free, phospholipid-free, protein impurities-free,
heat stable, non-polymeric crosslinked tetrameric hemoglobin is
then added to a pharmaceutically acceptable carrier.
[0018] Thereafter, the heat stable, crosslinked tetrameric
hemoglobin is formulated and packaged in a custom-made and
air-tight polyethylene, ethylene-vinyl-acetate, and ethylene-vinyl
alcohol (PE, EVA, EVOH) infusion bag. The packaging prevents oxygen
contamination which results in the formation of inactive
met-hemoglobin.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 is a flow-chart depicting an overview of a process of
the present invention.
[0020] FIG. 2 schematically depicts an instant cytolysis apparatus
used in one embodiment of the process of the present invention.
[0021] FIG. 3 depicts high performance liquid chromatography
analysis for (a) non-heat treated crosslinked tetrameric
hemoglobin, and (b) heat stable crosslinked tetrameric hemoglobin
which has undergone a heat treatment at 90.degree. C. for 45
seconds to 2 minutes or 80.degree. C. for 30 minutes.
[0022] FIG. 4 is an elution profile for flowthrough column
chromatography; the hemoglobin solution is in the flowthrough
fraction.
[0023] FIG. 5 schematically depicts a flowthrough CM column
chromatography system with ultrafiltration for an industrial scale
operation.
[0024] FIG. 6 is a schematic depiction of an apparatus used for
HTST heat processing step.
[0025] FIG. 7 demonstrates the temperature profile in the HTST
processing apparatus and the time taken to remove unstabilized
tetramer (dimer) in the system at 85.degree. C. and 90.degree. C.
of the present invention.
[0026] FIG. 8 is a schematic depiction of an infusion bag for the
heat stable crosslinked tetrameric hemoglobin of the present
invention.
[0027] FIG. 9 depicts reverse phase HPLC chromatogram of .alpha.
and .beta. globin chains of bovine hemoglobin before (dashed line)
and after (solid line) reaction with DBSF under deoxygenated
environment.
[0028] FIG. 10 depicts 15% SDS-PAGE analysis of (A) native bovine
hemoglobin and (B) hemoglobin crosslinked with DBSF under
deoxygenated condition.
[0029] FIG. 11 depicts the peptide mass fingerprint of
trypsin-digested peptides from the dimeric protein band (B6)
generated by MALDI-TOF analysis.
DETAILED DESCRIPTION OF INVENTION
[0030] Hemoglobin is an iron-containing oxygen-transport protein in
red blood cells of the blood of mammals and other animals.
Hemoglobin exhibits characteristics of both the tertiary and
quaternary structures of proteins. Most of the amino acids in
hemoglobin form alpha helices connected by short non-helical
segments. Hydrogen bonds stabilize the helical sections inside the
hemoglobin causing attractions within the molecule thereto folding
each polypeptide chain into a specific shape. A hemoglobin molecule
is assembled from four globular protein subunits. Each subunit is
composed of a polypeptide chain arranged into a set of
.alpha.-helix structural segments connected in a "myoglobin fold"
arrangement with an embedded heme group.
[0031] The heme group consists of an iron atom held in a
heterocyclic ring, known as a porphyrin. The iron atom binds
equally to all four nitrogen atoms in the center of the ring which
lie in one plane. Oxygen is then able to bind to the iron center
perpendicular to the plane of the porphyrin ring. Thus a single
hemoglobin molecule has the capacity to combine with four molecules
of oxygen.
[0032] In mammals, the most common type of hemoglobin is a
tetramer; in humans, it is called hemoglobin A and consists of two
.alpha. and two .beta. non-covalently bound subunits designated as
.alpha.2.beta.2, each made of 141 and 146 amino acid residues
respectively. The size and structure of .alpha. and .beta. subunits
are very similar to each other. Each subunit has a molecular weight
of about 16 kDa for a total molecular weight of the tetramer of
about 65 kDa. The four polypeptide chains are bound to each other
by salt bridges, hydrogen bonds and hydrophobic interaction. The
structure of bovine hemoglobin is similar to human hemoglobin
(90.14% identity in a chain; 84.35% identity in .beta. chain). The
difference is the two sulfhydryl groups in the bovine hemoglobin
positioned at .beta. Cys 93, while the sulfhydryls in human
hemoglobin are at positioned at a Cys 104, .beta. Cys 93 and .beta.
Cys 112 respectively. Human hemoglobin shares high similarity with
bovine, canine, porcine and equine hemoglobin when comparing their
amino acid sequences.
[0033] In naturally-occurring hemoglobin inside the red blood
cells, the association of an .alpha. chain with its corresponding
.beta. chain is very strong and does not disassociate under
physiological conditions. However, the association of one
.alpha..beta. dimer with another .alpha..beta. dimer is fairly weak
outside red blood cells. The bond has a tendency to split into two
.alpha..beta. dimers each approximately 32 kDa. These undesired
dimers are small enough to be filtered by the kidneys and be
excreted, with the result being potential renal injury and
substantially decreased intravascular retention time.
[0034] Therefore, it is necessary to stabilize any hemoglobin that
is used outside of red blood cells both for efficacy and safety.
The process for producing the stabilized hemoglobin is outlined
below; an overview of the process of the present invention is
presented in the flow chart of FIG. 1.
[0035] Initially, a whole blood source is selected as a source of
hemoglobin from red blood cells. Mammalian whole blood is selected
including, but not limited to, human, bovine, porcine, equine, and
canine whole blood. The red blood cells are separated from the
plasma, filtered, and washed to remove plasma protein
impurities.
[0036] In order to release the hemoglobin from the red blood cells,
the cell membrane is lysed. Although various techniques can be used
to lyse red blood cells, the present invention uses lysis under
hypotonic conditions in a manner which can be precisely controlled
at volumes suitable for industrial-scale production. To this end,
an instant cytolysis apparatus as seen in FIG. 2 is used to lyse
the red blood cells. Hypotonic lysis creates a solution of lysate
including hemoglobin and a waste retentate. To enable
industrial-scale production, the lysis is carefully controlled such
that only red blood cells are lysed without lysing white blood
cells or other cells. In one embodiment, the size of the instant
cytolysis apparatus is selected such that the red blood cells
traverse the apparatus in 2 to 30 seconds or otherwise a time
sufficient to lyse the red blood cells and preferably, 30 seconds.
The instant cytolysis apparatus includes a static mixer. Deionized
and distilled water is used as a hypotonic solution. Of course it
is understood that the use of other hypotonic solutions having
different saline concentrations would result in different time
periods for red blood cell lysis. Because the controlled lysis
procedure lyses the red blood cells only, not white blood cells or
cellular matter, it minimizes the release of toxic proteins,
phospholipids or DNA from white blood cells and other cellular
matter. A hypertonic solution is added immediately after 30
seconds, that is, after the red blood-cell containing solution has
traversed the static mixer portion of the instant cytolysis
apparatus. The resultant hemoglobin has a higher purity and lower
levels of contaminants such as undesired DNA and phospholipids than
hemoglobin resulted from using other lysis techniques. Undesired
nucleic acids from white blood cells and phospholipids impurities
are not detected in the hemoglobin solution by polymerase chain
reaction (detection limit=64 pg) and high performance liquid
chromatography (HPLC, detection limit=1 .mu.g/ml) method
respectively.
[0037] At this stage in the process, the hemoglobin solution is
purified to remove various protein and other impurities. This
purification can be ultrafiltration based, chromatography based, or
a combination of one or more ultrafiltration and/or chromatography
processes. In an exemplary embodiment, two ultrafiltration
processes are performed: one which removes impurities having
molecular weights greater than hemoglobin before flowthrough column
chromatography, and another which removes impurities having
molecular weights less than hemoglobin after flowthrough column
chromatography. The latter ultrafiltration process concentrates the
hemoglobin. In some embodiments, a 100 kDa filter is used for the
first ultrafiltration, while a 30 kDa filter is used for the second
ultrafiltration.
[0038] Flowthrough column chromatography is used to remove protein
impurities in the purified hemoglobin solution such as
immunoglobin-G, albumin and carbonic anhydrase. In some
embodiments, column chromatography is carried out by using one or a
combination of commercially available ion exchange columns such as
a DEAE column, CM column, hydroxyapatite column, etc. The pH for
column chromatography is typically from 6 to 8.5. In one
embodiment, a flowthrough CM column chromatography step is used to
remove protein impurities at pH 8.0. Enzyme-linked immunosorbent
assay (ELISA) and HPLC method are performed to detect the protein
impurities and phospholipids remaining in the sample after elution
from the column chromatography. This unique flowthrough column
chromatography separation enables a continuous separation scheme
for industrial-scale production. The ELISA result shows that the
amount of these impurities is substantially low in the eluted
hemoglobin (immunoglobin-G: 44.3 ng/ml; albumin: 20.37 ng/ml;
carbonic anhydrase: 81.2 .mu.g/ml). The protein impurities removal
results using different kinds of columns with different pH values
are shown in Table 1 below.
TABLE-US-00001 TABLE 1 Removal percentage (%) Carbonic Column (pH
condition) anhydrase Albumin Immunoglobin-G DEAE (at pH 7.5) -- 68
29.8 DEAE (at pH 7.8) -- 60 50.9 CM (at pH 6.2) -- 32 21.8 CM (at
pH 8.0) 5.6 53.2 66.4 Hydroxyapatite (at pH 7.5) 4.5 23.5 22.8
[0039] Following the column chromatographic process, the hemoglobin
is subjected to cross-linking by DBSF. The conditions are selected
such that cross-linking occurs between the beta-beta subunits is
favored. For cross-linking under deoxygenated condition, the
resulting hemoglobin has a low oxygen affinity with a higher p50
value compared with the native hemoglobin of the same species
measured under substantially similar conditions. For example, for
bovine hemoglobin, the native bovine hemoglobin has a p50 value on
the order of 23-29 mm Hg. The crosslinked bovine hemoglobin formed
under deoxygenated conditions in the present invention has a p50
value on the order of 38-50 mm Hg. Lower oxygen affinity means that
the tetramer can "offload" oxygen to a target more easily than a
material with a higher oxygen affinity.
[0040] For cross-linking of bovine hemoglobin under oxygenated
conditions, a material with a higher oxygen affinity is formed with
a lower p50 value, less than approximately 23 mm Hg, compared with
native bovine hemoglobin which has a p50 value on the order of
23-29 mm Hg. Lower oxygen affinity compositions are used when rapid
oxygenation is desired as in cases of tissue hypoxia resulting from
extensive blood loss (e.g., hemorrhagic shock). Higher oxygen
affinity compositions are useful for oxygenation adjunct therapies
in cancer treatment where a slower delivery rate of oxygen is
desirable.
[0041] For human hemoglobin, cross-linking under deoxygenated
condition typically produces a majority of alpha-alpha crosslinked
hemoglobin with lower oxygen affinity, that is, an oxygen affinity
that is decreased on the order of at least 2-fold from native human
hemoglobin.
[0042] Human hemoglobin cross-linked under oxygenated conditions
tends to favor production of beta-beta crosslinked hemoglobin with
a higher oxygen affinity (that is, a lower p50, less than
approximately 23 mm Hg), compared with the native human hemoglobin
under the same condition (a p50 value on the order of approximately
23-30 mm Hg).
[0043] For deoxygenated cross-linking condition preferably less
than 0.1 ppm dissolved oxygen level, it is maintained with a molar
ratio of hemoglobin to DBSF from 1:2.5 to 1:4.0 for a period of
time from 3 to 16 hours at ambient temperature (15-25.degree. C.),
at a pH of preferably around 8-9. The resultant crosslinked
hemoglobin is tetrameric hemoglobin having a molecular weight of
60-70 kDa, demonstrating that polymeric hemoglobin is not present.
The yield of the DBSF reaction is high, >99% and the dimer
concentration in the final product is low. Optionally, the present
process does not require sulfhydryl treatment reagents such as
iodoacetamide to react with the hemoglobin before cross-linking as
used in various prior art processes.
[0044] For cross-linking under oxygenated conditions, an oxygenated
environment (such as air, pO.sub.2 is around 149 mmHg; or pure
O.sub.2, pO.sub.2 is nearly 760 mmHg) is used while the remaining
conditions above are substantially the same.
[0045] For bovine hemoglobin, the beta-beta cross-linking is
greater than 50%, and preferably greater than 60% for cross-linking
under deoxygenated conditions (less than 0.1 ppm dissolved oxygen
level).
[0046] For bovine hemoglobin crosslinked under oxygenated
condition, beta-beta cross-linking is also favored, typically at a
level greater that 40% beta-beta crosslinking.
[0047] For human hemoglobin, cross-linking under oxygenated
conditions favors beta-beta cross-linking typically at a level
greater that 40% beta-beta crosslinking.
[0048] Following cross-linking, phosphate buffered saline (PBS), a
physiological buffer, is exchanged for the cross-linking solution
and any residual chemicals are removed by tangential flow
filtration.
[0049] Following cross-linking, the present invention provides a
heat processing step (High Temperature Short Time, HTST) for the
crosslinked tetrameric hemoglobin solution. The heat treatment
takes place in a deoxygenated environment. Prior to heat treatment,
N-acetyl cysteine is optionally added to prevent formation of
met-hemoglobin (inactive hemoglobin). After the heat processing
step, the solution is cooled and N-acetyl cysteine is optionally
added to maintain a low level of met-hemoglobin. If N-acetyl
cysteine is added before and after heat treatment, the amount added
before heat treatment is approximately 0.2%, while the amount added
after heat treatment is approximately 0.2 to 0.4%. However, if
N-acetyl cysteine is added only after heat treatment, then the
amount added is 0.4%.
[0050] In some embodiments, the crosslinked tetrameric hemoglobin
solution is heated in a deoxygenated environment (less than 0.1 ppm
dissolved oxygen level) under a range of temperatures from
50.degree. C. to 95.degree. C. for durations from 0.5 minutes to 10
hours. In some embodiments, the crosslinked tetrameric hemoglobin
solution is heated under a range of temperatures from 70.degree. C.
to 95.degree. C. and for durations from 30 seconds to 3 hours. In
some preferred embodiments, the crosslinked tetrameric hemoglobin
solution is heated under 80.degree. C. for 30 minutes. And yet in
other preferred embodiments, the crosslinked hemoglobin solution is
heated to 90.degree. C. for 30 seconds to 3 minutes, then rapidly
cooled down to approximately 25.degree. C. in approximately 15 to
30 seconds, and the N-acetyl cysteine is optionally added as set
forth above.
[0051] To analyze the outcome of the HTST heat processing step, a
HPLC analytical method is used to detect the amount of dimer after
this heat processing step. The mobile phase for HPLC analysis
contains magnesium chloride (0.75M) which can separate dimer
(non-stabilized tetramer) and heat stable crosslinked tetrameric
hemoglobin. For promoting hemoglobin dissociation into dimers,
magnesium chloride is approximately 30 times more effective than
sodium chloride at the same ionic strength. The heat processing
step also acts as a denaturation step to dramatically remove
unwanted protein impurities in the crosslinked tetrameric
hemoglobin (undetectable in immunoglobin-G; 96.15% decrease in
albumin; 99.99% decrease in carbonic anhydrase). Enzyme-linked
immunosorbent assay (ELISA) is performed to detect the protein
impurities in the sample. Thus the purified, heat stable
crosslinked tetrameric hemoglobin solution has an undetectable
level of dimer (below detection limit: 0.043%), and immunoglobin-G,
and a very low amount of albumin (0.02 .mu.g/ml) and carbonic
anhydrase (0.014 .mu.g/ml). FIG. 3 shows that the dimeric form of
hemoglobin is undetectable in a HPLC system. Table 2 shows the
experimental results regarding the protein impurities and dimer
removal by the HTST heat processing step. This HTST heat processing
step enables the selective separation of heat stable crosslinked
tetramer from unstable tetramer (e.g., non crosslinked tetramer)
and dimer.
TABLE-US-00002 TABLE 2 Immunoglobin-G Albumin Carbonic Tetramer
Dimer (.mu.g/ml) (.mu.g/ml) anhydrase (.mu.g/ml) (%) (%) No heat
0.36 0.57 355.41 90.1 5.4 treatment 80.degree. C. for 10 min Not
detectable 0.33 0.032 92.7 3.4 80.degree. C. for 15 min Not
detectable 0.14 0.022 93.3 2.9 80.degree. C. for 30 min Not
detectable 0.03 0.014 96.6 Not detectable No heat 0.29 0.52 261.80
91.8 5.3 treatment 90.degree. C. for 1.0 min Not detectable 0.21
>0.063 93.4 2.0 90.degree. C. for 1.5 min Not detectable 0.04
0.022 94.9 0.6 90.degree. C. for 2.0 min Not detectable 0.02 0.016
96.1 Not detectable
[0052] Following the heat processing step for the crosslinked
hemoglobin under a deoxygenated condition, the heat stable
crosslinked tetrameric hemoglobin is ready for pharmaceutical
formulation and packaging. The present invention describes an
air-tight packaging step of the heat stable crosslinked tetrameric
hemoglobin solution in a deoxygenated environment. Heat stable
crosslinked tetrameric hemoglobin in the present invention is
stable when maintained in a deoxygenated condition for more than
two years.
[0053] In this invention, the oxygen carrier-containing
pharmaceutical composition is primarily intended for intravenous
injection application. Traditionally, prior products use
conventional PVC blood bag or Stericon blood bag which has high
oxygen permeability which will eventually shorten the life span of
the product since it turns into inactive met-hemoglobin rapidly
(within a few days) under oxygenated conditions.
[0054] The packaging used in the present invention results in the
heat stable crosslinked tetrameric hemoglobin being stable for more
than two years. A multi-layer package of EVA/EVOH material is used
to minimize the gas permeability and to avoid the formation of
inactive met-hemoglobin. A 100 ml infusion bag designed for use
with the purified and heat stable crosslinked tetrameric hemoglobin
of the present invention is made from a five layers EVA/EVOH
laminated material with a thickness of 0.4 mm that has an oxygen
permeability of 0.006-0.132 cm.sup.3 per 100 square inches per 24
hours per atmosphere at room temperature. This material is a Class
VI plastic (as defined in USP<88>), which meets the in-vivo
Biological Reactivity Tests and the Physico-Chemical Test and is
suitable for fabricating an infusion bag for intravenous injection
purpose. This primary bag is particularly useful to protect the
heat stable crosslinked tetrameric hemoglobin solution from long
term oxygen exposure that causes its instability and eventually
affects its therapeutic properties.
[0055] For secondary protection of blood products, it has been
known to use aluminum overwrap to protect against potential air
leakage and to maintain the product in a deoxygenated state.
However, there is a potential of pin holes in the aluminum overwrap
that compromises its air tightness and makes the product unstable.
Therefore the present invention uses as secondary packaging an
aluminum overwrap pouch which prevents oxygenation and also
prevents light exposure. The composition of the overwrap pouch
includes 0.012 mm of polyethylene terephthalate (PET), 0.007 mm of
aluminum (Al), 0.015 mm of nylon (NY) and 0.1 mm of polyethylene
(PE). The overwrap film has a thickness of 0.14 mm and an oxygen
transmission rate of 0.006 cm.sup.3 per 100 square inches per 24
hours per atmosphere at room temperature. This secondary packaging
lengthens the stability time for the hemoglobin, extending the
product shelf-life.
[0056] The process in this invention is applicable to large scale
industrial production of the heat stable crosslinked tetrameric
hemoglobin. In addition, the heat stable crosslinked tetrameric
hemoglobin in combination with a pharmaceutical carrier (e.g.
water, physiological buffer, in capsule form) is suitable for
mammalian use.
[0057] The oxygen carrier-containing pharmaceutical composition of
the present invention is useful in improving tissue oxygenation, in
cancer treatment, in the treatment of oxygen-deprivation disorders
such as hemorrhagic shock, and in heart preservation under a low
oxygen content environment (e.g. heart transplant). In exemplary
embodiments, the dosage is selected to have a concentration range
of approximately 0.2-1.3 g/kg with an infusion rate of less than 10
ml/hour/kg body weight.
[0058] For the use in the treatment of oxygen-deprivation disorders
and for heart preservation, the oxygen carrier-containing
pharmaceutical composition with a lower oxygen affinity of the
present invention serves as a blood substitute providing oxygen to
a target organ. Lower oxygen affinity crosslinked hemoglobin is
useful for applications requiring rapid tissue oxygenation (e.g.
hemorrhagic shock and ex vivo organ preservation).
[0059] For applications in cancer treatment, the oxygen
carrier-containing pharmaceutical composition with a higher oxygen
affinity of the present invention serves as a tissue oxygenation
agent to improve the oxygenation in tumor tissues, thereby
enhancing chemo- and radiation sensitivity. A higher oxygen
affinity hemoglobin is useful for applications requiring a slower
rate of oxygenation (e.g. cancer adjunct therapy).
EXAMPLES
[0060] The following examples are provided by way of describing
specific embodiments of this invention without intending to limit
the scope of this invention in any way.
Example 1
[0061] Process Overview
[0062] A schematic flow diagram of the process of the present
invention is illustrated in FIG. 1. Bovine whole blood is collected
into an enclosed sterile container/bag containing 3.8% (w/v)
tri-sodium citrate solution as anti-coagulant. Blood is then
immediately mixed well with tri-sodium citrate solution to inhibit
blood clotting. Red blood cells (RBC) are isolated and collected
from plasma and other smaller blood cells by an apheresis
mechanism. A "cell washer" is used for this procedure with gamma
sterilized disposable centrifuge bowl. RBC are washed with an equal
volume of 0.9% (w/v sodium chloride) saline.
[0063] Washed RBC are lysed to release hemoglobin content by
manipulating hypotonic shock to the RBC cell membrane. A
specialized instant cytolysis apparatus for RBC lysis device
depicted in FIG. 2 is used for this purpose. Following RBC lysis,
hemoglobin molecules are isolated from other proteins by
tangential-flow ultrafiltration using a 100 kDa membrane.
Hemoglobin in the filtrate is collected for flowthrough column
chromatography and further concentrated to 12-14 g/dL by a 30 kDa
membrane. Column chromatography is carried out to remove the
protein impurities.
[0064] The concentrated hemoglobin solution is first reacted with
DBSF to form heat stable crosslinked tetrameric hemoglobin
molecules. A heat processing step is then performed under
deoxygenated conditions at 90.degree. C. for 30 seconds to three
minutes before final formulation and packaging.
Example 2
[0065] Time & Controlled Hypotonic Lysis and Filtration
[0066] Bovine whole blood is freshly collected and transported
under a cool condition (2 to 10.degree. C.). The red blood cells
are separated from the plasma via a cell washer and subsequently
with a 0.65 .mu.m filtration. After washing the red blood cells
(RBC) filtrate with 0.9% saline, the filtrate is disrupted by
hypotonic lysis. The hypotonic lysis is performed by using the
instant cytolysis apparatus depicted in FIG. 2. The instant
cytolysis apparatus includes a static mixer to assist in cell
lysis. A RBC suspension with controlled hemoglobin concentration
(12-14 g/dL) is mixed with 4 volumes of purified water to generate
a hypotonic shock to RBC cell membranes. The period of hypotonic
shock is controlled to avoid unwanted lysis of white blood cells
and platelets. The hypotonic solution passes through the static
mixer portion of the instant cytolysis apparatus for 2 to 30
seconds or otherwise a time sufficient to lyse the red blood cells
and preferably, 30 seconds. The shock is terminated after 30
seconds by mixing the lysate with 1/10 volume of hypertonic buffer
as it exits the static mixer. The hypertonic solution used is 0.1M
phosphate buffer, 7.4% NaCl, pH 7.4. The instant cytolysis
apparatus of FIG. 2 can process at 50 to 1000 liters of lysate per
hour and, preferably at least 300 liters per hour in a continuous
manner.
[0067] Following the RBC lysis, the lysate of red blood cells is
filtered by a 0.22 .mu.m filter to obtain a hemoglobin solution.
Nucleic acids from white blood cells and phospholipids impurities
are not detected in the hemoglobin solution by polymerase chain
reaction (detection limit=64 pg) and HPLC (detection limit=1
.mu.g/ml) method respectively. A first 100 kDa ultrafiltration is
performed to remove impurities having a higher molecular weight
than hemoglobin. A flowthrough column chromatography is followed to
further purify the hemoglobin solution. A second 30 kDa
ultrafiltration is then performed to remove impurities having a
lower molecular weight than hemoglobin and for concentration.
Example 3
[0068] Viral Clearance Study on Stroma-Free Hemoglobin Solution
[0069] In order to demonstrate the safety of the product from this
invention, the virus removal abilities of (1) 0.65 .mu.m
diafiltration step and (2) 100 kDa ultrafiltration step are
demonstrated by virus validation study. This is done by the
deliberate spiking of a down-scaled version of these two processes
with different model viruses (encephalomyocarditis virus,
pseudorabies virus, bovine viral diarrhea virus and bovine
parvovirus). In this study, four types of viruses (see Table 3) are
used. These viruses vary in their biophysical and structural
features and they display a variation in resistance to physical and
chemical agents or treatments.
TABLE-US-00003 TABLE 3 Target Size Virus Model Virus Taxonomy
Genome Structure [nm] Stability* Hepatitis Bovine viral diarrhea
Flaviviridae ssRNA enveloped 40-60 low C virus virus (BVDV) --
Encephalomyocarditis Picornavirus ssRNA non- 25-30 medium virus
(EMCV) enveloped Parvovirus Bovine parvovirus Parvoviridae ssDNA
non- 18-26 very high B19 (BPV) enveloped Hepatitis Pseudorabies
virus Herpesviridae dsDNA enveloped 120-200 Low to B virus (PRV)
medium
[0070] The validation scheme is briefly shown in the following
Table 4.
TABLE-US-00004 TABLE 4 Diafiltration Ultrafiltration Cell Washing
Virus spiking .dwnarw. .dwnarw. Virus spiking Ultrafiltration
.dwnarw. .dwnarw. Diafiltration Virus tests .dwnarw. Virus
tests
[0071] The summary of the log reduction results of the 4 viruses in
(1) 0.65 .mu.m diafiltration and (2) 100 kDa ultrafiltration is
shown in the following Table 5. All four viruses, BVDV, BPV, EMCV
and PRV, are effectively removed by 0.65 .mu.m diafiltration and
100 kDa ultrafiltration.
TABLE-US-00005 TABLE 5 Viruses BVDV BPV EMCV PRV Run 1 2 1 2 1 2 1
2 0.65 .mu.m Diafiltration 2.69 3.20 3.73 3.53 3.25 .gtoreq.3.90
2.67 2.63 100 kDa Ultrafiltration .gtoreq.4.68 .gtoreq.4.38 5.87
5.92 3.60 3.43 .gtoreq.6.05 3.27 Cumulative maximum .gtoreq.7.88
9.65 .gtoreq.7.50 .gtoreq.8.72 Cumulative minimum .gtoreq.7.07 9.40
6.68 5.90 Annotation: .gtoreq.no residual infectivity
determined
Example 4
[0072] Flowthrough Column Chromatography
[0073] A CM column (commercially available from GE healthcare) is
used to further remove any protein impurities. The starting buffer
is 20 mM sodium acetate (pH 8.0), and the elution buffer is 20 mM
sodium acetate, 2M NaCl (pH 8.0). After the equilibration of the CM
column with starting buffer, the protein sample is loaded into the
column. The unbound protein impurities are washed with at least 5
column volume of starting buffer. The elution is performed using
25% elution buffer (0-0.5M NaCl) in 8 column volume. The elution
profile is shown in FIG. 4; the hemoglobin solution is in the
flowthrough fraction. The purity of the flowthrough fraction is
analyzed by ELISA. The results are indicated in the following Table
6.
TABLE-US-00006 TABLE 6 Protein impurities Carbonic Immunoglobin-G
anhydrase Albumin Before CM column 1320 ng/ml 860.3 .mu.g/ml 435.2
ng/ml Flowthrough 44.3 ng/ml 81.2 .mu.g/ml 20.4 ng/ml (containing
hemoglobin)
[0074] As the hemoglobin solution is in the flowthrough from the CM
column chromatography at pH 8 (not in the eluate), it is a good
approach for continuous industrial scale operation. The first
ultrafiltration set-up is connected directly to the flowthrough CM
column chromatography system, and the flowthrough tubing can be
connected to the second ultrafiltration set-up for industrial scale
operation. The schematic industrial process configuration is shown
in FIG. 5.
Example 5
[0075] Preparation of Heat Stable Crosslinked Tetrameric
Hemoglobin
[0076] (5a) Cross-Linking Reaction with DBSF Under a Deoxygenated
Condition
[0077] The cross-linking reaction is carried out in a deoxygenated
condition, that is, less than 0.1 ppm dissolved oxygen level. DBSF
is added to the hemoglobin solution to form crosslinked tetrameric
hemoglobin without formation of polymeric hemoglobin. DBSF
stabilization procedure stabilizes the tetrameric form of
hemoglobin (65 kDa) and prevents dissociation into dimers (32 kDa)
which are excreted through the kidneys. In this embodiment, a molar
ratio of hemoglobin to DBSF of 1:2.5 is used and the pH is 8.6.
This process is carried out for a period of 3-16 hours at ambient
temperature (15-25.degree. C.) in an inert atmosphere of nitrogen
to prevent oxidation of the hemoglobin to form ferric
met-hemoglobin which is physiologically inactive (dissolved oxygen
level maintained at less than 0.1 ppm). The completeness of DBSF
reaction is monitored by measuring the residual DBSF using HPLC.
The yield of the DBSF reaction is high, >99%. The production of
beta-beta crosslinks is on the order of at least about 40%.
[0078] (5b) HTST Heat Processing Step
[0079] A High Temperature Short Time (HTST) processing apparatus is
shown in FIG. 6. A heat processing step using the HTST processing
apparatus is performed on the crosslinked tetrameric hemoglobin. In
this example, the condition for heat treatment is 90.degree. C. for
30 seconds to 3 minutes, and preferably 45 to 60 seconds although
other conditions can be selected as discussed above and the
apparatus modified accordingly. A solution containing crosslinked
hemoglobin optionally with 0.2% of N-acetyl cysteine added thereto
is pumped into a HTST processing apparatus (first section of the
HTST heat exchanger is pre-heated and maintained at 90.degree. C.)
at a flow rate of 1.0 liter per minute, the residence time of the
first section of the apparatus is between 45 to 60 seconds, then
the solution is passed through at the same flow rate into another
section of the heat exchanger that is maintained at 25.degree. C.
The time required for cooling is between 15 to 30 seconds. After
cooling down to 25.degree. C., N-acetyl cysteine is immediately
added at a concentration of 0.2% to 0.4%, preferably at 0.4%. The
set-up of the heat processing apparatus is easily controlled for
industrial operation. A temperature profile with dimer content is
shown in FIG. 7. If the hemoglobin is not crosslinked, it is not
heat stable and forms a precipitate after the heat processing step.
The precipitate is then removed by a centrifugation or a filtration
to form a clear solution thereafter.
[0080] The following Table 7 shows that protein impurities such as
immunoglobin-G, albumin, carbonic anhydrase and undesirable
non-stabilized tetramer or dimers are removed after the heat
processing step. The amount of immunoglobin-G, albumin and carbonic
anhydrase are measured using an ELISA method, while the amount of
dimer is determined by an HPLC method. The purity of heat stable
crosslinked tetrameric hemoglobin is extremely high after the HTST
heat processing step, in the range of 98.0 to 100%.
TABLE-US-00007 TABLE 7 Protein impurities (by ELISA) Immunoglobin-
Al- Carbonic By HPLC Sample G bumin anhydrase Tetramer Dimer
condition (.mu.g/ml) (.mu.g/ml) (.mu.g/ml) (%) (%) No heat 0.29
0.52 261.80 91.8 5.3 treatment 90.degree. C. Not detectable 0.02
0.016 96.1 Not for 2 min detectable Removal 100.0 96.15 99.99 --
100.0 (%)
Example 6
[0081] Packaging
[0082] Because the product of the present invention is stable under
deoxygenated conditions, the packaging for the product is important
to minimize gas permeability. For intravenous application, a custom
designed, 100 ml infusion bag is made from a five-layer EVA/EVOH
laminated material with a thickness of 0.4 mm that has an oxygen
permeability of 0.006 to 0.132 cm.sup.3 per 100 square inches per
24 hours per atmosphere at room temperature. This specific material
is a Class VI plastic (as defined in USP<88>), which meets
the in-vivo biological reactivity tests and the physico-chemical
tests and are suitable for fabricating containers for intravenous
injection purpose (note that other forms of packaging can be made
from this material as well depending upon the desired application).
A secondary packaging aluminum overwrap pouch is also applied to
the primary packaging infusion bag that provides an additional
barrier, minimizing light exposure and oxygen diffusion. The layers
of the pouch comprise: 0.012 mm of Polyethylene terephthalate
(PET), 0.007 mm of Aluminum (Al), 0.015 mm of Nylon (NY) and 0.1 mm
of Polyethylene (PE). The overwrap film has a thickness of 0.14 mm
and oxygen transmission rate of 0.006 cm.sup.3 per 100 square
inches per 24 hours per atmosphere at room temperature. A schematic
depiction of the infusion bag is depicted in FIG. 8. The overall
oxygen permeability for each infusion bag according to the present
invention is 0.0025 cm.sup.3 per 24 hours per atmosphere at room
temperature.
Example 7
[0083] Characterization of Crosslinked Bovine Hb (Deoxygenated
Cross-Linking Condition)
[0084] (7a) Separation of Globin Chains by Reverse Phase High
Performance Liquid Chromatography (HPLC)
[0085] The globin chains of native bovine hemoglobin and
crosslinked globin chains of DBSF crosslinked bovine hemoglobin are
resolved on a VYDAC C4 column using the gradients developed by
Shelton et al., 1984 with minor modification.
[0086] (7b) Sodium Dodecyl Sulfate Polyacrylamide Gel
Electrophoresis (SDS-PAGE) Analysis of DBSF Crosslinked Bovine
Hemoglobin
[0087] Native bovine hemoglobin and DBSF crosslinked bovine
hemoglobin solution are prepared by mixing with reducing sample
buffer (62 mM Tris-HCl (pH 6.8), 10% (v/v) glycerol, 5% (v/v)
mercaptoethanol and 2.3% (w/v) SDS), and heated at 95.degree. C.
for 10 min. The sample mixture is resolved using a 15% acrylamide
slab gel with a 4% stacking gel. The electrophoresis is run with a
constant current of 60 mA. After electrophoresis, the SDS-PAGE gel
is stained with 0.1% (w/v) Coomassie Blue R350, 20% (v/v) methanol
and 10% (v/v) acetic acid. To estimate the percentage of different
types of cross-linking in DBSF crosslinked bovine hemoglobin, the
intensities of the resolved protein bands expressed in Black Light
Unit (BLU) are quantified using Lumi-Analyst 3.1 Software.
[0088] (7c) Trypsin Digestion of Reduced Globin Chain
[0089] The protein band corresponding to the major crosslinked
globin chain is excised from the SDS-PAGE gel, cut into cubes
(1.times.1 mm), and de-stained with 10% methanol/10% acetic acid.
The de-stained gel cubes are reduced with 10 mM DTT in 25 mM
NH.sub.4CO.sub.3 and alkylated with 55 mM idoacetamide in 25 mM
NH.sub.4CO.sub.3 for 45 min in dark, and then in-gel digested with
20 ng/.mu.l modified trypsin in 25 mM NH.sub.4CO.sub.3 at
37.degree. C. overnight. After trypsin digestion, the
trypsin-digested peptides are extracted by diffusion into 50% (v/v)
acetonitrile (ACN) and 1% (v/v) trifluoroacetic acid (TFA).
[0090] (7d) Matrix Assisted Laser Desorption/Ionization
Time-of-Flight (MALDI-TOF) Mass Spectrometry (MS) Analysis
[0091] The trypsin digested peptides extracted from the protein
band are spotted onto an Anchorchip plate, which is pre-spotted
with 1 .mu.l of matrix solution (2 mg/ml cyano-4-hydroxycinnamic
acid, saturated in 50% ACN/0.1% TFA, and allowed to air-dry. After
drying, the sample spot is washed with 10 mM monophosphate buffer
and recrystallized using a solution of ethanol: acetone: 0.1% TFA
(6:3:1 ratio). MALDI-TOF MS analysis is performed with a Bruker
Autoflex III (Bruker Daltonic GmbH, Bremen, Germany) operated in
the reflectron mode over the m/z range of 800-3500 Da and the
parameters are set as follows: ion source 25 kV for peptide mass
fingerprint (PMF), and reflector 26.3 kV for PMF. External
calibration is performed using a Bruker Peptide Mix Calibration
Standard. The peaks with a S/N ratio over 4 are automatically
labeled by Flex-Analysis (Bruker Daltonic GmbH, Bremen, Germany).
The MS data is further analyzed through MASCOT 2.2.04 and Biotools
2.1 software (Bruker Daltonic GmbH, Bremen, Germany), and these
data were searched against Mammalian proteins in NCBI nonreduntant
(NCBInr) database. The following parameters are used for database
searches: monoisotopic mass accuracy <250 ppm, parent charge+1,
missed cleavages 1, carbamidomethylation of cysteine as fixed
modification, oxidation of methionine as variable modification.
[0092] (7e) Liquid Chromatography--Electrospray Ionization (LC-ESI)
Tandem Mass Spectrometry (MS/MS) Analysis
[0093] Nano-LC MS/MS analysis of the trypsin digested peptides from
the protein band is performed using a capillary HPLC coupled
directly to HCT Ultra ESI-ion trap mass spectrometer (Bruker
Daltonic GmbH, Bremen, Germany). Peptide digests are dissolved in
0.1% formic acid/2% ACN prior to column injection. A gradient from
4-90% (0.001% formic acid and 0.001% formic acid in 80% ACN) is
used for peptide separation using a C18 column (15 cm.times.75 nm,
LC PACKINGS). The flow rate is 250 ng/min at 25.degree. C. Eluates
from a C18 column are entered into the HCT Ultra ESI-ion trap mass
spectrometer, operated in linear mode for online analysis. The ion
trap mass spectrometer is optimized with the nanosource with a
spray voltage of 137V and a heated capillary temperature of
160.degree. C. The accumulation time for peptide ions in the ion
trap is set to be 200 ms, and the mass to charge ratio selected for
MS/MS analysis is from 100 to 1800 Da with a charge state 1-3.
[0094] The reverse phase HPLC on a VYDAC C4 column, monitored at a
wavelength of 220 nm, is employed to separate different types of
cross-linking occurring between .alpha. and .beta. chains in the
DBSF crosslinked bovine hemoglobin. The chromatographic patterns
obtained using bovine hemoglobin before and after cross-linking
with DBSF are shown in FIG. 9. In FIG. 9, the .alpha. chains are
more mobile than the .beta. chains of native bovine hemoglobin (as
shown with dashed line). Their identities are confirmed by
MALDI-TOF analysis. After the reaction with DBSF, the .beta. chains
are crosslinked while a large majority of .alpha. chains are left
alone (as shown with solid line). As a consequence of cross-linking
with DBSF, 6 major globin peaks with greater hydrophobicity than
the native .beta. chains are formed. The crosslinked globin chains
in the DBSF crosslinked bovine hemoglobin are also resolved by 15%
SDS-PAGE, as shown in FIG. 10. The major crosslinked globin chain
(B6 in FIG. 10) is subjected to trypsin digestion and subsequent
MALDI-TOF analysis, and it is identified as beta globin chain only,
based on its peptide mass fingerprint, as shown in FIG. 11.
[0095] While the foregoing invention has been described with
respect to various embodiments, such embodiments are not limiting.
Numerous variations and modifications would be understood by those
of ordinary skill in the art. Such variations and modifications are
considered to be included within the scope of the following
claims.
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