NZ617344B2 - Method for removing unmodified hemoglobin from cross-linked hemoglobin solutions including polymeric hemoglobin with high temperature short time heat treatment apparatus - Google Patents
Method for removing unmodified hemoglobin from cross-linked hemoglobin solutions including polymeric hemoglobin with high temperature short time heat treatment apparatus Download PDFInfo
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- NZ617344B2 NZ617344B2 NZ617344A NZ61734412A NZ617344B2 NZ 617344 B2 NZ617344 B2 NZ 617344B2 NZ 617344 A NZ617344 A NZ 617344A NZ 61734412 A NZ61734412 A NZ 61734412A NZ 617344 B2 NZ617344 B2 NZ 617344B2
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- hemoglobin
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- haemoglobin
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- red blood
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- 108010054147 Hemoglobins Proteins 0.000 title claims description 151
- 102000001554 Hemoglobins Human genes 0.000 title claims description 151
- 238000010438 heat treatment Methods 0.000 title claims description 25
- 210000003743 Erythrocytes Anatomy 0.000 claims abstract description 32
- 210000003324 RBC Anatomy 0.000 claims abstract description 32
- 239000001301 oxygen Substances 0.000 claims abstract description 21
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 21
- MYMOFIZGZYHOMD-UHFFFAOYSA-N oxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 claims abstract description 20
- 210000004369 Blood Anatomy 0.000 claims abstract description 15
- 239000008280 blood Substances 0.000 claims abstract description 15
- 239000002244 precipitate Substances 0.000 claims abstract description 14
- 238000001914 filtration Methods 0.000 claims abstract description 13
- 210000002381 Plasma Anatomy 0.000 claims abstract description 12
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 8
- 239000000969 carrier Substances 0.000 claims abstract description 8
- 238000001816 cooling Methods 0.000 claims abstract description 8
- 239000000539 dimer Substances 0.000 claims abstract description 8
- 238000000746 purification Methods 0.000 claims abstract description 8
- 238000005119 centrifugation Methods 0.000 claims abstract description 7
- 239000012535 impurity Substances 0.000 claims abstract description 7
- 238000005755 formation reaction Methods 0.000 claims abstract description 6
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 6
- 238000004132 cross linking Methods 0.000 claims abstract description 5
- 230000002934 lysing Effects 0.000 claims abstract description 5
- 238000002360 preparation method Methods 0.000 claims abstract description 4
- 239000006166 lysate Substances 0.000 claims abstract 8
- 238000000034 method Methods 0.000 claims description 17
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 claims description 8
- 210000001519 tissues Anatomy 0.000 claims description 8
- 229960004308 ACETYLCYSTEINE Drugs 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 5
- 108010061951 Methemoglobin Proteins 0.000 claims description 4
- 238000000108 ultra-filtration Methods 0.000 claims description 3
- XUJNEKJLAYXESH-REOHCLBHSA-N L-cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 claims description 2
- 238000004587 chromatography analysis Methods 0.000 claims description 2
- 235000018417 cysteine Nutrition 0.000 claims description 2
- 101700014779 GLB1 Proteins 0.000 claims 1
- 239000000243 solution Substances 0.000 description 88
- 239000003633 blood substitute Substances 0.000 description 13
- 239000000463 material Substances 0.000 description 10
- 239000008215 water for injection Substances 0.000 description 9
- 238000006116 polymerization reaction Methods 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 5
- 229910000033 sodium borohydride Inorganic materials 0.000 description 5
- YOQDYZUWIQVZSF-UHFFFAOYSA-N sodium borohydride Substances [BH4-].[Na+] YOQDYZUWIQVZSF-UHFFFAOYSA-N 0.000 description 5
- ODGROJYWQXFQOZ-UHFFFAOYSA-N sodium;boron(1-) Chemical compound [B-].[Na+] ODGROJYWQXFQOZ-UHFFFAOYSA-N 0.000 description 5
- 238000009826 distribution Methods 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 230000014759 maintenance of location Effects 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 241000283690 Bos taurus Species 0.000 description 3
- 206010021143 Hypoxia Diseases 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 238000005349 anion exchange Methods 0.000 description 3
- 238000011026 diafiltration Methods 0.000 description 3
- 238000003379 elimination reaction Methods 0.000 description 3
- 230000001146 hypoxic Effects 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 230000003134 recirculating Effects 0.000 description 3
- 230000000268 renotropic Effects 0.000 description 3
- 230000003068 static Effects 0.000 description 3
- 206010002199 Anaphylactic shock Diseases 0.000 description 2
- 108010003320 Carboxyhemoglobin Proteins 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 108010064719 Oxyhemoglobins Proteins 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 238000007792 addition Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- UGFAIRIUMAVXCW-UHFFFAOYSA-N carbon monoxide Chemical compound [O+]#[C-] UGFAIRIUMAVXCW-UHFFFAOYSA-N 0.000 description 2
- 229910002091 carbon monoxide Inorganic materials 0.000 description 2
- 210000004027 cells Anatomy 0.000 description 2
- 239000000356 contaminant Substances 0.000 description 2
- 238000006392 deoxygenation reaction Methods 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- 238000011045 prefiltration Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000000717 retained Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 230000002194 synthesizing Effects 0.000 description 2
- 210000004881 tumor cells Anatomy 0.000 description 2
- 230000003612 virological Effects 0.000 description 2
- 206010000565 Acquired immunodeficiency syndrome Diseases 0.000 description 1
- 208000003455 Anaphylaxis Diseases 0.000 description 1
- 208000007502 Anemia Diseases 0.000 description 1
- 241000272517 Anseriformes Species 0.000 description 1
- 230000037227 Blood Loss Effects 0.000 description 1
- 210000002421 Cell Wall Anatomy 0.000 description 1
- 230000036826 Excretion Effects 0.000 description 1
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- 206010018987 Haemorrhage Diseases 0.000 description 1
- 208000006454 Hepatitis Diseases 0.000 description 1
- 241000282619 Hylobates lar Species 0.000 description 1
- 210000003734 Kidney Anatomy 0.000 description 1
- GSDSWSVVBLHKDQ-UHFFFAOYSA-N Levofloxacin Chemical compound FC1=CC(C(C(C(O)=O)=C2)=O)=C3N2C(C)COC3=C1N1CCN(C)CC1 GSDSWSVVBLHKDQ-UHFFFAOYSA-N 0.000 description 1
- 241000283898 Ovis Species 0.000 description 1
- 206010034695 Pernicious anaemia Diseases 0.000 description 1
- 230000037289 Plasma half life Effects 0.000 description 1
- 230000037240 Plasma half-life Effects 0.000 description 1
- 206010061481 Renal injury Diseases 0.000 description 1
- 206010040070 Septic shock Diseases 0.000 description 1
- 208000007056 Sickle Cell Anemia Diseases 0.000 description 1
- 229940005581 Sodium Lactate Drugs 0.000 description 1
- NGSFWBMYFKHRBD-UHFFFAOYSA-M Sodium lactate Chemical compound [Na+].CC(O)C([O-])=O NGSFWBMYFKHRBD-UHFFFAOYSA-M 0.000 description 1
- KKCBUQHMOMHUOY-UHFFFAOYSA-N Sodium oxide Chemical compound [O-2].[Na+].[Na+] KKCBUQHMOMHUOY-UHFFFAOYSA-N 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K Trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 230000002429 anti-coagulation Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000036770 blood supply Effects 0.000 description 1
- 238000009582 blood typing Methods 0.000 description 1
- CROBTXVXNQNKKO-UHFFFAOYSA-N borohydride Chemical compound [BH4-] CROBTXVXNQNKKO-UHFFFAOYSA-N 0.000 description 1
- UIIMBOGNXHQVGW-UHFFFAOYSA-M buffer Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 238000005352 clarification Methods 0.000 description 1
- 230000001808 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 108010002255 deoxyhemoglobin Proteins 0.000 description 1
- 230000001419 dependent Effects 0.000 description 1
- 230000001627 detrimental Effects 0.000 description 1
- XGZRAKBCYZIBKP-UHFFFAOYSA-L disodium;dihydroxide Chemical compound [OH-].[OH-].[Na+].[Na+] XGZRAKBCYZIBKP-UHFFFAOYSA-L 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 150000004679 hydroxides Chemical class 0.000 description 1
- 230000001631 hypertensive Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
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- 230000000813 microbial Effects 0.000 description 1
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- 230000004048 modification Effects 0.000 description 1
- 238000006011 modification reaction Methods 0.000 description 1
- 230000003204 osmotic Effects 0.000 description 1
- 238000002496 oximetry Methods 0.000 description 1
- 238000006213 oxygenation reaction Methods 0.000 description 1
- 239000006174 pH buffer Substances 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000000651 prodrug Substances 0.000 description 1
- 229940002612 prodrugs Drugs 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000036303 septic shock Effects 0.000 description 1
- 231100000486 side effect Toxicity 0.000 description 1
- KEAYESYHFKHZAL-UHFFFAOYSA-N sodium Chemical compound [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- 239000001540 sodium lactate Substances 0.000 description 1
- 235000011088 sodium lactate Nutrition 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 108010001708 stroma free hemoglobin Proteins 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
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- 239000011778 trisodium citrate Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
- C07K14/805—Haemoglobins; Myoglobins
Abstract
Disclosed is a method for the preparation of a highly purified and heat stable oxygen carrier containing pharmaceutical composition, the oxygen carrier-containing pharmaceutical composition including haemoglobin, the haemoglobin including cross-linked polymeric haemoglobin, the method comprising: (a) providing whole blood including at least red blood cells and plasma; (b) separating the red blood cells from the plasma in the whole blood; (c) filtering the red blood cells that were separated from the plasma to obtain a filtered red blood cell fraction; (d) lysing the red blood cells to create a solution comprising a lysate of disrupted red blood cells; (e) extracting a first haemoglobin solution from the lysate; (f) performing one or more purification processes to produce a purified haemoglobin solution; (g) cross-linking haemoglobin tetramers to create a solution including cross-linked polymeric haemoglobin, the polymeric haemoglobin including two or more cross-linked haemoglobin tetramers; (h) heat treating the solution including the cross-linked polymeric haemoglobin in a deoxygenated environment at a temperature greater than 85°C and less than or equal to approximately 95°C for a period of less than approximately 40 minutes to denature and precipitate any residual non-cross-linked tetrameric haemoglobin, any dimeric haemoglobin, and any other impurities such to form a heat stable solution including a cross-linked polymeric haemoglobin; (i) cooling the heat-treated solution to a temperature approximately less than or equal to 25 °C in approximately two minutes or less to prevent formation of met-haemoglobin; (j) removing precipitate by a centrifugation or a filtration apparatus to form a solution of purified, heat stable haemoglobin including cross-linked polymeric haemoglobin having an undetectable concentration of dimer. (a) providing whole blood including at least red blood cells and plasma; (b) separating the red blood cells from the plasma in the whole blood; (c) filtering the red blood cells that were separated from the plasma to obtain a filtered red blood cell fraction; (d) lysing the red blood cells to create a solution comprising a lysate of disrupted red blood cells; (e) extracting a first haemoglobin solution from the lysate; (f) performing one or more purification processes to produce a purified haemoglobin solution; (g) cross-linking haemoglobin tetramers to create a solution including cross-linked polymeric haemoglobin, the polymeric haemoglobin including two or more cross-linked haemoglobin tetramers; (h) heat treating the solution including the cross-linked polymeric haemoglobin in a deoxygenated environment at a temperature greater than 85°C and less than or equal to approximately 95°C for a period of less than approximately 40 minutes to denature and precipitate any residual non-cross-linked tetrameric haemoglobin, any dimeric haemoglobin, and any other impurities such to form a heat stable solution including a cross-linked polymeric haemoglobin; (i) cooling the heat-treated solution to a temperature approximately less than or equal to 25 °C in approximately two minutes or less to prevent formation of met-haemoglobin; (j) removing precipitate by a centrifugation or a filtration apparatus to form a solution of purified, heat stable haemoglobin including cross-linked polymeric haemoglobin having an undetectable concentration of dimer.
Description
METHOD FOR REMOVING UNMODIFIED HEMOGLOBIN FROM CROSS—
LINKED HEMOGLOBIN SOLUTIONS INCLUDING POLYMERIC
HEMOGLOBIN WITH A HIGH TEIVIPERATURE SHORT TIME HEAT
TREATMENT APPARATUS
FIELD OF THE INVENTION
The invention relates to the field of hemoglobin-based oxygen carriers and, more
particularly, to heat treatment ques for purifying hemoglobin—based oxygen carriers
including polymeric hemoglobin.
BACKGROUND OF THE INVENTION
There exists a need for a blood-substitute to treat or prevent hypoxia resulting from
blood loss (e. g., from acute hemorrhage or during surgical operations), resulting from anemia
(e.g., pernicious anemia or sickle cell anemia) or resulting from shock (e.g., volume deficiency
shock, anaphylactic shock, septic shock or allergic shock).
The use ofblood and blood fractions as in this capacity as a blood-substitute is fraught
with disadvantages. For example, the use e blood often is accompanied by the risk of
transmission of hepatitis—producing viruses and AIDS—producing Viruses which can complicate
patient recovery or result in t fatalities. onally, the use of whole blood requires
blood-typing and cross-matching to avoid immunohematological ms and inter donor
incompatibility.
Hemoglobin, as a blood-substitute, possesses osmotic activity and the ability to transport
and transfer oxygen. r, aqueous hemoglobin exists in brium between the
eric (65 KDa) and dimeric (32 KDa) forms. Hemoglobin dimers are excreted by the
kidney and result in rapid intravascular elimination of hemoglobin solutions with such solutions
typically having a 2-4 hour plasma half—life.
Efforts have been directed to overcome the inherent limitations of hemoglobin solutions
by molecularly modifying the hemoglobin. Intramolecularly and intermolecularly cross—linking
hemoglobin has generally d renal elimination and increased intravascular retention time.
r, solutions of cross-linked hemoglobin still typically contain a significant
fraction ofunmodified tetrameric obin. This fied tetrameric hemoglobin can
convert to dirneric hemoglobin and then be excreted from the body, thereby ng the
average intravascular retention time for cross-linked obin blood-substitutes. Furthermore,
current means for separation, such as standard filtration, do not adequately guish between
unmodified tetrameric hemoglobin and modified tetramerie obin,
Thus, in spite of the recent advances in the preparation of cross-linked hemoglobin
blood-substitutes, the need continues to exist for a method to effectively separate unmodified
hemoglobin from a solution of an intramolecularly and/or intermolecularly cross-linked
hemoglobin blood—substitute to improve the e intravascular retention time of the blood-
substitute and to prevent significant levels of renal excretion of hemoglobin.
Prior approaches to removal of various impurities from hemoglobin solutions has
focused on relatively low temperature long term r than one hour) heat treatment processes.
US. Patent No. 5,281,579 describes heat treatment from 45 to 85 °C, and particularly 60-66 °C
for 1 to 30 hours. US. Patent No. 5,741,894 describes a process for l of impurities from
partially oxygenated hemoglobin solutions in a range of 45 to 85 °C, and particularly 76 °C for
90 minutes. r, such long term heat treatment conditions can lead to the formation of
PCT/U82012/034608
met-hemoglobin, which cannot be used to oxygenate tissues. Further, such long term heat
treatment processes are not compatible with commercial—scale production processes.
SUMMARY OF THE INVENTION
The present invention relates to a method for separating unmodified hemoglobin from
cross-linked hemoglobin in a hemoglobin solution including ric hemoglobin. The
method es contacting the hemoglobin solution with a high temperature short time
apparatus wherein unmodified tetrameric hemoglobin is thermally denatured to form a
precipitate. The denatured and precipitated hemoglobin dimers are then separated from the
solution, while retaining the cross—linked hemoglobin in the solution.
The advantages of this invention include ing a substitute with an improved
intrayascular retention time, a reduction or ation of significant renal elimination of
hemoglobin and the side effects ated therewith, a suitable oncotic pressure, and reduced
hypertensive effects.
BRIEF DESCRIPTION OF THE DRAWINGS
represents a schematic flow m of a high temperature short time apparatus
method for denaturing unmodified hemoglobin from d hemoglobin blood-substitute
ing to the present invention.
depicts high performance liquid chromatography analysis for non-heat treated
polymeric hemoglobin.
PCT/U82012/034608
depicts high performance liquid chromatography analysis for (a) at treated
polymeric hemoglobin with spiked hemoglobin dimer and (b) heat stable polymeric hemoglobin
in which has undergone short term heat treatment at 80°C, 85°C, 90°C and 95°C respectively.
DETAILED DESCRIPTION OF THE INVENTION
Hemoglobin (Hb) suitable for Hb solutions of this invention can be derived from new,
old or outdated blood from humans and other vertebrates, such as cattle, pigs, sheep, ducks and
chickens ,
The blood can be collected from live or freshly slaughtered donors. es of suitable
methods for obtaining hemoglobin, derived from red blood cells, are described in US. Patent
Nos. 5,084,558 and 5,296,465, issued to Rausch et al, US. Patent No. 6498141, issued to
DeWoskin et al, and US. Patent No. 7291592, issued to Gould et al. The teachings of U.S.
Patent Nos. 5,084,558, 5,296,465, 6498141 and 2 are incorporated herein by reference in
their entirety.
In a preferred ment, hemoglobin is derived from red blood cells as described in
US. Patent No. 581, the teachings of which are incorporated herein by reference in their
entirety.
Suitable hemoglobin solutions se s solutions of dissolved Hb wherein the
dissolved Hb includes unmodified Hb in addition to modified tetrameric Hb and/or polymeric
Unmodified hemoglobin, as defined , is hemoglobin in a non-dissociated and
uncross-linked tetrameric form which can dissociate into Hb dimers in vitro or in vivo;
WO 48832
unmodified hemoglobin may also include dissociated Hb dimers. Hb dimers can further
dissociate into Hb subunits (monomers). Modified hemoglobin may be intramolecularly cross-
linked into stable tetramers as well as intermolecularly cross-linked into a r chain within
the Hb solution. A r-containing hemoglobin solution used as the starting solution of the
present invention can e cross-linked tetrameric hemoglobin along with intermolecularly
cross-linked polymeric hemoglobin, and also include undesirable unmodified hemoglobin in
tetrameric or c form.
Polymeric hemoglobin may e only hemoglobin components as set forth in US.
Patent Nos. 5,753,616, 5,895,810 and 6,288,027, the disclosures h are incorporated by
reference herein; it may include non-hemoglobin molecules conjugated with hemoglobin such
as polyethylene glycol (PEG). Such materials are described in U.S. Patent Nos. 900,
7,501,499, and 7,795,401, the disclosures of which are incorporated by reference . All of
the above materials can be used as starting hemoglobin-containing solutions in the dimer-
removal processes described below. Commercially available hemoglobin-based oxygen carriers
can also be used in the dimer-removal process of the present invention, including
HEMOPURE®, OXYGLOBIN®, POLYHEME®, HEMOLINKTM and MP4.
In the process of the present invention, a polymeric obin-containing solution
prepared according to any ofthe above ngs is subjected to a heat treatment process from
approximately 80°C to approximately 95 °C, and, more preferably, greater than 85 °C to 95 °C
to sfully remove uncross—linked tetrameric and dimeric forms of hemoglobin from the
polymeric hemoglobin—containing solution. Any precipitates formed during the heat treatment
are removed by centrifugation or a filtration apparatus to form a clear hemoglobin-containing
solution. The high temperature short time heat treatment is preferably carried out using the
PCT/U82012/034608
apparatus ed in and described in more detail in Example 1. In this temperature
than one hour.
range, all of the heat treatments can take place for durations of ntially less
In the lower range of 80-85 °C, a time of about 20 to 40 minutes is sufficient. In a temperature
minutes is sufficient.
range of greater than 85 °C and less than 90 °C a period from 8 to about 30
However at higher temperatures, such as 90-95 °C, the heat treatment can be performed in an
exemplary embodiment in 5 minutes or less, and more preferably in less than three minutes.
Cooling preferably takes less than two minutes, and more preferably less than one minute to
minimize formation ofmet-hemoglobin.
The high temperature short time process for heat treating the hemoglobin solutions in
the purification process also removes other impurities, for example immunoglobin—G and
harmful microbial materials and viruses, so that renal injury, ar detrimental effects and
other toxicity reactions can be avoided.
Heat treatment oftetrameric hemoglobin is described in US. Patent No. 7,932,356 and
US. Patent Application Nos. 12/821,214 and 13/013,850 all of the sures h are
incorporated herein by reference. The heat treated polymeric hemoglobin of the present
invention can be packaged as described in US. 356 and can be used in various tissue
ation techniques sed in the above patents and applications. The highly purified
and heat stable oxygen carrier-containing pharmaceutical composition is used in methods of
ating tissue in which the composition is provided to animal tissue either in vivo or ex
vivo as described in the ‘356 patent.
EXAMPLE 1
HTST heat processing
PCT/U82012/034608
A High Temperature Short Time (HTST) processing apparatus is shown in A
heating process using the HTST sing apparatus is performed on the polymeric
hemoglobin—containing ng material. In this e, the condition for heat treatment is
90°C for 30 seconds to 3 minutes, and preferably 45 to 60 seconds; although other conditions
can be selected as discussed above and the tus modified ingly. A solution
containing polymeric hemoglobin, that is commercially available Oxyglobin®, is optionally
treated with 0.2% of N—acetyl cysteine and pumped into a HTST processing apparatus (first
section of the HTST heat exchanger is pre—heated and ined at 90°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°C. The time required for cooling is between 15 to 30
s. After cooling down to approximately 25°C, N—acetyl cysteine is ally added
immediately afterward at a concentration of 0.2% to 0.4%, preferably at 0.4%: This chemical
addition after the HTST heating process maintains met-hemoglobin (inactive hemoglobin) at a
low level. The set-up of the processing apparatus is easily controlled for industrial operation. If
the hemoglobin is not linked, it is not heat stable and forms a precipitate after the heat
treatment step. The precipitate is then removed by a centrifugation or a filtration apparatus to
form a substantially clear solution thereafter.
shows the molecular weight distribution of polymeric hemoglobin by size—
ion High Performance Liquid Chromatography (HPLC). The molecular weight
distribution for polymeric hemoglobin solution ranges from 32 KDa to >500 KDa. Following
2012/034608
the HTST heat process step (from 80°C to 95°C) in our invention, the spiked dimeric form of
hemoglobin can be removed successfully from the polymeric hemoglobin solution (shown in
. Any precipitates formed during the HTST heat process step are removed by
centrifugation or a filtration apparatus to form a substantially clear cross-linked hemoglobin
solution.
Hemox Analyzer for p50 (oxygen partial pressure, at which the hemoglobin solution is
50% saturated) measurement is used thereafter to analyze the (a) non-heat treated polymeric
hemoglobin-containing solution, and (b) a heat treated polymeric hemoglobin-containing
solution (undergo 80°C treatment). No significant change in hemoglobin content (as measured
by Co-Oximetry) is detected between (1) before HTST heat process step, and (2) after HTST
heat process step. However, the p50 value is shifted to a lower value (from 37.5 mmHg to 23.3
Hg) after HTST heat s step, This indicates that the hemoglobin-oxygen g affinity is
higher. The lowering of p50 value is an advantage to upload oxygen in c tissues and
hypoxic tumor cells. As a tumor grows, it rapidly outgrows its blood supply, leaving portions of
the tumor with regions where the oxygen concentration is significantly lower than in y
tissues. Denny (Denny W. A., Prodrug gies in cancer therapy, Eur. J. Med. Chem, 2001,
36, 5) reported that hypoxic tumor cells are usually resistant to radiotherapy and
chemotherapy; r, they can be made more susceptible to treatment by increasing their
oxygen t.
Table 1
p50 value tmmHg)
Non-heat treated polymeric hemoglobin-containing 37.5
solution
Heat treated lobin—containin_ solution 23.3
2012/034608
EXAMPLE 2
Oxyglobin® and/or Hemopure ® polymeric hemoglobin
Synthesis of Stable Polymeric Hemoglobin Blood-Substitute based on the description of
US Patent Nos. 5,895,810, 5,296,465, 5084558, 6 and 5955581, is also known as
Oxyglobin® and/or Hemopure® product, the disclosures ofwhich are incorporated by reference
herein.
The following example relates to a method for making polymeric—containing
hemoglobin solutions which are suitable for treatment by the heat treatment apparatus and
method ofthe present invention.
Bovine whole blood is collected, mixed with a sodium citrate anticoagulant to form a
blood solution. The red blood cells (RBCs) are isolated from bovine whole blood. The RBCs
are then washed to separate extracellular plasma proteins, such as BSA or IgG, from the RBCs.
Following separation of the RBCs, the RBCs are lysed to form a hemoglobin-containing
solution.
The concentrated Hb solution is then directed from the ultrafiltrate tank onto the media
contained in parallel chromatographic columns to te the Hb by high performance liquid
chromatography. The chromatographic s n an anion exchange medium suitable to
separate Hb from nonhemoglobin proteins. The anion exchange media is a quaternary
ammonium anion exchange medium on silica gel. This method of ng silica gel is described
in the Journal of Chromatography, 120:321-333 (1976).
The Hb solution is then deoxygenated to a level where oxyhemoglobin or HbOz t
is about 101. During deoxygenation, temperature of the Hb on is maintained between
PCT/U82012/034608
about 19°C and about 31°C. Also during deoxygenation, and uently throughout the
process, the Hb is maintained in a low oxygen environment to minimize oxygen absorption by
the Hb and to maintain an oxygenated Hb (HbOz) content of less than about 10% in the deoxy—
Prior to the polymerization process, depyrogenated and oxygen-depleted “water for
injection” (WFI) is added to the Hb solution to a concentration of about 40 g Hb/L. The
polymerization is conducted in a 12 mM phosphate buffer with a pH of 7.8, having a chloride
concentration less than or equal to about 35 mM.
The oxidation-stabilized deoxy-Hb and yl cysteine (NAC) are subsequently
slowly mixed with the cross-linking agent glutaraldehyde, specifically 29.4 grams of
glutaraldehyde for each am of Hb over a five hour period, While heating at 42°C and
recirculating the Hb solution through a Kenics l-1/2 inch static mixer with 6 elements
(Chemineer, Inc), to form a polymeric Hb solution (hereinafter Hb)"). After
polymerization, the temperature of the poly(Hb) in the polymerization reactor is d to a
temperature between about 18°C to about 22°C,
The poly(Hb) is then concentrated by recirculating the poly(Hb) through the ultrafilter
until the tration of the poly(Hb) is increased to about 85 g/L. A suitable ultrafilter is a 30
KDa ultra filter. Subsequently, the b) solution is then mixed with 66.75 g sodium
dride, and then recirculated through the static mixer at a flow rate of 10-20 liters per
minute.
After the pH and electrolytes are restored to physiologic levels, the stable polymeric Hb
blood-substitute is then diluted to a concentration of 50 g/L by adding the filtered,
deoxygenated low pH buffer to the polymerization reactor. The diluted blood-substitute is then
diafiltered by recirculating from the polymerization reactor through the static mixer and a 100
KDa purification filter against a filtered deoxygenated buffer containing 27 mM sodium lactate,
12 mM NAC, 115 mM NaCl, 4 mM KCl and 1.36 mM CaClz in WFI, (pH 7.8). Diafiltration
continues until the blood-substitute ns less than or equal to about 10% modified
tetrameric and unmodified tetrameric species.
A polymeric Hb solution is formed according to the method described in this Example 2
(according to the description ofUS. Patent No. 5,084,558, issued to Rausch et al.). This Hb
solution is analyzed by gel permeation chromatography and found to comprise about 45% Hb
dimers, about 15% unmodified Hb tetramers, and about 40% polymeric Hb les which are
larger than unmodified tetramers.
The polymeric hemoglobin-containing material may then be subjected to the heat
treatment as discussed in Example 1 to remove dimer and unmodified Hb tetramer.
Polyheme® polymeric obin
Synthesis of Stable Polymeric Hemoglobin Blood—Substitute based on the description of
US Patent Nos. 6498141 and 7291592, is also known as Northfield laboratories Inc. product
(Polyheme®), the sures of which are incorporated by reference .
The following example s to a method for making polymeric-containing
hemoglobin solutions le for treatment by the heat treatment apparatus and method of the
present invention.
2012/034608
(3a) Preparation of Red Blood Cells, Cell wash and Lysis
Mix a blood solution with a 1% aqueous sodium chloride solution to form a 4% total
hemoglobin solution; carbon monoxide is then introduced into the mixing tank so that the tank
contains an here of carbon monoxide.
By coupling to a 0.65 um tangential flow filter, this 4% total hemoglobin solution is
washed with about 8 volumes of the 1% sodium chloride solution to remove plasma protein
contaminants. Subsequent to washing, the solution is concentrated to about 16% total
hemoglobin, and "water for inj ection" (WFI) is added to bring the volume of the on up to
about 2.5 times volume. With the addition of the WFI, the cells swell and rupture releasing
obin into solution. The concentration of the resulting obin solution is about 7%
total hemoglobin.
The resulting solution is clarified; red blood cells stroma contaminants and cell wall
material is retained and removed by the filter. The remaining solution is about 3.3% total
hemoglobin solution.
(3b) Heat Treatment, Clarification and Viral Reduction
The resulting solution of stroma-free hemoglobin is then heat treated at a ature of
about 60-62°C over a period of about 10 hours. During this time, the on is moderately
agitated. As the solution is heated and passes a temperature of about 55°C, a precipitate forms.
The resulting 3.3% total hemoglobin (w/v) stroma-free, heat treated hemoglobin solution
is then filtered through a 0.2 um pre-filter ed by a 0.1 urn pre—filter and then pumped
through a 100 KDa viral reduction ultra filter.
2012/034608
(3c) Ultra-filtration Concentration
The filtered hemoglobin on is then concentrated to about 14% total Hb
concentration and subsequently washed and diafilter with 4 volumes of WFI. The concentration
and diafiltration is lished using a 10 KDa ultra filter. This hemoglobin in the solution is
primarily carboxyhemoglobin.
(3d) Gas exchange with Oxygen and Nitrogen
The resulting yhemoglobin solution is sparged with a flow of oxygen for 18 hours
at 10°C. The resulting solution contains less than 5% carboxyhemoglobin based on the weight
of total hemoglobin.
[005 0] After oxygenation, the solution is sparged with a similar flow of nitrogen for about 3 to
3.5 hours at 10°C. until less than 10% oxyhemoglobin based on the weight of total hemoglobin
remains in the solution.
(Be) Chemical Modification
The deoxyhemoglobin (at about 4°C) solution is then added an aqueous solution of
pyridoxyl—S-phosphate (P5P) (93.75 g/L) at a 2:1 PSP to hemoglobin molar ratio. The
pyridoxylation is conducted at a ature of about 4°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 ratio of 0.8 g of
sodium hydroxide and 90.8 g of sodium borohydride per 2 liters of hemoglobin on. The
borohydride solution is added as rapidly as possible over a period of about 1 minute and then
PCT/U82012/034608
stirred for one hour. The resulting solution of pyridoxylated obin is subsequently
diafiltered using 10 KDa ultra filter to remove excess reactants with 4 volumes ofWFI.
(St) Polymeric Hemoglobin solution
The pyridoxylated hemoglobin is added sufficient WFI to prepare a 4.5% total
hemoglobin solution. A glutaraldehyde solution is added to the xylated 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 15-18 hours.
The target molecular weight distribution is about 75% polymer and 25% er. The target
polymers have molecular weights of less than about 600 KDa with a predominant fraction of the
lar weights residing in the 100 KDa-350KDa range.
When the polymerization reaction reaches the target molecular weight distribution (after
about 15-18 hours), aqueous glycine is added (as a quench) to the hemoglobin solution at a
140:1 molar ratio of glycine to hemoglobin. The solution pH at this point is 8.9. The resulting
solution is then mixed for about 30-40 s 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 by ultrafiltration and
washed with 4 volumes 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 s ofWFI resulting in polymeric, pyridoxylated,
-free obin that has been heat treated.
PCT/U82012/034608
The resulting solution is oxygenated by allowing the solution to stand under an oxygen
atmosphere. The hemoglobin is then diluted to about 4% total hemoglobin. The 4% total
hemoglobin solution is then diafiltered using 10 mM NaCl/20 mM NaOH and a 300 KDa ultra-
filter. The filtration is continued until about 97% of the hemoglobin material passes through the
filter and is continuously concentrated to 4-8% total hemoglobin using a 70 KDa ultrafilter.
(About 3% of the material, i.e., high molecular weight polymers is retained).
The resulting material is about 4-8% total hemoglobin and ns about 25% tetramer.
Subsequently, the polymeric hemoglobin-containing material may then be subjected to the heat
treatment as sed in Example 1 to remove dimer and fied Hb tetramer.
Although various aspects of the ion are set out in the independent claims, other
aspects of the invention comprise other combinations of features from the described
embodiments and/or the dependent claims with the features of the independent claims, and not
solely the combinations itly set out in the claims.
It is also noted herein that while the above describes exemplary embodiments of the
invention, these descriptions should not be viewed in a ng sense. Rather, variations and
modifications may be made without departing from the scope of the present invention as
defined in the appended claims.
Claims (12)
1. An ex Vivo method for the preparation of a highly purified and heat stable oxygen carrier-containing pharmaceutical composition, the oxygen r—containing pharmaceutical composition including hemoglobin, the hemoglobin including cross- linked ric hemoglobin, the method comprising: (a) separating red blood cells from plasma in whole blood; (b) filtering the red blood cells that were separated from the plasma to obtain a filtered red blood cell fraction; (0) lysing the red blood cells to create a solution sing a lysate of disrupted red blood cells; (d) extracting a first hemoglobin solution from the lysate; (e) performing one or more purification processes to produce a purified hemoglobin solution; (i) cross—linking hemoglobin tetramers to create a solution including cross-linked polymeric hemoglobin, the polymeric hemoglobin including two or more cross—linked hemoglobin tetramers; (g) heat ng the solution including the cross—linked polymeric hemoglobin in or equal to a deoxygenated environment at a temperature greater than 85°C and less than approximately 95°C for a period of less than imately 40 minutes to denature and precipitate any residual oss—linked tetrameric hemoglobin, any dimeric hemoglobin, and any other impurities such to form a heat stable solution including a linked polymeric hemoglobin; (h) cooling the heat—treated solution to a temperature approximately less than or equal to 25°C in approximately two minutes or less to prevent formation of met- hemoglobin; (i) removing precipitate by a centrifugation or a filtration apparatus to form a solution of purified, heat-stable hemoglobin including cross—linked polymeric hemoglobin having an undetectable concentration of dimer.
2. The method of claim 1 r comprising adding N—acetyl cysteine ately following heat treating.
3. The method of claim 1 wherein the g is performed in less than one minute.
4. The method of claim 1 wherein the heat treatment occurs at a temperature 8 minutes to about range of r than 85°C and less than 90°C for a period from about 30 minutes.
5. The method of claim 1 wherein the heat ent occurs at approximately 90 0C for a period from about 45 seconds to about 150 seconds.
6. The method of claim 1 wherein the heat treatment occurs at approximately 95 0C for a period from about 30 to about 100 seconds.
7. The method of claim 1 further sing adding N-acetyl cysteine immediately prior to heat treating.
8. The method of claim 2 fuither comprising adding yl cysteine immediately prior to heat treating.
9. The method of claim 1 n the performing one or more purification ultrafiltration. processes to produce a d hemoglobin solution includes
10. The method of claim 1 wherein the performing one or more ation processes to produce a purified hemoglobin solution includes purification by chromatography.
11. A highly purified and heat stable oxygen carrier—containing pharmaceutical composition including hemoglobin and N—acetyl cysteine, the hemoglobin including cross—linked polymeric hemoglobin, the linked polymeric hemoglobin prepared by: (a) separating red blood cells from plasma in whole blood; (b) filtering the red blood cells that were separated from the plasma to obtain a filtered red blood cell fraction; (c) lysing the red blood cells to create a solution comprising a lysate of disrupted red blood cells; (d) extracting a first hemoglobin solution from the lysate; (e) performing one or more purification processes on the first hemoglobin solution; (t) cross—linking hemoglobin tetramers in the first hemoglobin solution to create a second hemoglobin solution ing cross—linked ric hemoglobin, the polymeric hemoglobin including two or more cross—linked hemoglobin tetramers; (g) heat treating the second hemoglobin solution including the cross-linked ric hemoglobin in a deoxygenated environment at a temperature greater than 85°C and less than or equal to imately 95°C for a period of less than approximately 40 minutes to denature and precipitate any residual non—reacted hemoglobin, non—stabilized hemoglobin, dimer, and any other impurities such that the resulting hemoglobin solution has an undetectable amount of dimer when measured using high performance liquid chromatography; (h) cooling the heat—treated solution to a temperature approximately less than or equal to 25°C in approximately two minutes or less to prevent formation of met- hemoglobin; (i) adding N—acetyl cysteine either immediately prior to or after heat treating and cooling the second hemoglobin solution; (j) ng precipitate formed during the heat treating by a centrifugation or a filtration apparatus.
12. A method of ating tissue comprising providing the composition of claim ll to animal tissue ex vivo.
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201113097183A | 2011-04-29 | 2011-04-29 | |
US13/097,183 | 2011-04-29 | ||
US13/217,337 | 2011-08-25 | ||
US13/217,337 US8084581B1 (en) | 2011-04-29 | 2011-08-25 | Method for removing unmodified hemoglobin from cross-linked hemoglobin solutions including polymeric hemoglobin with a high temperature short time heat treatment apparatus |
PCT/US2012/034608 WO2012148832A2 (en) | 2011-04-29 | 2012-04-23 | Method for removing unmodified hemoglobin from cross-linked hemoglobin solutions including polymeric hemoglobin with high temperatures short time heat treatment apparatus |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ617344A NZ617344A (en) | 2015-12-24 |
NZ617344B2 true NZ617344B2 (en) | 2016-03-30 |
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