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.

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° C. to 95° 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.