Method of forming a polymerized hemoglobin solution from stabilized hemoglobin

Abstract

A stabilized hemoglobin solution is contacted with polymerizing agent. The stabilized hemoglobin solution includes stabilized tetrameric hemoglobin. At least a portion of the stabilized tetrameric hemoglobin is polymerized by reaction with the polymerizing agent, thereby producing a polymerized hemoglobin solution. In one embodiment, the stabilized hemoglobin solution includes a filtrate formed by filtrating polymerized solution of native hemoglobin through a filter having a molecular weight cut off of about 100 kD.

1 . A method for forming a polymerized hemoglobin solution, comprising contacting a stabilized hemoglobin solution with a polymerizing agent, said stabilized hemoglobin solution including tetrameric and polymeric hemoglobin, whereby at least a portion of the hemoglobin solution is further polymerized, thereby producing the polymerized hemoglobin solution. 
   
   
       2 . A method of  claim 1 , wherein the stabilized hemoglobin is present in a physiological buffer when contacted with the polymerizing agent. 
   
   
       3 . A method of  claim 2 , wherein the physiological buffer has a pH of about 7.6 to about 7.9. 
   
   
       4 . A method of  claim 2 , wherein the physiological buffer includes at least one component selected from the group consisting of: sodium lactate, N-acetyl-L-cysteine, sodium chloride, potassium chloride, and calcium chloride.2H 2 O. 
   
   
       5 . A method of  claim 3 , wherein the physiological buffer includes sodium lactate at a concentration of about 290 to about 330 mg/100 ml. 
   
   
       6 . A method of  claim 3 , wherein the physiological buffer includes N-acetyl-L-cysteine at a concentration of about 130 to about 220 mg/100 ml. 
   
   
       7 . A method of  claim 3 , wherein the physiological buffer includes sodium chloride at a concentration of about 570 to about 620 mg/100 ml. 
   
   
       8 . A method of  claim 3 , wherein the physiological buffer includes potassium chloride at a concentration of about 27 to about 33 mg/100 ml. 
   
   
       9 . A method of  claim 3 , wherein the physiological buffer includes calcium chloride.2H 2 O at a concentration of about 18 to about 22 mg/100 ml. 
   
   
       10 . A method of  claim 1 , wherein the stabilized hemoglobin solution comprises bovine hemoglobin. 
   
   
       11 . A method of  claim 1 , wherein the concentration of the hemoglobin in the stabilized hemoglobin solution during the further polymerization is about 40 grams per liter. 
   
   
       12 . The method of  claim 1 , further including the step of forming the stabilized hemoglobin solution by contacting native hemoglobin with a cross-linking and/or polymerizing agent. 
   
   
       13 . The method of  claim 12 , wherein forming the stabilized hemoglobin solution includes directing a solution of polymerized, native hemoglobin through a filter having a molecular weight cut off of at least about 100 kD, whereby resulting filtrate is the stabilized hemoglobin solution. 
   
   
       14 . A method of  claim 13 , wherein the polymerizing agent is glutaraldehyde. 
   
   
       15 . A method of  claim 1 , wherein the concentration of glutaraldehyde added to the stabilized hemoglobin solution is about 1 to about 20 grams of glutaraldehyde per kilogram of total hemoglobin present in the stabilized hemoglobin solution. 
   
   
       16 . A method of  claim 15 , wherein the concentration of glutaraldehyde in the stabilized hemoglobin solution is about 10 grams of glutaraldehyde per kilogram of total hemoglobin present in the stabilized hemoglobin solution. 
   
   
       17 . A method of  claim 1 , wherein no more than about 10% by weight of the hemoglobin present in the polymerized hemoglobin solution has a molecular weight of at least about 500 kD. 
   
   
       18 . A method of  claim 1 , wherein between about 45% and about 65% by weight of total hemoglobin present in the polymerized hemoglobin solution is tetrameric and octameric hemoglobin. 
   
   
       19 . A method of  claim 1 , wherein no more than about 40% by weight of total hemoglobin present in the polymerized hemoglobin solution has as molecular weight of about 65 kD or less. 
   
   
       20 . A method of  claim 1 , wherein no more than 10% by weight of total hemoglobin present in the polymerized hemoglobin solution is methemoglobin. 
   
   
       21 . The method of  claim 1 , further comprising directing the polymerized hemoglobin through a filter having a molecular weight cut off of at least about 100 kD, whereby the resulting retentate comprises a polymerized hemoglobin, wherein no more than about 15% by weight of the hemoglobin present in the retentate has a molecular weight of at least about 500 kD, and no more than about 10% by weight of the hemoglobin present in the retentate has a molecular weight of about 65 kD or less. 
   
   
       22 . A method for forming a polymerized hemoglobin solution, comprising contacting a stabilized hemoglobin solution with glutaraldehyde at a concentration of about 1 to about 20 grams of glutaraldehyde per kilogram of hemoglobin present in the stabilized hemoglobin solution, said stabilized hemoglobin solution including a physiological buffer, whereby at least a portion of the hemoglobin is polymerized, thereby forming the polymerized hemoglobin solution. 
   
   
       23 . A method of  claim 22 , wherein the physiological buffer has a pH of about 7.6 to about 7.9. 
   
   
       24 . A method of  claim 22 , wherein the physiological buffer includes at least one component selected from the group consisting of: sodium lactate, N-acetyl-L-cysteine, sodium chloride, potassium chloride, and calcium chloride.2H 2 O. 
   
   
       25 . A method of  claim 22 , wherein the physiological buffer includes sodium lactate at a concentration of about 290 to about 330 mg/100 ml. 
   
   
       26 . A method of  claim 22 , wherein the physiological buffer includes N-acetyl-L-cysteine at a concentration of about 130 to about 220 mg/100 ml. 
   
   
       27 . A method of  claim 22 , wherein the physiological buffer includes sodium chloride at a concentration of about 570 to about 620 mg/100 ml. 
   
   
       28 . A method of  claim 22 , wherein the physiological buffer includes potassium chloride at a concentration of about 27 to about 33 mg/100 ml. 
   
   
       29 . A method of  claim 22 , wherein the physiological buffer includes calcium chlorideÿ2H 2 O at a concentration of about 18 to about 22 mg/100 ml. 
   
   
       30 . A method of  claim 22 , wherein the stabilized hemoglobin solution comprises bovine hemoglobin. 
   
   
       31 . A method of  claim 22 , wherein the concentration of the hemoglobin in the stabilized hemoglobin solution during the further polymerization is about 40 grams per liter. 
   
   
       32 . The method of  claim 22 , further includes the step of forming the stabilized hemoglobin solution. 
   
   
       33 . The method of  claim 22 , further including the step of forming the stabilized hemoglobin solution by contacting native hemoglobin with either or both of a cross-linking and polymerizing agent, resulting in polymerized, native hemoglobin. 
   
   
       34 . The method of  claim 33 , wherein forming the stabilized hemoglobin solution includes directing a solution of polymerized, native hemoglobin through a filter having a molecular weight cut off of at least about 100 kD, whereby resulting filtrate is the stabilized hemoglobin solution. 
   
   
       35 . A method of  claim 22 , wherein the concentration of glutaraldehyde added to the stabilized hemoglobin solution is about 10 grams of glutaraldehyde per kilogram of total hemoglobin present in the stabilized hemoglobin solution. 
   
   
       36 . A method of  claim 22 , wherein no more than about 10% by weight of the hemoglobin present in the polymerized hemoglobin solution has a molecular weight of greater than about 500 kD. 
   
   
       37 . A method of  claim 22 , wherein between about 45% and about 65% by weight of total hemoglobin present in the polymerized hemoglobin solution is tetrameric and octameric hemoglobin. 
   
   
       38 . A method of  claim 22 , wherein no more than about 40% by weight of total hemoglobin present in the polymerized hemoglobin solution has a molecular weight of about 65 kDa or less. 
   
   
       39 . A method for forming a polymerized hemoglobin solution, comprising
 contacting a stabilized hemoglobin solution with glutaraldehyde at a concentration of about 1 to about 20 grams of glutaraldehyde per kilogram of hemoglobin present in the stabilized hemoglobin solution, said stabilized hemoglobin solution including hemoglobin tetramers, wherein the stabilized hemoglobin solution includes N-acetyl-L-cysteine at a concentration of about 130 to about 220 mg/100 ml, sodium lactate at a concentration of about 290 to about 330 mg/100 ml, sodium chloride at a concentration of about 570 to about 620 mg/100 ml, potassium chloride at a concentration of about 27 to about 33 mg/100 ml, calcium chloride.2H 2 O at a concentration of about 18 to about 22 mg/100 ml and having a pH of about 7.6 to about 7.9, whereby at least a portion of the hemoglobin is polymerized, thereby forming a polymerized hemoglobin solution wherein no more than about 10% by weight of total hemoglobin present in the hemoglobin solution has a molecular weight of at least about 500 kDa, no more than about 40% by weight of total hemoglobin has a molecular weight of about 65 kD or less, and no more than about 10% by weight of total hemoglobin is methemoglobin.   
   
   
       40 . A method of  claim 39 , wherein the stabilized hemoglobin solution comprises bovine hemoglobin. 
   
   
       41 . A method of  claim 39 , wherein the concentration of the hemoglobin in the stabilized hemoglobin solution during the further polymerization is about 40 grams per liter. 
   
   
       42 . The method of  claim 39 , further including the step of forming the stabilized hemoglobin solution by contacting native hemoglobin with a cross-linking and/or polymerizing agent, resulting in polymerized, native hemoglobin. 
   
   
       43 . The method of  claim 42 , wherein forming the stabilized hemoglobin solution includes directing a solution of polymerized, native hemoglobin through a filter having a molecular weight cut off of at least about 100 kD, whereby resulting filtrate is the stabilized hemoglobin solution. 
   
   
       44 . A method for forming a polymerized hemoglobin solution, comprising:
 a) contacting a stabilized hemoglobin solution with glutaraldehyde at a concentration of about 1 to about 20 grams of glutaraldehyde per kilogram of hemoglobin present in the stabilized hemoglobin solution, said stabilized hemoglobin solution including hemoglobin tetramers, wherein the stabilized hemoglobin solution includes N-acetyl-L-cysteine at a concentration of about 130 to about 220 mg/100 ml, sodium lactate at a concentration of about 290 to about 330 mg/100 ml, sodium chloride at a concentration of about 570 to about 620 mg/100 ml, potassium chloride at a concentration of about 27 to about 33 mg/100 ml, calcium chloride.2H 2 O at a concentration of about 18 to about 22 mg/100 ml and having a pH of about 7.6 to about 7.9, whereby at least a portion of the hemoglobin is polymerized; and   b) directing the polymerized hemoglobin of a) through a filter having a molecular weight cut off of at least about 100 kD, and obtaining the retentate;   thereby forming a polymerized hemoglobin solution, wherein no more than about 15% by weight of the hemoglobin present in the retentate has a molecular weight of at least about 500 kD, and no more than about 10% by weight of the hemoglobin present in the retentate has a molecular weight of about 65 kD or less.