Biopure Corporation

Virtech Bio, Inc.

William Richard Light

Maria S Gawryl

Robert A Houtchens

Anthony J Laccetti

Thomas C Page

Carl W Rausch

Edward E Jacobs, Jr

Jose O Torres

Javed Baqai

Joseph Tucker

Methods for making hemoglobin based blood substitute preparations and hemoglobin based blood substitute preparations. The methods involve preparing a low purity erythrocyte protein fraction comprising hemoglobin protein and endogenous non-hemoglobin protein complement, and chemically modifying the proteins in the protein fraction to form a cross-linked hemoglobin containing blood substitute preparation. The low purity erythrocyte protein preparation can contain from at least about 0.2% (mole/mole) up to about 20% (mole/mole) endogenous non-hemoglobin protein complement. At least about 90% (mole/mole) of the hemoglobin proteins can be cross-linked, so that the average molecular mass of cross-linked proteins comprising hemoglobin protein molecules in the preparation is at least about 300 kDa. The preparations can be used to prepare finished blood substitute formulations for in-vivo and ex-vivo use.

Blood substitutes comprising hemoglobin and methods of making

Red blood cells are purified by defibrinating whole blood and then filtering the defibrinated whole blood, whereby at least a portion of a plasma component is separated from the red blood cells to form a suspension of red blood cells, thereby purifying the red blood cells. Whole blood is defibrinated by, for example, using a chemical coagulating agent or mechanical agitation. Separation of the plasma component from red blood cells can be completed by, for example, diafiltration. The suspension of red blood cells can then be employed to produce a hemoglobin-based oxygen carrier.

Use of defibrinated blood for manufacture of hemoglobin-based oxygen carrier

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.

Method of forming a polymerized hemoglobin solution from stabilized hemoglobin

A method of treatment of a patient includes administering to the patient stored red blood cells and a hemoglobin solution. The stored red blood cells and the hemoglobin solution can be administered to the patient simultaneously. Alternatively, the hemoglobin solution can be administered to the patient prior to administrating the stored red blood cells or the stored red blood cells can be administered to the patient prior to administering the hemoglobin solution. A composition of the invention includes stored red blood cells and a hemoglobin solution.

Method for improving oxygen transport by stored red blood cells

The invention relates to a method for preserving the stability of a hemoglobin blood substitute comprising maintaining the hemoglobin blood substitute in an atmosphere substantially free of oxygen. The method for preserving a deoxygenated hemoglobin blood substitute comprises maintaining the deoxygenated hemoglobin blood substitute in an oxygen barrier film overwrap package. In one embodiment, the package comprises a transparent laminate material comprising an oxygen barrier layer and a polyolefin layer, wherein the laminant has a thickness of between about 0.001 and about 0.01 inches (or about 0.0254 to about 0.254 millimeters) and an oxygen permeability of less than about 0.01 cubic centimeters per 100 square inches (or about 0.01 cc per 645 square centimeters) over 24 hours at 1 atmosphere and at about 23&#xb0; C. The oxygen barrier layer comprises ethylene vinyl alcohol. Aso provided herein is a preserved deoxygenated hemoglobin blood substitute comprises a deoxygenated hemoglobin blood substitute and an oxygen barrier film overwrap package. The package comprises a transparent laminant material comprising an oxygen barrier layer and a polyolefin layer. Said oxygen barrier layer has an oxygen permeability of less than about 0.01 cubic centimeters per 100 square inches (or about 0.01 cc per 645 square centimeters) over 24 hours at 1 atmosphere and at about 27&#xb0; C. room temperature, wherein the deoxygenated hemoglobin blood substitute is sealed within the overwrap package. In one embodiment, the oxygen barrier layer comprises ethylene vinyl alcohol.

Preserving a hemoglobin blood substitute with a transparent overwrap

The invention relates to a method for preserving the stability of a hemoglobin blood substitute comprising maintaining the hemoglobin blood substitute in an atmosphere substantially free of oxygen. The method for preserving the deoxygenated hemoglobin blood substitute comprises maintaining the deoxygenated blood substitute in an oxygen barrier film overwrap package, wherein at least one face of the overwrap package comprises a transparent laminate material and wherein at least one other face of the overwrap package comprises a foil laminate material. The preserved deoxygenated hemoglobin blood substitute comprises a deoxygenated hemoglobin blood substitute and an oxygen barrier film overwrap package wherein at least one face of the overwrap package comprises a transparent laminate material and wherein at least one other face of the overwrap package comprises a foil laminate material.

Red blood cells are purified by separating whole blood, such as by centrifugation, to form a red blood cell fraction and a liquid fraction. The whole blood can be defibrinated or treated to prevent coagulation prior to separation. Preferably, the whole blood is bovine blood. The red blood cell fraction is then diafiltered to purify the red blood cells. The purified red blood cells can then be lysed to form a lysate of purified red blood cells. The purified red blood cells and the lysate of purified red blood cells are suitable for use in producing hemoglobin blood substitute.

Purification of red blood cells by separation and diafiltration

Use of defibrinated blood for manufacture of a hemoglobin-based oxygen carrier

The invention relates to a method for preserving the stability of a hemoglobin blood substitute comprising maintaining the hemoglobin blood substitute in an atmosphere substantially free of oxygen. The invention also involves a method for producing a stable polymerized hemoglobin blood-substitute from blood. The method of this invention includes mixing blood with an anticoagulant to form a blood solution, washing the red blood cells in the blood solution and then separating the washed red blood cells from the white blood cells. This method also includes disrupting the red blood cells to release hemoglobin and form a hemoglobin solution, which is then treated by high performance liquid chromatography to form a hemoglobin eluate. The hemoglobin eluate is then deoxygenated, contacted with a first sulfhydryl compound to form an oxidation-stabilized deoxygenated hemoglobin solution, and mixed with a cross-linking agent to form a polymerization reaction mixture, which is then polymerized. The polymerized hemoglobin solution is then diafiltered with a physiologic solution and with a sulfhydryl compound, whereby the polymerized hemoglobin solution is made physiologically acceptable, and whereby the sulfhydryl compound scavenges oxygen, to form a stable polymerized hemoglobin blood-substitute, which is then packaged and stored in an atmosphere substantially free of oxygen.

The invention relates to a method for preserving the stability of a hemoglobin blood substitute comprising maintaining the hemoglobin blood substitute in an atmosphere substantially free of oxygen. The invention also involves a method for producing a stable polymerized hemoglobin blood-substitute from blood. The method of this invention includes mixing blood with an anticoagulent to form a blood solution, washing the red blood cells in the blood solution and then separating the washed red blood cells from the white blood cells. This method also includes disrupting the red blood cells to release hemoglobin and form a hemoglobin solution, which is then treated by high performance liquid chromatography to form a hemoglobin eluate. The hemoglobin eluate is then deoxygenated, contacted with a first sulfhydryl compound to form an oxidation-stabilized deoxygenated hemoglobin solution, and mixed with a cross-linking agent to form a polymerization reaction mixture, which is then polymerized. The polymerized hemoglobin solution is then diafiltered with a physiologic solution and with a sulfhydryl compound, whereby the polymerized hemoglobin solution is made physiologically acceptable, and whereby the sulfhydryl compound scavenges oxygen, to form a stable polymerized hemoglobin blood-substitute, which is then packaged and stored in an atmosphere substantially free of oxygen.

The invention relates to a method for preserving the stability of a hemoglobin blood substitute comprising maintaining the hemoglobin blood substitute in an atmosphere substantially free of oxygen. The invention also involves a method for producing a stable polymerized hemoglobin blood-substitute from blood. The method of this invention includes mixing blood with an anticoagulent to form a blood solution, washing the red blood cells in the blood solution and then separating the washed red blood cells from the white blood cells. This method also includes disrupting the red blood cells to release hemoglobin and form a hemoglobin solution, which is then treated by high performance liquid chromatography to form a hemoglobin eluate. The hemoglobin eluate is then deoxygenated, contacted with a first sulfhydryl compound to form an oxidation-stabilized deoxygenated hemoglobin solution, and mixed with with a cross-linking agent to form a polymerization reaction mixture, which is then polymerized. The polymerized hemoglobin solution is then diafiltered with a physiologic solution and with a sulfhydryl compound, whereby the polymerized hemoglobin solution is made physiologically acceptable, and whereby the sulfhydryl compound scavanges oxygen, to form a stable polymerized hemoglobin blood-substitute, which is then packaged and stored in an atmosphere substantially free of oxygen.

Method for preserving a hemoglobin blood substitute

A method for producing a stable polymerized hemoglobin blood-substitute from blood. The method of this invention includes mixing blood with an anticoagulant to form a blood solution, washing the red blood cells in the blood solution and then separating the washed red blood cells from the white blood cells. This method also includes disrupting the red blood cells to release hemoglobin and form a hemoglobin solution, which is then treated by high performance liquid chromatography to form a hemoglobin eluate. The hemoglobin eluate is then deoxygenated, contacted with a first sulfhydryl compound to form an oxidation-stabilized deoxygenated hemoglobin solution, and mixed with a cross-linking agent to form a polymerization reaction mixture, which is then polymerized. The polymerized hemoglobin solution is then diafiltered with a physiologic solution and with a sulfhydryl compound, whereby the polymerized hemoglobin solution is made physiologically acceptable, and whereby the sulfhydryl compound scavenges oxygen, to form a stable polymerized hemoglobin blood-substitute.

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Natick, MA, United States of America

William Richard Light