Biopure Corporation

Maria S Gawryl

William Richard Light

Robert A Houtchens

Anthony J Laccetti

Carl W Rausch

Javed Baqai

Curtis E Wertz

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

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.

Method for producing a stable polymerized hemoglobin blood-substitute

A method for separating unmodified hemoglobin from cross-linked hemoglobin in a hemoglobin solution. The method involves contacting the hemoglobin solution with a least one dissociating agent to form a dissociation solution wherein unmodified tetrameric hemoglobin is dissociated to form hemoglobin dimers. The hemoglobin dimers are then separated from the dissociation solution, while retaining the cross-linked hemoglobin in the dissociation solution.

Method for separating unmodified hemoglobin from cross-linked hemoglobin

The present invention relates to a composition of matter comprising a stable polymerized hemoglobin solution, useful for forming blood-substitutes, and to a method for forming said stable polymerized hemoglobin solution. The stable polymerized hemoglobin solution, and derived blood-substitutes, of this invention comprise polymerized hemoglobin and a sulfhydryl compound, both in solution, wherein the sulfhydryl compound stabilizes the polymerized hemoglobin. The method of this invention comprises deoxygenating hemoglobin in a hemoglobin solution and then mixing the deoxygenated hemoglobin with a sulfhydryl compound to form an oxidation-stabilized, deoxygenated hemoglobin solution. Subsequently, the oxidation-stabilized deoxygenated hemoglobin solution is mixed with a cross-linking agent to form a polymerization reaction mixture, which is then polymerized to form a stable polymerized hemoglobin solution.

Stable polymerized hemoglobin and use thereof

The present invention relates to a method of therapeutically, or prophylactically, treating a vertebrate to increase tissue oxygenation, or maintain issue oxygenation, in tissue of a vertebrate wherein the tissue has a reduced red blood cell flow, and wherein the vertebrate has a normovolemic blood volume and at least a normal systemic vascular resistance. The method comprises introducing into the circulatory system of the vertebrate at least one dose of hemoglobin.

Method for producing ultrapure stable polmerized hemoglobin

A method for removing a prion from a solution comprising the prion and at least one additional biomolecule, comprising directing the solution through an anion-exchange chromatography column under conditions that cause a gradient elution, whereby the prion is separated from at least one of the biomolecules, thereby causing said biomolecule to be collected in an eluate fraction that is distinct from an eluate fraction that includes the prion. In one embodiment, the gradient is a pH gradient, for example, a step gradient. The prion can be a causal agent for a spongiform encephalopathy, such as Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinken syndrome, scrapie, or bovine spongiform encephalopathy.

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East Boston, MA, United States of America

Maria S Gawryl