Lyophilized and reconstituted red blood cell and hemosome compositions

• US 5,043,261 A
• Filed: 06/02/1989
• Issued: 08/27/1991
• Est. Priority Date: 04/10/1989
• Status: Expired due to Fees

• Current Assignee: Cobe Cardiovascular Incorporated (LivaNova Plc)
• Original Assignee: Cryopharm Corp.
• Inventors: Goodrich, Raymond, Williams, Christine
• Primary Examiner(s): Rosen, Sam
• Application Number:
  US07/360,386
• Time in Patent Office: 816 Days
• Field of Search: 435/2, 424/532, 424/533
• US Class Current: 435/2
• CPC Class Codes: A61K 35/18 Erythrocytes haemoglobin A61K38/42

Abstract

A process and medium are disclosed for the lyophilization of red blood cells which comprises the use of solutions including monosaccharide hexoses and pentoses, and biocompatible polymers to permit the reconstitution of viable red blood cells.

Claims

1. A lyophilized erythrocyte- and/or hemosome-containing composition containing about 3% by weight or less of moisture prepared by lyophilization of an aqueous suspension of erythrocytes in a phosphate-buffered saline solution at a pH in the range of 7.0 to 7.4 comprising a final concentration of about 7 to 37.5% by weight of a monosaccharide or mixture of monosaccharides capable of permeating the membranes of erythrocytes, and a final concentration of about 0.7 by weight percent up to the saturation point of the solution of a polymer or mixture of polymers having a molecular weight in the range of about 2.5K to 360K.

2. A composition according to claim 1 wherein said monosaccharide is selected from the group consisting of xylose, glucose, ribose, mannose and fructose.

3. A composition according to claim 2 wherein said monosaccharide comprises glucose.

4. A composition according to claim 3 wherein the final glucose concentration in said solution is about 26% by weight.

5. A composition according to claim 1 wherein said polymer is selected from the group consisting of polyvinylpyrrolidone, hydroxyethyl starch and dextran.

6. A composition according to claim 5 wherein said polymer comprises polyvinylpyrrolidone.

7. A composition according to claim 6 wherein the final concentration of said polyvinylpyrrolidone in said solution is in the range of 12 to 20% by weight and has an average molecular weight of 10-40K.

8. A composition according to claim 7 wherein said solution comprises about 14.4% by weight of polyvinylpyrrolidone.

9. A process for reconstituting lyophilized erythrocytes and/or hemosomes to a transfusably-useful state, wherein said lyophilized erythrocytes or hemosomes consist essentially of a composition according to claim 1, comprising the step of:

mixing said composition with a sufficient volume of a phosphate-buffered saline reconstitution solution having a pH in the range of about 7.0-7.4 at a temperature in the range of about 15°

-50°

C., said reconstitution solution comprising a final concentration of about 0.7% by weight up to the saturation concentration of a polymer or mixture of polymers having a molecular weight in the range of about 2.5K to 360K, to form erythrocytes in a transfusably-useful state characterized by 80-85% hemoglobin recovery compared to the pre-lyophilized state, and normal erythrocyte morphology.

10. A process according to claim 9 further comprising the steps of:

separating said erythrocytes from said reconstitution solution by centrifugation and washing said erythrocytes by at least one wash cycle by resuspending said erythrocytes in a washing buffer solution comprising phosphate buffer, 16% weight by volume of said polymer at pH in the range of about 7.0-7.4 and separating said erythrocytes from said washing buffer solution by centrifugation.

11. A process according to claim 10 further comprising the steps of resuspending and incubating for approximately one hour at 37°

C. said erythrocytes in said washing buffer containing an additional 10% vol/vol of a glycolytic intermediary solution comprising phosphate buffer, calcium chloride, magnesium chloride, ADP, NAD, adenine, adenosine and inosine at pH of about 7.0-7.4 and separating said erythrocytes from said washing buffer and glycolytic intermediary solution by centrifugation; and

resuspending and incubating said erythrocytes at about 20°

-40°

C. for less than about 10 minutes in an iso-osmotic solution comprising a final concentration of about 0.1 to 10% weight by volume of a polyanion selected from the group consisting of inositol hexaphosphate, adenosine triphosphate, pyrophosphate, and tripolyphosphate, 0.1 to 5.0% weight by volume of a 3-5KD polyethylene glycol at a pH in the range of about 7.0-7.2 at a dilution factor of 10-50;

separating said erythrocytes from said isoosmotic solution by centrifugation;

then incubating said erythrocytes at about 37°

C. for about 30 minutes;

resuspending said erythrocytes in a phosphate buffered saline or dextrose-saline solution, and separating said erythrocytes by centrifugation.

12. A method according to claim 11 further comprising the step of resuspending said erythrocytes in a solution comprising phosphate buffered solution or dextrose-saline solution and a monosaccharide at a temperature of about 20°

-40°

C., and separating said erythrocytes by centrifugation.

13. A transfusably useful erythrocyte-containing composition made according to the process of any of claims 9 through 12.

14. A process according to claim 11 wherein said polyanion comprises inositol hexaphosphate.

15. A composition according to any of claims 1 through 8 comprising hemosomes.

16. A composition according to any of claims 1 through 8 comprising erythrocytes.

18. A process according to claim 14 wherein said polymer is selected from the group consisting of polyvinylpyrrolidone, hydroxyethyl starch and dextran.

22. A process according to any of claims 9-12 or 17-18 wherein said composition comprises hemosomes.

23. A process according to any of claims 9-12 or 17-18 wherein said composition comprition comprises erythrocytes.

17. A process for reconstituting a lyophilized composition comprising erythrocytes and/or hemosomes comprising the step of contacting said erythrocytes and/or hemosomes at a temperature greater than about 17°

C. with an aqueous solution of a polymer or mixture of polymers having a molecular weight of from about 2.5K to about 360K which is present in a final concentration in the range of 12 to 30% by weight.

19. A process according to claim 17 wherein said polymer comprises polyvinylpyrrolidone of average molecular weight of 40K.

20. A process according to claim 19 wherein said polyvinylpyrrolidone is present in said solution in a final concentration of about 14.4% by weight.

21. A process according to claim 17 wherein said solution further comprises a monosaccharide in a final concentration of about 7.0 to 37.5% by weight.