Lyophilized and reconstituted blood platelet compositions

• US 5,213,814 A
• Filed: 05/17/1990
• Issued: 05/25/1993
• Est. Priority Date: 04/10/1989
• Status: Expired due to Fees

• Current Assignee: COBE Laboratories Incorporated (Terumo Corporation)
• Original Assignee: Cryopharm Corp.
• Inventors: Wong, Victoria A., Goodrich, Raymond P. Jr.
• Primary Examiner(s): Rosen, Sam
• Application Number:
  US07/525,392
• Time in Patent Office: 1,104 Days
• Field of Search: 424/532, 435/2
• US Class Current: 424/532
• CPC Class Codes: A01N 1/02 Preservation of living parts
• CPC Class Codes: A01N 1/0215 Disinfecting agents, e.g. antimicrobials for preserving living parts
• CPC Class Codes: A01N 1/0221 Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
• CPC Class Codes: A61K 35/19 Platelets; Megacaryocytes

Abstract

A process and medium are disclosed for the lyophilization of cells, specifically platelets, and cell-like matter, which comprises the use of solutions including monosaccharide hexoses and pentoses, and biocompatible amphipathic polymers to permit the reconstitution of transfusably useful cells, specifically platelets, and cell-like matter.

Claims

1. A lyophilized platelet-containing composition containing about 3% by weight or less of moisture prepared by lyophilization of an aqueous suspension of platelets 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 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 1K to 600K.

2. A composition according to claim 1 wherein said polymers are amphipathic.

3. A composition according to claim 1 wherein said polymers have a molecular weight in the range of about 2.5K to 360K.

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

5. A composition according to claim 4 wherein said monosaccharide comprises glucose.

6. A composition according to claim 3 wherein said polymer is selected from the group consisting of polyvinylpyrrolidone, hydroxyethyl starch, dextran and mixtures thereof.

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

8. A composition according to claim 7 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.

9. A composition according to claim 7 wherein said polyvinylpyrrolidone has an average molecular weight of about 24K.

10. A composition according to claim 7 wherein said polyvinylpyrolidone has an average molecular weight of about 360K.

11. A composition according to claim 9 or 10 wherein said composition comprises a mixture of polyvinylpyrrolidone and hydroxyethyl starch.

12. A composition according to claim 11 wherein said hydroxyethyl starch has an average molecular weight of about 500K.

13. A process of reconstituting lyophilized platelets to a transfusably-useful state, wherein said lyophilized platelets 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 1K to 360K, to form platelets in a transfusably-useful state.

14. A process according to claim 13 wherein said polymers are amphipathic.

15. A process according to claim 13 wherein said polymers have a molecular weight in the range of about 2.5K to 360K.

16. A process according to claim 13, 14 or 15 further comprising the steps of:

separating said platelets from said reconstitution solution by centrifugation and washing said platelets by at least one wash cycle by resuspending said platelets in a dextrose-saline buffer solution at a pH in the range of about 7.0-7.4 and separating said platelets from said buffer solution by centrifugation.

17. A transfusably useful platelet-containing composition made according to the process of claim 13.

18. A transfusably useful platelet-containing composition made according to the process of claim 16.

19. A process of reconstituting a lyophilized composition comprising platelets comprising the step of contacting said platelets 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 1K to about 600K which is present in a final concentration in the range of 10 to 30% by weight.

20. A process according to claim 19 wherein said polymers are amphipathic.

21. A process according to claim 19 where said polymers have a molecular weight in the range of about 2.5K to 360K.

22. A process according to claim 19, 20 or 21 wherein said polymer is selected from the group consisting of polyvinylpyrrolidone, hydroxyethyl starch, dextran and mixtures thereof.

23. A process according to claim 20 wherein said polymer comprises polyvinylpyrrolidone of average molecular weight of about 10K.

24. A process according to claim 19, 20 or 21 wherein said solution further comprises a monosaccharide in a final concentration of about 7.0 to 37.5% by weight.

25. A transfusably-useful platelet composition reconstituted from a dried state comprising

platelets characterized by a reconstituted recovery of at least about 20% by weight, based on the number of the platelets prior to drying;

which aggregate about 25-80% upon stimulation with ADP and are capable of phagocytosis;

and further comprising a polymer or mixture of polymers having a molecular weight of from about 1K to about 600K in a concentration of about 10 to 30% by weight, wherein said polymer is selected from the group consisting of polyvinylpyrrolidone, hydroxyethyl starch, dextran and mixtures thereof.

26. A composition according to claim 25 wherein said polymer comprises polyvinylpyrrolidone of average molecular weight of about 10K.

27. A composition according to claim 25 wherein said solution further comprises a monosaccharide in a final concentration of about 7.0 to 37.5% by weight.

28. A process for the lyophilization of platelets comprising:

immersing a plurality of platelets in a buffered solution which includes;

a monosaccharide which is present in the solution in a concentration of from about 7.0 to 37.5%, and

a polymer, or a mixture of polymers, each of said polymers having a number average molecular weight in the range of about 1K to about 600K, wherein the total concentration of said polymers is of from about 0.7% up to saturation in the solution;

freezing the solution; and

drying the cells by sublimation of the water.

29. The process of claim 28 wherein said polymers are amphipathic.

30. The process of claim 28 wherein one of said polymers has a molecular weight in the range of about 2.5K to about 360K.

31. The process of claim 28 wherein the monosaccharide is selected from the group consisting of xylose, glucose, ribose, mannose and fructose.

32. The process of claim 28 wherein said mixture of polymers comprises polyvinylpyrrolidone and hydroxyethyl starch.

33. The process of claim 28 wherein said polymer comprises polyvinylpyrrolidone.

34. The process of claim 32 or 33 wherein said polyvinylpyrrolidone has an average molecular weight of about 24K.

35. The process of claim 32 or 33 wherein said polyvinylpyrrolidone has an average molecular weight of about 360K.

36. The process according to claim 32 wherein said hydroxyethyl starch has an average molecular weight of about 500K.