Ultrapurification of factor VIII using monoclonal antibodies

Abstract

Description

Claims

1. An improved method of preparing factor viii procoagulant activity protein comprising the steps of

(a) adsorbing a viii:C/viii:RP complex from a plasma or commercial concentrate source onto particles bound to a monoclonal antibody specific to viii:RP,

(b) eluting the viii:C,

(c) adsorbing the viii:C obtained in step (b) in another adsorption to concentrate and further purify same,

(d) eluting the adsorbed viii:C, and

(e) recovering highly purified and concentrated viii:C.

2. A method according to claim 1, wherein the elutant used in each of steps (b) and (d) is a saline solution.

3. The method according to claim 2, wherein the saline solution is calcium chloride.

4. The method according to claim 3, wherein the concentration of said calcium chloride solution used in steps (b) and (d) ranges from about 0.25 M to about 0.5 M.

5. The method according to claim 1, wherein said adsorbent particles in step (a) are agarose.

6. The method according to claim 1, wherein aminohexyl agarose is employed in step (c) as the adsorbent.

7. The method according to claim 6, wherein calcium chloride solution is employed as the elutant in steps (b) and (d), concentration of said calcium chloride solution ranging from about 0.25 M to about 0.5 M in step (b) and from about 0.25 M to about 0.5 M in step (d).

8. An improved immunoadsorbent for isolation and purification of viii:C from viii:C/viii:RP comprising a monoclonal antibody specific to viii:RP bound to solid particles.

9. The improved immunoadsorbent of claim 8, wherein said solid particles comprise a resin.

10. The improved immunoadsorbent of claim 9, wherein said resin comprises agarose.

11. The improved immunoadsorbent of claim 10, wherein said agarose is cross-linked agarose.

12. The improved immunoadsorbent of claim 11, wherein said immunoadsorbent has a coupling density of 3 to 4 g of monoclonal antibody per liter of agarose.

13. Highly purified and concentrated human or porcine viii:C prepared in accordance with the method of claim 1.

14. Highly purified and concentrated human or porcine viii:C prepared in accordance with the method of claim 6.

15. In a method for purifying factor viii procoagulant activity protein from plasma or concentrate, the improvement comprising the step of passing said plasma or concentrate through a chromatographic type column having adsorbent to which is bound monoclonal antibodies which is specific to viii:RP and eluting the viii-C therefrom.

16. The method according to claim 15, wherein said adsorbent is agarose and said elutant is a saline solution.

17. A viii:C of claim 13, wherein said viii:C is human viii:C. 18. A viii:C of claim 14, wherein said viii:C is human viii:C. 19. An improved immunoadsorbent of claim 8 wherein said monoclonal antibody remains bound to viii:RP during elution of viii:C. 20. An improved immunoadsorbent of claim 19, wherein said elution is carried out with a saline solution. 21. An improved immunoadsorbent of claim 20 wherein said solution contains calcium ions. 22. An improved immunoadsorbent of claim 21, wherein said solution is calcium chloride. 23. An improved immunoadsorbent of claim 22, wherein the concentration of said calcium chloride is from 0.25 M to about 0.5 M. 24. A human viii:C preparation having a potency in the range of 134 to 1172 units per ml, and

being substantially free of viii:RP. 25. A human viii:C preparation of claim 24, wherein the viii:C concentration is at least 160,000 fold purified relative to viii:C in plasma. 26. A human viii:C preparation of claim 24, wherein the ratio of viii:C to viii:RP is greater than 100,000 times the ratio in plasma. 27. A human viii:C preparation of claim 24, wherein said viii:C is isolated from viii:C/viii:RP and 90-100 percent of the viii:RP has been

removed. 28. A human viii:C preparation having a specific activity greater than 2240 units/mg. 29. A human viii:C preparation of claim 28 wherein the potency is in the range of 134 to 1172 units/ml. 30. An improved method of preparing factor viii procoagulant activity protein comprising the steps of

(a) adsorbing a viii:C/viii:RP complex from a plasma or commercial concentrate source onto a substrate to which is bound a monoclonal antibody specific to viii:RP,

(b) eluting the viii:C,

(c) adsorbing the viii:C obtained in step (b) in another adsorption to concentrate and further purify same,

(d) eluting the adsorbed viii:C, and

(e) recovering highly purified and concentrated viii:C.

A method according to claim 30, wherein said substrate is a resin. 32. A method according to claim 30, wherein said substrate is comprised of particles. 33. An improved immunoadsorbent for isolation and purification of viii:C from viii:C/viii:RP comprising a monoclonal antibody specific to viii:RP bound to a substrate. 34. Highly purified and concentrated human or porcine viii:C prepared in accordance with the method of claim 30. 35. In a method for purifying factor viii procoagulant activity protein from plasma or concentrate, the improvement comprising the step of passing said plasma or concentrate over a substrate to which is bound monoclonal antibodies which are specific to viii:RP and eluting the viii:C therefrom. 36. The method according to claim 35, wherein said substrate is agarose and said elutant is a saline solution.