New factor VIII coagulant polypeptides

Claim 2

A factor VIII:C coagulant containing one or more polypeptides characterized in that: (i) one or more of the polypeptides exhibit a band at a point corresponding to an M of about 92,000; or bands at points corresponding to M values of about 92,000, about 80,000, and about 79,000; of about 92,000, about 72,000, and about 71,000; or of about 92,000, about 80,000, about 79,000, about 72,000, and about 71,000; when subjected to sodium dodecyl sulfate- polyacrylamide gel electrophoresis; (ii) the coagulent exhibits specific coagulant activity higher than 1800 Units/mg; (iii) the coagulant exhibits the activity in step (ii) over a continuous period of at least about 10 minutes; and (iv) the coagulant exhibits human factor VIII:C antibody binding.capacity.

Claim 3

The coagulant of claim 1 wherein in part (ii) the specific activity is more than 5400 Units/mg.

Claim 4

The coagulant of claim 1 or 2 further characterized in that it is free from α-thrombin and equivalent proteases in active form.

Claim 5

The coagulant of any of claims 1 to 3 wherein the coagulant is prepared by the digestion of human factor VIII:C by α-thrombin or an equivalent protease.

Claim 6

The coagulant of any of claims 1 to 4 wherein the coagulant is essentially free of polypeptides exhibiting bands at M r above 92,000.

Claim 7

A biological preparation containing a factor VIII:C coagulant of any of claims 1 to 5 in an amount of about at least 1% by weight.

Claim 8

A biological preparation of claim 6 containing from 30 to 95% by weight of the coagulant.

Claim 9

A biological preparation of claim 6 or 7 containing from 60 to 90% by weight.

Claim 10

A biological preparation of any of claims 6 to 8 dissolved in a pharmaceutically acceptable saline solution.

Claim 11

The method of making a factor VIII:C coagulant, comprising the steps of (a) digesting human factor VIII:C in a digestion mixture with an effective amount of a protease under digestion conditions to form the coagulant of claim 1; (b) discontinuing the digestion of step (a) while the coagulant is still present in the digestion mixture; and (c) recovering the resultant coagulant.

Claim 12

The process of claim 10 wherein the human factor VIII:C has previously been purified.

Claim 13

A process for recovering the coagulant of claim 1 characterized by adsorbing the coagulant in active form to an immunoadsorbent containing a plurality of monoclonal antibodies to human factor VIII:C epitopes and eluting the coagulant from the immunoadsorbent with an aqueous solution containing calcium ions under elution conditions effective to desorb the coagulant from the immunoadsorbent without denaturing the coagulant.

Claim 14

The process of Claim 12 wherein the solution contains u p to about 1.0 M of calcium ion and the solution is applied to the imminoadsorbent at up to about 8 bed volumes per hour.

Claim 15

A process according to Claim 12 or 13 wherein the monoclonal antibodies are of class IgG and are produced by a hybridoma formed by fusion of cells from a mouse myeloma and spleen cells from a mouse previously immunized with human factor VIII:C, the antibodies react with human factor VIII:C and exhibit one of the following reaction profiles with coagulant derived from human factor VIII:C: (A) reacts with those polypeptides so derived of M r =92,000; M r =180,000 and greater; and 54,000; and does not. react with those polypeptides so derived of M =44,000; 71,000; 72,000; 79,000; nor 80,000; (B) reacts with those polypeptides so derived of M r =92,000; M =108,000 and greater; and 44,000; and does not react with those polypeptides so derived of M r =54,000; 71,000; 72,000; 79,000; nor 80,000; (C) reacts with those polypeptides so derived of M r =79,000 and 80,000; and M =108,000 and greater; and does not react with those polypeptides so derived of M =44,000; 54,000, 71,000; 72,000; nor 92,000; (D) reacts with those polypeptides so derived of M r =108,000 and higher; and does not react with those polypeptides so derived of M r less than 108,000; wherein the M r values are determined by subjecting the polypeptides to sodium dodecyl sulfate- polyacrylamide gel electrophoresis.

Claim 16

A monoclonal antibody of class IgG produced by a hybridoma formed by fusion of cells from a mouse myeloma and spleen cells from a mouse previously immunized with human factor VIII:C, which antibody reacts with human factor VIII:C and exhibits one of the following reaction profiles with polypeptides derived from human factor VIII:C: (A) reacts with those polypeptides so derived of M r =92,444; M r =180,000 and greater; and 54,000; and does not react with those polypeptides so derived of M =44,000; 71,000; 72,000; 79,000; nor 80,000; (B) reacts with those polypeptides so derived of Mr =92,000; M r =108,000 and greater; and 44,000; and does not react with those polypeptides so derived of M r =54,000; 71,000; 72,000; 79,000; nor 80,000; (C) reacts with those polypeptides so derived of M =79,000 and 80,000; and M =108,000 and greater; and does not react with those polypeptides so derived of M r =44,000; 54,000, 71,000; 72,000; nor 92,000; (D) reacts with those polypeptides so derived of M =108,000 and higher; and does not react with those polypeptides so derived of M r less than 108,000; wherein the M r values are determined by subjecting the polypeptides to soduim dodecyl sulfate- polyacrylamide gel electrophoresis.

Claim 17

A monoclonal antibody of class IgG produced by a hybridoma formed by fusion of cells from a mouse myeloma and spleen cells from a mouse previously immunized with human factor VIII:C, which antibody reacts with human factor VIII:C and exhibits one of the following reaction profiles with polypeptides derived from human factor VIII:C: (A) reacts with those polypeptides so derived of M r =92,444; M r =180,000 and greater; and 54,000; and does not react with those polypeptides so derived of M =44,000; 71,000; 72,000; 79,000; nor 80,000; (B) reacts with those polypeptides so derived of Mr =92,000; M r =108,000 and greater; and 44,000; and does not react with those polypeptides so derived of M r =54,000; 71,000; 72,000; 79,000; nor 80,000; (C) reacts with those polypeptides so derived of M =79,000 and 80,000; and M =108,000 and greater; and does not react with those polypeptides so derived of M r =44,000; 54,000, 71,000; 72,000; nor 92,000; (D) reacts with those polypeptides so derived of M =108,000 and higher; and does not react with those polypeptides so derived of M r less than 108,000; wherein the M r values are determined by subjecting the polypeptides to soduim dodecyl sulfate- polyacrylamide gel electrophoresis.

Claim 18

A monoclonal antibody according to claim 15 having profile (B) or (C) recited therein, further characterized in that it neutralizes the coagulant activity of human factor VIII:C when said factor VIII:C is bound to the antibody.

Claim 19

A biological preparation comprising an antibody exhibiting (a) reaction profile A of claim 15 which is essentially free from antibodies exhibiting the other three of said profiles, or (b) reaction profile B of claim 15 which is essentially free from antibodies exhibiting the other three of said profiles or (c) reaction profile C of claim 15 which is essentially free from antibodies exhibiting the other three of said profiles or (d) reaction profile D of claim 15 which is essentially free from antibodies exhibiting the other three of said profiles.

Claim 20

A biological preparation comprising a mixture of antibodies according to claim 15 which exhibit reaction profiles A and B, which is essentially free from antibodies according to claim 15 which exhibit reaction profiles C and,D.

Claim 21

A biological preparation comprising a mixture of antibodies according to claim 15 which exhibit reaction profiles A, B and C, which is essentially free from antibodies according to claim 15 which exhibit reaction profile D.

Claim 22

An immunoadsorbent column comprising particles of a solid immunologically inert adsorbent material bound to an antibody exhibiting profile (A), (B), (C), or ( D ) of claim 15.

Claim 23

A column according to claim 20 wherein the adsorbent material is agarose.