Chemistry of carbon compounds – Miscellaneous organic carbon compounds – C-metal
Patent
1983-12-21
1985-10-22
Schain, Howard E.
Chemistry of carbon compounds
Miscellaneous organic carbon compounds
C-metal
424101, 514 8, 514 21, C07G 700, A61K 3514
Patent
active
RE0320110
DESCRIPTION:
BRIEF SUMMARY
10-20 units of VIII:C per ml of eluant are obtained with the present invention, in contract to 0.5-1.25 units per ml of eluant with the Tuddenham et al method.
The present method also permits the selection of a monoclonal antibody having a high affinity for VIII:RP; however, the use of polyclonal antibodies results in varying affinities. It should be realized that there is an indirect relationship between the affinity of the bound antibody for VIII:RP and the elution of VIII:RP. Thus, the higher the affinity of the antibody for VIII:RP, the less VIII:RP will be present with VIII:C in the eluant. The present invention also makes it possible to produce an unlimited supply of the specified monoclonal antibody, thus eliminating variations among different batches.
Although Austen, as earlier described, has reported the use of aminohexyl-agarose to separate VIII:C from VIII:RP, such a material has not heretofore been used to concentrate VIII:C following a separation and purification step. Heretofore, the highest VIII:C concentrations achieved by using aminohexyl agarose in chromatography were 0.53 units per ml of eluant for human protein and 2.38 per ml of eluant for porcine VIII:C. The present method permits concentrations several orders of magnitude greater than these. Perhaps of even greater significance, is the fact that the present invention provides for a greater purification of human VIII:C than has ever been reported (164,000 vs 17,000 fold over plasma). The present method, which is described in more detail hereinafter, yields VIII:C with a specific activity of 2,300 units/mg when commercial concentrate is used. This corresponds to a 164,000 fold purification from plasma. The ratio of VIII:C to VIII:RP is greater than 10.sup.5 as compared to the ratio in plasma.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The following description provides details of the manner in which the embodiments of the present invention may be made and used in order to achieve the separation, purification and concentration of VIII:C to a degree of purity and concentration not known heretofore. This description, while exemplary of the present invention, is not to be construed as specifically limiting the invention and such variations which would be within the purview of one skilled in this art are to be considered to fall within the scope of this invention.
A. Preparation of Monoclonal Antibody to VIII:RP
The monoclonal antibody to VIII:RP which is subsequently bound to the separation substrate may be prepared in a stepwise procedure starting with a highly purified preparation of factor VIII/von Willebrand factor (VIII:C/VIII:RP complex). The purification for immunization is accomplished with material obtained from a plasma source. Less highly purified material for coating polyvinyl plates is obtained in higher concentration from commercial extracts such as FACTORATE (trademark of Armour Pharmaceutical Co., Tuckahoe, N.Y.) or Hemophil (trademark of Hyland Laboratories, Costa Mesa, California). Purification is performed by a standard agarose-gel filtration of cryoprecipitate, such as that described by Zimmerman and Roberts, "Factor VIII Related Antigen", appearing in IMMUNOASSAYS: CLINICAL LABORATORY TECHNIQUES FOR THE 1980's, R. M. Nakamura et al, eds., Alan R. Liss, Inc., New York, pp. 339-349 (1980). Mice were injected with highly purified factor VIII/von Willebrand factor obtained from plasma according to the following procedure. On day zero, the mice are injected intraperitoneally with a composition prepared by dissolving (or suspending) 10 Mg of the protein in 0.1 ml of buffer containing 0.05 M Tris, 0.15 M sodium chloride, 0.02 % sodium azide, 1 mM phenyl methyl sulfonyl fluoride, traysylol 10 units/ml at pH7.3. and shaking with an equal volume of complete Freund's adjuvant. On day 14, the mice are again injected with the same material except that incomplete Freund's adjuvant is substituted for complete Freund's adjuvant. On day 21, the injection of day 14 is repeated. On day 38, the mice are injected with purified VIII:C/
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Fulcher Carol A.
Zimmerman Theodore S.
Schain Howard E.
Scripps Clinic and Research Foundation
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