Changes in Factor VIII Complex Activities During the Production of a Clinical Intermediate Purity Factor VIII Concentrate

Abstract

SummarySamples taken at various stages of preparation of intermediate purity factor VIII concentrate were assayed for factor VIII coagulant activity (VIII:C), factor VIII coagulant antigen (VIIIC: Ag) and factor VIII related antigen (VIIIR: Ag). The antigen results were used to assess mechanical loss during fractionation as these markers are relatively stable. In contrast VIII:C is sensitive to both mechanical and inactivation losses.The major loss of factor VIII occurred during the cryoprecipitation and extraction step and was due to both mechanical loss and inactivation. Losses before and after this step were largely due to inactivation of the factor VIII.Assay of VIIIR: Ag in concentrates presented problems and a modified technique is suggested.

Studies on the Stability of VIII:C during the Manufacture of a Factor VIII Concentrate for Clinical Use

“…Results from the preliminary study suggested that the loss of VIII:C activity previously observed over product finishing [4] might be almost fully explained by a citrateinduced inactivation of VIII:C. Similar inactivation behaviour had been observed in plasma [13,14] with one suggestion being that the presence of calcium ions is essential for VIILC stability. Therefore a number of for mulations were examined in which the concentration of ionised calcium was controlled by the addition of calcium chloride to factor VIII solutions at different citrate con centrations.…”

“…There have been few reports investigating the points of VIIFC loss during routine manufacturing pro cesses: however, the fact that significant loss of VIIFC can occur over the sterile filtration or finishing procedures has been appreciated for some time [21][22][23]. This was believed to be largely due to the retention of factor VIII on the filter itself, but the development of immunoassays lead instead to the suggestion that some form of inacti vation may provide the predominant mechanism of loss [4]. Previous studies concerning VIIFC inactivation dur ing processing have tended to concentrate on various types of proteolytic degradation [24], whereas our prelim inary stability study drew attention to the importance of product formulation and especially to the role of citrate.…”

“…The patient was a severely affected haemovWf:Ag was assayed by the standard immunoelectrophoretic method as previously described [4] and factor VIII polypeptide solution, required up to 2-3 h to complete in the full-scale process.…”

“…Previously [4], we concluded that finishing losses com prised a 1 % loss by adsorption onto the membrane filter, a 4% volume loss and a 17% loss due to inactivation of VIII:C. To pursue this latter finding we have examined the stability of VIII:C at key points in the manufacturing 1…”

“…In an earlier study concerning factor VIII yield [4] we monitored a routine manufacturing process over a 2-year period, using both clotting and immunological assays, to help to define points of VIII:C loss and the mechanisms involved. While cryoprecipitation was confirmed as the principal point of loss, a further yield loss of almost 22% VIII:C was noted over the process-finishing steps (i.e.…”