Background: Therapeutic products with coagulation factor VIII (FVIII) have wide ranges of specific activity (IU/mg), indicating the presence of significant amounts of protein with altered structure and properties. In particular, the products with recombinant FVIII (rFVIII) contain a protein fraction (FVIII*) with reduced activity and inability to bind von Willebrand factor (vWF) (Lin et al, 2004; Ofosu et al 2012). As the ability to bind vWF is important for reduction of FVIII immunogenicity (Hartholt et al, 2017), FVIII* may contribute to the development of FVIII inhibitors. Thus, it represents a product-related impurity and risk factor for product efficacy.

Aims: To develop a method to separate the FVIII* fraction from FVIII preparations and characterize this protein in various rFVIII products.

Methods: Chromatography of FVIII samples on immobilized vWF and testing the fractions by PAGE with Coomassie/silver-staining/Western blotting and chromogenic FVIII activity assay.

Results: The conditions of chromatography assay were elaborated and proven to be robust for 55 column cycles. Ten rFVIII products available in the U.S. including those with extended half-life were tested. The protein unable to bind vWF (FVIII*) was found in all products at level of 0.5-23% (Figure 1) and activity 0.4-17% compared to starting samples. In some products, the band pattern of FVIII* by PAGE differed from that of protein bound to the column (Figure 2).

Conclusions: We developed a robust method to separate the FVIII* fraction from FVIII samples. The applicability of the method was demonstrated by characterization of ten rFVIII products for the content and molecular weight distribution of FVIII* provided better knowledge of these products. The method can be useful to achieve higher purity and efficacy of FVIII products for the care of Hemophilia A. The study was funded by FDA. This is an informal communication and it represents the authors’ own best judgment. These comments do not bind or obligate FDA.

[Figure 1]

[Figure 2]

« Back to ISTH 2020 Congress

ISTH Congress Abstracts - https://abstracts.isth.org/abstract/evaluation-of-structurally-altered-protein-in-factor-viii-therapeutic-products-using-chromatography-on-immobilized-von-willebrand-factor/