Identification of aggregates in therapeutic formulations of recombinant full‐length factor VIII products by sedimentation velocity analytical ultracentrifugation

Essentials Factor VIII inhibitors are the most serious complication in patients with hemophilia A. Aggregates in biopharmaceutical products are an immunogenic risk factor. Aggregates were identified in recombinant full-length factor VIII products. Aggregates in recombinant factor VIII products are identified by analytical ultracentrifugation. SUMMARY Background The development of inhibitory anti-factor VIII antibodies is the most serious complication in the management of patients with hemophilia A. Studies have suggested that recombinant full-length FVIII is more immunogenic than plasma-derived FVIII, and that, among recombinant FVIII products, Kogenate is more immunogenic than Advate. Aggregates in biopharmaceutical products are considered a risk factor for the development of anti-drug antibodies. Objective To evaluate recombinant full-length FVIII products for the presence of aggregates. Methods Advate, Helixate and Kogenate were reconstituted to their therapeutic formulations, and subjected to sedimentation velocity (SV) analytical ultracentrifugation (AUC). Additionally, Advate and Kogenate were concentrated and subjected to buffer exchange by ultrafiltration to remove viscous cosolvents for the purpose of measuring s20,w values and molecular weights. Results The major component of all three products was a population of ~7.5 S heterodimers with a weight-average molecular weight of ~230 kDa. Helixate and Kogenate contained aggregates ranging from 12 S to at least 100 S, representing ≈ 20% of the protein mass. Aggregates greater than 12 S represented < 3% of the protein mass in Advate. An approximately 10.5 S aggregate, possibly representing a dimer of heterodimers, was identified in buffer-exchanged Advate and Kogenate. SV AUC analysis of a plasma-derived FVIII product was confounded by the presence of von Willebrand factor in molar excess over FVIII. Conclusions Aggregate formation has been identified in recombinant full-length FVIII products, and is more extensive in Helixate and Kogenate than in Advate. SV AUC is an important method for characterizing FVIII products.