Sedimentation Velocity Analytical Ultracentrifugation of Oxidized Recombinant Full-Length Factor VIII.

Philip M Zakas,David Lillicrap,Pete Lollar,John F Healey,Ian W Smith

doi:10.3389/fimmu.2020.00150

Abstract

Anti-drug antibodies to coagulation factor VIII (fVIII), often termed inhibitors, present the greatest economical and treatment related obstacle in the management of hemophilia A. Although several genetic and environmental risk factors associated with inhibitor development have been identified, the precise mechanisms responsible for the immune response to exogenous fVIII therapies remain undefined. Clinical trials suggest there is an increased immunogenic potential of recombinant fVIII compared to plasma-derived products. Additional biochemical and immunological studies have demonstrated that changes in recombinant fVIII production and formulation can alter fVIII structure and immunogenicity. Recently, one study demonstrated increased immunogenicity of the recombinant fVIII product Helixate in hemophilia A mice following oxidation with hypochlorite (ClO−). It is widely reported that protein aggregates within drug products can induce adverse immune reactions in patients. Several studies have therefore investigated the prevalence of molecular aggregates in commercial recombinant products with and without use-relevant stress and agitation. To investigate the potential link between oxidation-induced immunogenicity and molecular aggregation, we analyzed the recombinant fVIII product, Helixate, via sedimentation velocity analytical ultracentrifugation following oxidation with ClO−. At 80 μM ClO−, a concentration that reduced the specific-activity by 67%, no detectable increase in large molecular aggregates (s > 12 S) was observed when compared to non-oxidized fVIII. This lack of aggregates was demonstrated both in commercial excipient as well as a HEPES buffered saline formulation. These data suggest that oxidation induced immunogenicity is independent of aggregate-mediated immune response. Therefore, our data support multiple, independent mechanisms underlying fVIII immunogenicity.

Highlights

The standard of care for patients with hemophilia A is prophylactic treatment with concentrated exogenous factor VIII products to prevent and control bleeding and mitigate joint damage
Peyron et al recently demonstrated that oxidation of Helixate reduced procoagulant function and increased immunogenicity without affecting binding to von Willebrand factor (VWF) [16]
This study demonstrates that oxidation of Helixate, damaging to procoagulant function, does not result in increased large molecular weight aggregates

Summary

Introduction

The standard of care for patients with hemophilia A is prophylactic treatment with concentrated exogenous factor VIII (fVIII) products to prevent and control bleeding and mitigate joint damage. Analytical Ultracentrifugation of Oxidized Factor VIII prevent fVIII activation [1], interfere with co-factor function to factor IX [1], abrogate phospholipid and/or platelet binding [2], or enhance drug clearance [3]. These inhibitors can occur in upwards of 40% of previously untreated patients [4,5,6] and recent clinical studies have suggested that recombinant products may be more immunogenetic [7,8,9]. The concentration of coagulation factors at sites of injury and inflammation may result in undesirable structural changes which alter the immunogenicity of the protein

Methods

Results

Conclusion