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

• Type of Material: Article
• Authors: Philip M. Zakas, Queens University; John F. Healey, Emory University; Ian W. Smith, Emory University; David Lillicrap, Queens University; Pete Lollar, Emory University
• Language: English
• Date: 2020-02-07
• Publisher: FRONTIERS MEDIA SA
• Publication Version: Final Published Version
• Copyright Statement: © 2020 Zakas, Healey, Smith, Lillicrap and Lollar.
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.3389/fimmu.2020.00150
• Title of Journal or Parent Work: FRONTIERS IN IMMUNOLOGY
• Volume: 11
• Start Page: 150
• End Page: 150
• Grant/Funding Information: This work was supported by the National Institutes of Health/National Heart, Lung and Blood Institute (award number U54HL141981 to PL) and the Canadian Institute of Health Research (award number FDN 154285 to DL).
• Keywords: NONGENETIC RISK-FACTORS; oxidation; Life Sciences & Biomedicine; hemophilia A; DOMAIN; Immunology; MICE; analytical ultracentrifugation; Science & Technology; factor VIII; INHIBITOR DEVELOPMENT; PRODUCTS; PREVIOUSLY UNTREATED PATIENTS; SEVERE HEMOPHILIA-A; CHILDREN; MODEL; immunogenicity; IMMUNOGENICITY
• Research Categories: Biology, Genetics; Health Sciences, Immunology

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.