Characterization and Quantitation of Aggregates and Particles in Interferon-β Products: Potential Links Between Product Quality Attributes and Immunogenicity

ABSTRACT Interferon- β (IFN-β) products have been used for many years in the treatment of multiple sclerosis and include recombinant IFN-β-1b (Betaseron®) and IFN-β-1a (Avonex® and Rebif®). All three products lead to the formation of neutralizing antibodies (NAbs) and resulting loss of efficacy in patients but to different extents. Across several clinical trials, the reported rates ofneutralizing-antibody formation were 22%–47% (Betaseron®), 5%–35% (Rebif®), and 2%–13% (Avonex®). In the current study, all products were purchased from the pharmacy and aggregates were characterized and/or quantified using size-exclusion chromatography (SEC), analytical ultracentrifugation, gel electrophoresis, and dot-blotting immunoassays. Particle characterization and counting were performed using microflow imaging, particle tracking analysis, and resonant mass measurement. Betaseron® and Rebif®, which are formulated with human serum albumin, had the greatest amount of aggregated protein and particles (e.g., 9%–15% high molecular weight species by SEC and > 100,000 particles/mL by flow imaging). Avonex® was found to have the least amount of aggregated protein, with > 95% monomer content by both SEC and analytical ultracentrifugation, and the particles detected in Avonex® were determined to be primarily silicone oil droplets. These results strongly suggest that protein aggregate and particle contents are key product quality attributes in a given product’s propensity to elicit the production of NAbs in patients. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:915–928, 2013