Characterization and quantitation of aggregates and particles in interferon-β products: potential links between product quality attributes and immunogenicity.

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 of neutralizing-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.