Purification of Protein Charge Variants and Determination of their Potency

Abstract

Monoclonal antibodies (mAbs) related biological drugs are fastest growing in therapeutic industries across the globe. mAbs were in top best-selling categories of antibody related therapies in 2015. The interest towards mAbs biosimilar development is increasing day by day as there are many patents filed by innovators are supposed to be expire soon. Biosimilars are highly complex and similar biological drugs are developed with different manufacturing processes which are not similar to originator manufacturing process. Due to this, biosimilar product inherently has quality differences in comparison to innovator molecule which may be related to size, charge and glycosylation. Despite these differences they are supposed to demonstrate similar behaviour in safety and efficacy profile to the reference product and these differences should not be clinically meaningful. Charge variants are one of the critical quality attributes and sources of heterogeneity. Omalizumab (Xolair) is a humanized monoclonal antibody derived by recombinant DNA technology. It binds specifically to immunoglobulin E (IgE) which plays a major role in allergic reaction. In this study, biosimilar product of Xolair was expressed in mammalian cell culture process in laboratory to isolate charge variants (acidic and basic) and main peak. Isolated charge variants were purified with preparative cation exchange chromatography technique and characterized with different analytical tools includes size exclusion chromatography (SEC HPLC) and cation exchange chromatography (CEX-HPLC). Purity of acidic variants, main peak and basic variants was more than 90% estimated by SEC-HPLC and CEX-HPLC. Highly purified charge variants of Xolair biosimilar were also assessed for their impact on in vitro potency and stability at different thermal stress conditions (2-8 °C and -20 °C). The study data indicates purified charge variants (> 90%) have no impact on in-vitro potency and are stable at different thermal stress conditions up to a week. This is not only one factor related to charge heterogeneity which shows no impact, but also other factors can affect potency of mAbs. Hence, product safety and efficacy are dependent on other quality parameters those needs to be ensured throughout the product life cycle. This study also showed that biological activity of mAbs is totally dependent on mAb molecule interaction, either Fab or Fc is interacting and providing the drug response