Abstract
Antibody charge variants have gained considerable attention in the biotechnology industry due to their potential influence on stability and biological activity. Subtle differences in the relative proportions of charge variants are often observed during routine biomanufacture or process changes and pose a challenge to demonstrating product comparability. To gain further insights into the impact on biological activity and pharmacokinetics (PK) of monoclonal antibody (mAb) charge heterogeneity, we isolated the major charge forms of a recombinant humanized IgG1 and compared their in vitro properties and in vivo PK. The mAb starting material had a pI range of 8.7-9.1 and was composed of about 20% acidic variants, 12% basic variants, and 68% main peak. Cation exchange displacement chromatography was used to isolate the acidic, basic, and main peak fractions for animal studies. Detailed analyses were performed on the isolated fractions to identify specific chemical modification contributing to the charge differences, and were also characterized for purity and in vitro potency prior to being administered either subcutaneously (SC) or intravenously (IV) in rats. All isolated materials had similar potency and rat FcRn binding relative to the starting material. Following IV or SC administration (10 mg/kg) in rats, no difference in serum PK was observed, indicating that physiochemical modifications and pI differences among charge variants were not sufficient to result in PK changes. Thus, these results provided meaningful information for the comparative evaluation of charge-related heterogeneity of mAbs, and suggested that charge variants of IgGs do not affect the in vitro potency, FcRn binding affinity, or the PK properties in rats.
Acknowledgements
We would like to thank Michelle G. Schweiger and the In-Vivo Studies Group for carrying out the pharmacokinetic studies, Jennifer Visich for her support and for her helpful discussions of the pharmacokinetic data, Bert Gunter and Cherry Lei for statistical analysis of the FcRn binding and PK data, Will McElroy for skillfully carrying out the icIEF experiments, C. Andrew Boswell and Daniela Bumbaca for editorial efforts. We also would like to express appreciation to the many colleagues and peers who have contributed to expanding our knowledge of charge-related heterogeneity in antibodies and regret the absence herein of many valuable reference citations due to space limitations.
Financial Disclosure
All authors are employees of Genentech, Inc., a member of the Roche Group and hold financial interest in Roche.