Charge variants in IgG1

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.

Figures and Tables

Figure 1 Chromatographic profiles obtained from a (A) IEC shown in full scale and (B) SEC shown in expanded scale for all charge variant fractions, starting material and buffer blanks. See Table 2 for numerical values obtained from these analyses.

Figure 2 Representative sensorgrams of fitted data from the kinetic analysis of all charge variant fractions (A) acidic, (B) main, (C) basic and (D) starting material to immobilized rat FcRn. Black lines are fitted curves using a bivalent binding model. All sample concentrations (from bottom to top) are 62.5, 125, 250, 500, 1,000, 2,000 and 4,000 nM. Residual plot shows the difference between experimental and fitted data for every point in the sensorgram. Also see Table 4 for clarity.

Figure 3 Linear changes in serum concentrations versus time for all charge variant fractions following single (A) intraveneous or (B) subcutaneous administration of 10 mg/kg in normal rats. The assay minimum quantifiable concentration was 0.4 µg/mL in rat serum. Results are mean ± SD (n = 12 rats/time point).

Table 1 Major chemical degradation pathways which are a common source of charge-related heterogeneity of therapeutic IgG1 mAbs

Table 2 Analytical results to assess all mAb charge variant fractions

Table 3 Analytical characterization of isolated charge variant fractions

Table 4 Comparison of all charge variant fractions to evaluate their kinetics of binding to rat FcRn, binding responses (in response unit) and their in vitro binding specific activity

Table 5 Pharmacokinetic parameters of all mAb fractions in normal rats