Analytical protein A chromatography as a quantitative tool for the screening of methionine oxidation in monoclonal antibodies

The presence of oxidized methionine residues in therapeutic monoclonal antibod- ies can potentially impact drug efficacy, safety, as well as antibody half-life in vivo. Therefore, methionine oxidation of antibodies is a strong focus during pharmaceutical development and a well-known degradation pathway. The monitoring of methionine oxidation is currently rou- tinely performed by peptide mapping/liquid chromatography-mass spectrometry techniques, which are laborious and time consuming. We have established analytical protein A chromatog- raphy as a method of choice for fast and quantitative screening of total Fc methionine oxida- tion during formulation and process development. The principle of this method relies on the lower binding affinity of protein A for immunoglobulin G-Fc domains containing oxidized me- thionines, compared with nonoxidized Fc domains. Our data reveal that highly conserved Fc methionines situated close to the binding site to protein A can serve as marker for the oxidation of other surface-exposed methionine residues. In case of poor separation of oxidized species by protein A chromatography, analytical protein G chromatography is proposed as alternative. We demonstrate that analytical protein A chromatography, and alternatively protein G chromatog- raphy, is a valuable tool for the screening of methionine oxidation in therapeutic antibodies during formulation and process development. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:4248-4257, 2012