1. Analysis of monoclonal antibody oxidation by simple mixed mode chromatography
- Author
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Xiaojuan Li, Steven Chico, Jason K. Cheung, Xiaoyu Yang, Soundara Soundararajan, Umesh Kishnani, Jorge Alexander Pavon, Daisy Richardson, Huijuan Li, and Mohammed Shameem
- Subjects
0301 basic medicine ,Light ,medicine.drug_class ,Size-exclusion chromatography ,CHO Cells ,Mass spectrometry ,Monoclonal antibody ,Biochemistry ,Chemistry Techniques, Analytical ,Mass Spectrometry ,Analytical Chemistry ,Hydrophobic effect ,03 medical and health sciences ,chemistry.chemical_compound ,Cricetulus ,Methionine ,medicine ,Animals ,Chromatography ,biology ,Chemistry ,Organic Chemistry ,Tryptophan ,Antibodies, Monoclonal ,General Medicine ,Molecular Weight ,030104 developmental biology ,Mixed-mode chromatography ,Monomer ,biology.protein ,Antibody ,Oxidation-Reduction ,Chromatography, Liquid - Abstract
Analysis of oxidation of monoclonal antibodies (mAbs) in most cases relies on peptide mapping and LC-MS, which is time consuming and labor-intensive. A robust chromatography based method that is able to resolve and quantitate mAb oxidation variants due to oxidized methionine or tryptophan is highly desired. Here we developed a novel mixed mode chromatography method using the unique property of Sepax Zenix SEC-300MK column to analyze mAb oxidation levels. The separation of oxidized species relied upon the mixed mode of size exclusion and hydrophobic interaction between the resin and antibodies. The chromatography was performed in a regular SEC mobile phase, PBS, containing NaCl at a concentration (0-2.4M) specific for individual antibodies. This method was able to resolve and quantitate the oxidized antibodies as prepeaks, of either methionine-oxidized species induced by the common oxidants TBHP, tryptophan-oxidized species triggered by AAPH, or oxidized species by UV photo-irradiation. The prepeaks were further characterized by SEC-MALLS as monomers and confirmed by LC-MS as oxidized antibody variants with a mass increase of 16 or 32Da. This method has been successfully applied to monitor multiple monoclonal antibodies of IgG1, IgG2, and IgG4 subclasses.
- Published
- 2016