title: Analysis of monoclonal antibody oxidation by simple mixed mode chromatography.
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.
authors.affiliation: Bioprocess Development, Merck Research Laboratories, Merck & Co., Inc., Kenilworth, NJ 07033, USA.
authors.forename: Jorge Alexander
authors.initials: JA
authors.lastname: Pavon
authors.affiliation: Bioprocess Development, Merck Research Laboratories, Merck & Co., Inc., Kenilworth, NJ 07033, USA.
authors.forename: Xiaojuan
authors.initials: X
authors.lastname: Li
authors.affiliation: Bioprocess Development, Merck Research Laboratories, Merck & Co., Inc., Kenilworth, NJ 07033, USA.
authors.forename: Steven
authors.initials: S
authors.lastname: Chico
authors.affiliation: Bioprocess Development, Merck Research Laboratories, Merck & Co., Inc., Kenilworth, NJ 07033, USA.
authors.forename: Umesh
authors.initials: U
authors.lastname: Kishnani
authors.affiliation: Bioprocess Development, Merck Research Laboratories, Merck & Co., Inc., Kenilworth, NJ 07033, USA.
authors.forename: Soundara
authors.initials: S
authors.lastname: Soundararajan
authors.affiliation: Bioprocess Development, Merck Research Laboratories, Merck & Co., Inc., Kenilworth, NJ 07033, USA.
authors.forename: Jason
authors.initials: J
authors.lastname: Cheung
authors.affiliation: Bioprocess Development, Merck Research Laboratories, Merck & Co., Inc., Kenilworth, NJ 07033, USA.
authors.forename: Huijuan
authors.initials: H
authors.lastname: Li
authors.affiliation: Bioprocess Development, Merck Research Laboratories, Merck & Co., Inc., Kenilworth, NJ 07033, USA.
authors.forename: Daisy
authors.initials: D
authors.lastname: Richardson
authors.affiliation: Bioprocess Development, Merck Research Laboratories, Merck & Co., Inc., Kenilworth, NJ 07033, USA.
authors.forename: Mohammed
authors.initials: M
authors.lastname: Shameem
authors.affiliation: Bioprocess Development, Merck Research Laboratories, Merck & Co., Inc., Kenilworth, NJ 07033, USA. Electronic address: xiaoyu.yang@merck.com.
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Yang
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism*; D016466:CHO Cells; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D002853:Chromatography, Liquid; D003412:Cricetulus; D008027:Light; D013058:Mass Spectrometry; D008715:Methionine / Q000737:chemistry; D008970:Molecular Weight; D010084:Oxidation-Reduction; D014364:Tryptophan / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D014364:Tryptophan; D008715:Methionine
keywords: Methionine; Mixed mode chromatography; Monoclonal antibody; Oxidation; Tryptophan