title: High throughput peptide mapping method for analysis of site specific monoclonal antibody oxidation.
abstract: Oxidation of therapeutic monoclonal antibodies (mAbs) often occurs on surface exposed methionine and tryptophan residues during their production in cell culture, purification, and storage, and can potentially impact the binding to their targets. Characterization of site specific oxidation is critical for antibody quality control. Antibody oxidation is commonly determined by peptide mapping/LC-MS methods, which normally require a long (up to 24h) digestion step. The prolonged sample preparation procedure could result in oxidation artifacts of susceptible methionine and tryptophan residues. In this paper, we developed a rapid and simple UV based peptide mapping method that incorporates an 8-min trypsin in-solution digestion protocol for analysis of oxidation. This method is able to determine oxidation levels at specific residues of a mAb based on the peptide UV traces within <1h, from either TBHP treated or UV light stressed samples. This is the simplest and fastest method reported thus far for site specific oxidation analysis, and can be applied for routine or high throughput analysis of mAb oxidation during various stability and degradation studies. By using the UV trace, the method allows more accurate measurement than mass spectrometry and can be potentially implemented as a release assay. It has been successfully used to monitor antibody oxidation in real time stability studies.
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: Wei
authors.initials: W
authors.lastname: Xu
authors.affiliation: Bioprocess Development, Merck Research Laboratories, Merck & Co., Inc., Kenilworth, NJ, 07033, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Bioprocess Development, Merck Research Laboratories, Merck & Co., Inc., Kenilworth, NJ, 07033, USA.
authors.forename: Jia
authors.initials: J
authors.lastname: Zhao
authors.affiliation: Bioprocess Development, Merck Research Laboratories, Merck & Co., Inc., Kenilworth, NJ, 07033, USA.
authors.forename: Yan-Hui
authors.initials: YH
authors.lastname: Liu
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: Huijuan
authors.initials: H
authors.lastname: Li
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: D000911:Antibodies, Monoclonal / Q000032:analysis / Q000737:chemistry*; D002623:Chemistry Techniques, Analytical / Q000295:instrumentation* / Q000379:methods*; D002853:Chromatography, Liquid / Q000592:standards; D013058:Mass Spectrometry / Q000592:standards; D008715:Methionine / Q000737:chemistry; D010084:Oxidation-Reduction; D010449:Peptide Mapping / Q000379:methods*; D014357:Trypsin / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry; D014466:Ultraviolet Rays
chemical_list: D000911:Antibodies, Monoclonal; D014364:Tryptophan; D008715:Methionine; D014357:Trypsin
keywords: High throughput; Methionine; Monoclonal antibody; Oxidation; Tryptophan