title: Elucidating the effects of pH shift on IgG1 monoclonal antibody acidic charge variant levels in Chinese hamster ovary cell cultures.
abstract: Charge variants, especially acidic charge variants, in recombinant monoclonal antibodies are critical quality attributes, which can affect antibodies' properties in vitro and in vivo. Meanwhile, charge variants are cumulative effects of various post-translational modifications and chemical degradations on antibody. In this work, to investigate the effect of lowering culture pH in the stationary phase on acidic charge variant contents in fed-batch cultures and its mechanism, cell culture experiments in 2-L bioreactors were firstly performed to explore the changes in the charge distribution under the pH downshift condition using weak cation exchange chromatography. It is found that acidic charge variant contents were significantly decreased by pH downshift. Then, to reveal the mechanism by which the content of acidic charge variants is reduced under pH downshift condition, the variation of post-translational modifications and chemical degradations under the pH downshift condition was explored. Meanwhile, the structure of the acidic charge variants was characterized. Several analysis experiments including size exclusion chromatography, capillary electrophoresis-sodium dodecyl sulfate under non-reducing conditions, tryptic peptide map, and reduced antibody mass were applied in this study. The results show that the mechanism by which the content of acidic charge variants is reduced is that the contents of disulfide bond reduction, galactosylation, and asparagine deamination of the HC-N388 in the Fc domain were reduced by pH downshift.
authors.affiliation: The State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, No. 130 Meilong Road, Shanghai, 200237, China.
authors.forename: Panpan
authors.initials: P
authors.lastname: Xie
authors.affiliation: The State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, No. 130 Meilong Road, Shanghai, 200237, China.
authors.forename: Huijie
authors.initials: H
authors.lastname: Niu
authors.affiliation: The State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, No. 130 Meilong Road, Shanghai, 200237, China.
authors.forename: Xinning
authors.initials: X
authors.lastname: Chen
authors.affiliation: The State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, No. 130 Meilong Road, Shanghai, 200237, China.
authors.forename: Xintao
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Hisun Pharmaceutical (Hangzhou) Co., Ltd., Xialiancun, Xukou, Fuyang, Hangzhou, Zhejiang, 311404, China.
authors.forename: Shiwei
authors.initials: S
authors.lastname: Miao
authors.affiliation: Hisun Pharmaceutical (Hangzhou) Co., Ltd., Xialiancun, Xukou, Fuyang, Hangzhou, Zhejiang, 311404, China.
authors.forename: Xiancun
authors.initials: X
authors.lastname: Deng
authors.affiliation: The State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, No. 130 Meilong Road, Shanghai, 200237, China.
authors.forename: Xuping
authors.initials: X
authors.lastname: Liu
authors.affiliation: The State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, No. 130 Meilong Road, Shanghai, 200237, China.
authors.forename: Wen-Song
authors.initials: WS
authors.lastname: Tan
authors.affiliation: The State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, No. 130 Meilong Road, Shanghai, 200237, China. zhouyan@ecust.edu.cn.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: The State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, No. 130 Meilong Road, Shanghai, 200237, China. fanli@ecust.edu.cn.
authors.forename: Li
authors.initials: L
authors.lastname: Fan
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism*; D016466:CHO Cells; D055598:Chemical Phenomena; D003412:Cricetulus; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D011994:Recombinant Proteins
keywords: Acidic charge variants; Deamination; Disulfide bond reduction; Galactosylation; IgG1 monoclonal antibody; Liquid chromatography mass spectrometry