title: In-Depth Characterization of a Pro-Antibody-Drug Conjugate by LC-MS.
abstract: Pro-antibody-drug conjugate (PDC) is a hybrid structural format of immunoconjugate, where the structural complexity of pro-antibody and intrinsic heterogeneity of ADCs impose a prominent analytical challenge to the in-depth characterization of PDCs. In the present study, we successfully prepared and characterized PanP-DM1 as a model of PDCs, which is an anti-EGFR pro-antibody following conjugation with DM1 at lysine residues. The drug-to-antibody ratio (DAR) of PanP-DM1 was determined by LC-MS after deglycosylation, and verified by UV/vis spectroscopy. Following reduction or IdeS digestion, the pro-antibody fragments linked with DM1 were investigated by middle-down mass spectrometry. Furthermore, more than 20 modified lysine conjugation sites were determined by peptide mapping after trypsin digestion. Additionally, more than ten glycoforms of PanP-DM1 were also identified and quantified. In summary, critical quality attributes (CQAs) of PDCs including DAR, drug load distribution, and conjugation sites were fully characterized, which would contribute to the development of other PDCs for cancer treatment.
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology , 381 Wushan Road, Guangzhou 510641, China.|International Joint Cancer Institute, Second Military Medical University , 800 Xiangyin Road, Shanghai 200433, China.|State Key Laboratory of Antibody Medicine and Targeted Therapy, Shanghai Key Laboratory of Cell Engineering , 99 Libing Road, Shanghai 201203, China.
authors.forename: Boning
authors.initials: B
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Antibody Medicine and Targeted Therapy, Shanghai Key Laboratory of Cell Engineering , 99 Libing Road, Shanghai 201203, China.|Shanghai Zhangjiang Biotechnology Co. , 99 Libing Road, Shanghai 201203, China.
authors.forename: Huaizu
authors.initials: H
authors.lastname: Guo
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology , 381 Wushan Road, Guangzhou 510641, China.|International Joint Cancer Institute, Second Military Medical University , 800 Xiangyin Road, Shanghai 200433, China.|State Key Laboratory of Antibody Medicine and Targeted Therapy, Shanghai Key Laboratory of Cell Engineering , 99 Libing Road, Shanghai 201203, China.
authors.forename: Junjie
authors.initials: J
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Antibody Medicine and Targeted Therapy, Shanghai Key Laboratory of Cell Engineering , 99 Libing Road, Shanghai 201203, China.|Shanghai Zhangjiang Biotechnology Co. , 99 Libing Road, Shanghai 201203, China.|School of Life Sciences, Fudan University , 220 Handan Road, Shanghai 200433, China.
authors.forename: Jingya
authors.initials: J
authors.lastname: Xue
authors.affiliation: State Key Laboratory of Antibody Medicine and Targeted Therapy, Shanghai Key Laboratory of Cell Engineering , 99 Libing Road, Shanghai 201203, China.|School of Life Basic Medical Sciences, Xin Xiang Medical University , 601 Jinsui Road, Xinxiang 453003, China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Yang
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology , 381 Wushan Road, Guangzhou 510641, China.|International Joint Cancer Institute, Second Military Medical University , 800 Xiangyin Road, Shanghai 200433, China.|State Key Laboratory of Antibody Medicine and Targeted Therapy, Shanghai Key Laboratory of Cell Engineering , 99 Libing Road, Shanghai 201203, China.
authors.forename: Ting
authors.initials: T
authors.lastname: Qin
authors.affiliation: State Key Laboratory of Antibody Medicine and Targeted Therapy, Shanghai Key Laboratory of Cell Engineering , 99 Libing Road, Shanghai 201203, China.|Shanghai Zhangjiang Biotechnology Co. , 99 Libing Road, Shanghai 201203, China.
authors.forename: Jin
authors.initials: J
authors.lastname: Xu
authors.affiliation: International Joint Cancer Institute, Second Military Medical University , 800 Xiangyin Road, Shanghai 200433, China.|State Key Laboratory of Antibody Medicine and Targeted Therapy, Shanghai Key Laboratory of Cell Engineering , 99 Libing Road, Shanghai 201203, China.
authors.forename: Qingcheng
authors.initials: Q
authors.lastname: Guo
authors.affiliation: International Joint Cancer Institute, Second Military Medical University , 800 Xiangyin Road, Shanghai 200433, China.|State Key Laboratory of Antibody Medicine and Targeted Therapy, Shanghai Key Laboratory of Cell Engineering , 99 Libing Road, Shanghai 201203, China.
authors.forename: Dapeng
authors.initials: D
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Antibody Medicine and Targeted Therapy, Shanghai Key Laboratory of Cell Engineering , 99 Libing Road, Shanghai 201203, China.|Shanghai Zhangjiang Biotechnology Co. , 99 Libing Road, Shanghai 201203, China.
authors.forename: Weizhu
authors.initials: W
authors.lastname: Qian
authors.affiliation: International Joint Cancer Institute, Second Military Medical University , 800 Xiangyin Road, Shanghai 200433, China.|State Key Laboratory of Antibody Medicine and Targeted Therapy, Shanghai Key Laboratory of Cell Engineering , 99 Libing Road, Shanghai 201203, China.
authors.forename: Bohua
authors.initials: B
authors.lastname: Li
authors.affiliation: International Joint Cancer Institute, Second Military Medical University , 800 Xiangyin Road, Shanghai 200433, China.|State Key Laboratory of Antibody Medicine and Targeted Therapy, Shanghai Key Laboratory of Cell Engineering , 99 Libing Road, Shanghai 201203, China.
authors.forename: Sheng
authors.initials: S
authors.lastname: Hou
authors.affiliation: International Joint Cancer Institute, Second Military Medical University , 800 Xiangyin Road, Shanghai 200433, China.|State Key Laboratory of Antibody Medicine and Targeted Therapy, Shanghai Key Laboratory of Cell Engineering , 99 Libing Road, Shanghai 201203, China.
authors.forename: Jianxin
authors.initials: J
authors.lastname: Dai
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology , 381 Wushan Road, Guangzhou 510641, China.|State Key Laboratory of Antibody Medicine and Targeted Therapy, Shanghai Key Laboratory of Cell Engineering , 99 Libing Road, Shanghai 201203, China.|School of Pharmacy, Liaocheng University , 1 Hunan Road, Liaocheng 252000, China.
authors.forename: Yajun
authors.initials: Y
authors.lastname: Guo
authors.affiliation: International Joint Cancer Institute, Second Military Medical University , 800 Xiangyin Road, Shanghai 200433, China.|State Key Laboratory of Antibody Medicine and Targeted Therapy, Shanghai Key Laboratory of Cell Engineering , 99 Libing Road, Shanghai 201203, China.|School of Pharmacy, Liaocheng University , 1 Hunan Road, Liaocheng 252000, China.
authors.forename: Hao
authors.initials: H
authors.lastname: Wang
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods*; D018796:Immunoconjugates / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D011355:Prodrugs / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D018796:Immunoconjugates; D011355:Prodrugs
keywords: IdeS; charge heterogeneity; drug load distribution; drug occupation site; drug-loaded peptide mapping; drug-to-antibody ratio; liquid chromatography mass spectrometry; pro-antibody−drug conjugates; structural characterization