title: Characterization of alanine to valine sequence variants in the Fc region of nivolumab biosimilar produced in Chinese hamster ovary cells.
abstract: Nivolumab is a therapeutic fully human IgG4 antibody to programmed death 1 (PD-1). In this study, a nivolumab biosimilar, which was produced in our laboratory, was analyzed and characterized. Sequence variants that contain undesired amino acid sequences may cause concern during biosimilar bioprocess development. We found that low levels of sequence variants were detected in the heavy chain of the nivolumab biosimilar by ultra performance liquid chromatography (UPLC) and tandem mass spectrometry. It was further identified with UPLC-MS/MS by IdeS or trypsin digestion. The sequence variant was confirmed through addition of synthetic mutant peptide. Subsequently, the mixing base signal of normal and mutant sequence was detected through DNA sequencing. The relative levels of mutant A424V in the Fc region of the heavy chain have been detected and demonstrated to be 12.25% and 13.54%, via base peak intensity (BPI) and UV chromatography of the tryptic peptide mapping, respectively. A424V variant was also quantified by real-time PCR (RT-PCR) at the DNA and RNA level, which was 19.2% and 16.8%, respectively. The relative content of the mutant was consistent at the DNA, RNA and protein level, indicating that the A424V mutation may have little influence at transcriptional or translational levels. These results demonstrate that orthogonal state-of-the-art techniques such as LC- UV- MS and RT-PCR should be implemented to characterize recombinant proteins and cell lines for development of biosimilars. Our study suggests that it is important to establish an integrated and effective analytical method to monitor and characterize sequence variants during antibody drug development, especially for antibody biosimilar products.
authors.affiliation: a International Joint Cancer Institute, Second Military Medical University , Shanghai , China.|b State Key Laboratory of Antibody Medicine and Targeted Therapy , Shanghai , China.
authors.forename: Yantao
authors.initials: Y
authors.lastname: Li
authors.affiliation: a International Joint Cancer Institute, Second Military Medical University , Shanghai , China.|b State Key Laboratory of Antibody Medicine and Targeted Therapy , Shanghai , China.
authors.forename: Tuo
authors.initials: T
authors.lastname: Fu
authors.affiliation: a International Joint Cancer Institute, Second Military Medical University , Shanghai , China.|b State Key Laboratory of Antibody Medicine and Targeted Therapy , Shanghai , China.
authors.forename: Tao
authors.initials: T
authors.lastname: Liu
authors.affiliation: b State Key Laboratory of Antibody Medicine and Targeted Therapy , Shanghai , China.|c Shanghai Zhangjiang Biotechnology Co.
authors.forename: Huaizu
authors.initials: H
authors.lastname: Guo
authors.affiliation: a International Joint Cancer Institute, Second Military Medical University , Shanghai , China.|b State Key Laboratory of Antibody Medicine and Targeted Therapy , Shanghai , China.
authors.forename: Qingcheng
authors.initials: Q
authors.lastname: Guo
authors.affiliation: b State Key Laboratory of Antibody Medicine and Targeted Therapy , Shanghai , China.|c Shanghai Zhangjiang Biotechnology Co.
authors.forename: Jin
authors.initials: J
authors.lastname: Xu
authors.affiliation: a International Joint Cancer Institute, Second Military Medical University , Shanghai , China.|b State Key Laboratory of Antibody Medicine and Targeted Therapy , Shanghai , China.
authors.forename: Dapeng
authors.initials: D
authors.lastname: Zhang
authors.affiliation: b State Key Laboratory of Antibody Medicine and Targeted Therapy , Shanghai , China.|c Shanghai Zhangjiang Biotechnology Co.
authors.forename: Weizhu
authors.initials: W
authors.lastname: Qian
authors.affiliation: a International Joint Cancer Institute, Second Military Medical University , Shanghai , China.|b State Key Laboratory of Antibody Medicine and Targeted Therapy , Shanghai , China.
authors.forename: Jianxin
authors.initials: J
authors.lastname: Dai
authors.affiliation: a International Joint Cancer Institute, Second Military Medical University , Shanghai , China.|b State Key Laboratory of Antibody Medicine and Targeted Therapy , Shanghai , China.
authors.forename: Bohua
authors.initials: B
authors.lastname: Li
authors.affiliation: b State Key Laboratory of Antibody Medicine and Targeted Therapy , Shanghai , China.|d School of Pharmacy, Liaocheng University , Liaocheng , China.|e School of Bioscience and Bioengineering, South China University of Technology , Guangzhou , China.
authors.forename: Yajun
authors.initials: Y
authors.lastname: Guo
authors.affiliation: a International Joint Cancer Institute, Second Military Medical University , Shanghai , China.|b State Key Laboratory of Antibody Medicine and Targeted Therapy , Shanghai , China.
authors.forename: Sheng
authors.initials: S
authors.lastname: Hou
authors.affiliation: a International Joint Cancer Institute, Second Military Medical University , Shanghai , China.|b State Key Laboratory of Antibody Medicine and Targeted Therapy , Shanghai , China.|d School of Pharmacy, Liaocheng University , Liaocheng , China.
authors.forename: Hao
authors.initials: H
authors.lastname: Wang
mesh_terms: D000409:Alanine / Q000032:analysis* / Q000737:chemistry; D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000737:chemistry*; D059451:Biosimilar Pharmaceuticals / Q000032:analysis / Q000737:chemistry*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D014644:Genetic Variation; D007141:Immunoglobulin Fc Fragments / Q000032:analysis / Q000737:chemistry*; D000077594:Nivolumab; D010449:Peptide Mapping / Q000379:methods*; D014633:Valine / Q000032:analysis* / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D059451:Biosimilar Pharmaceuticals; D007141:Immunoglobulin Fc Fragments; D000077594:Nivolumab; D014633:Valine; D000409:Alanine
keywords: DNA sequencing; IdeS digests; Nivolumab; RT-PCR; UPLC-UV/MS/MS; quantitation; sequence variants; tryptic peptide mapping