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Highly sensitive HPLC analysis and biophysical characterization of N-glycans of IgG-Fc domain in comparison between CHO and 293 cells using Fc?RIIIa ligand.

ABSTRACT: Quality control of monoclonal antibodies is challenging due in part to the diversity of post-translational modifications present. The regulation of the N-glycans of IgG-Fc domain is one of the key factors to maintain the safety and efficacy of antibody drugs. The Fc?RIIIa affinity column is an attractive tool for the precise analysis of the N-glycans in IgG-Fc domain. We used the mutant Fc?RIIIa, which is produced in Escherichia coli and is therefore not glycosylated, as an affinity reagent to analyze the N-glycans of monoclonal antibodies expressed in Expi293 and ExpiCHO cells. The monoclonal antibodies expressed in these cells showed very different chromatograms, because of differences in terminal galactose residues on the IgG-Fc domains. We also carried out kinetic and thermodynamic analyses to understand the interaction between monoclonal antibodies and the mutant Fc?RIIIa. Expi293 cell-derived monoclonal antibodies had higher affinity for the mutant Fc?RIIIa than those derived from ExpiCHO cells, due to slower off rates and lower binding entropy loss. Collectively, our results suggest that the Fc?RIIIa column can be used to analyze the glycosylation of antibodies rapidly and specifically.

SUBMITTER: Kosuge H

PROVIDER: S-EPMC7757244 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Highly sensitive HPLC analysis and biophysical characterization of N-glycans of IgG-Fc domain in comparison between CHO and 293 cells using FcγRIIIa ligand.

Kosuge Hirofumi H Nagatoishi Satoru S Kiyoshi Masato M Ishii-Watabe Akiko A Tanaka Toru T Terao Yosuke Y Oe Seigo S Ide Teruhiko T Tsumoto Kouhei K

Biotechnology progress 20200706 6

Quality control of monoclonal antibodies is challenging due in part to the diversity of post-translational modifications present. The regulation of the N-glycans of IgG-Fc domain is one of the key factors to maintain the safety and efficacy of antibody drugs. The FcγRIIIa affinity column is an attractive tool for the precise analysis of the N-glycans in IgG-Fc domain. We used the mutant FcγRIIIa, which is produced in Escherichia coli and is therefore not glycosylated, as an affinity reagent to a ...[more]

PMID: 32390308

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Project description:The Fc-glycan profile of IgG1 anti-citrullinated peptide antibodies (ACPA) in rheumatoid arthritis (RA) patients has recently been reported to be different from non-ACPA IgG1, a phenomenon which likely plays a role in RA pathogenesis. Herein we investigate the Fc-glycosylation pattern of all ACPA-IgG isotypes and simultaneously investigate in detail the IgG protein-chain sequence repertoire. IgG from serum or plasma (S/P, n = 14) and synovial fluid (SF, n = 4) from 18 ACPA-positive RA-patients was enriched using Protein G columns followed by ACPA-purification on cyclic citrullinated peptide-2 (CCP2)-coupled columns. Paired ACPA (anti-CCP2 eluted IgG) and IgG flow through (FT) fractions were analyzed by LC-MS/MS-proteomics. IgG peptides, isotypes and corresponding Fc-glycopeptides were quantified and interrogated using uni- and multivariate statistics. The Fc-glycans from the IgG4 peptide EEQFNSTYR was validated using protein A column purification. Relative to FT-IgG4, the ACPA-IgG4 Fc-glycan-profile contained lower amounts (p = 0.002) of the agalacto and asialylated core-fucosylated biantennary form (FA2) and higher content (p = 0.001) of sialylated glycans. Novel differences in the Fc-glycan-profile of ACPA-IgG1 compared to FT-IgG1 were observed in the distribution of bisected forms (n = 5, p = 0.0001, decrease) and mono-antennnary forms (n = 3, p = 0.02, increase). Our study also confirmed higher abundance of FA2 (p = 0.002) and lower abundance of afucosylated forms (n = 4, p = 0.001) in ACPA-IgG1 relative to FT-IgG1 as well as lower content of IgG2 (p = 0.0000001) and elevated content of IgG4 (p = 0.004) in ACPA compared to FT. One λ-variable peptide sequence was significantly increased in ACPA (p = 0.0001). In conclusion, the Fc-glycan profile of both ACPA-IgG1 and ACPA-IgG4 are distinct. Given that IgG1 and IgG4 have different Fc-receptor and complement binding affinities, this phenomenon likely affects ACPA effector- and immune-regulatory functions in an IgG isotype-specific manner. These findings further highlight the importance of antibody characterization in relation to functional in vivo and in vitro studies.

Dataset Information

Highly sensitive HPLC analysis and biophysical characterization of N-glycans of IgG-Fc domain in comparison between CHO and 293 cells using Fc?RIIIa ligand.

Publications

Highly sensitive HPLC analysis and biophysical characterization of N-glycans of IgG-Fc domain in comparison between CHO and 293 cells using FcγRIIIa ligand.

Similar Datasets

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