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In-depth method for the characterization of glycosylation in manufactured recombinant monoclonal antibody drugs.

ABSTRACT: The glycosylation in recombinant monoclonal antibody (rMab) drugs is a major concern in the biopharmaceutical industry as it impacts the drugs' many attributes. Characterization is important but complicated by the intricate structures, microheterogeneity, and the limitations of current tools for structural analysis. In this study, we developed a liquid chromatography-mass spectrometry (LC-MS) N-glycan library based on eight commercial rMab drugs. A library of over 70 structures was developed for the rapid characterization of rMab. N-Glycans were separated on a porous graphitized carbon (PGC) column incorporated on a chip and then analyzed by an electrospray ionization hybrid quadrupole time-of-flight (ESI-Q-TOF) MS. The retention time and accurate mass for each N-glycan were recorded in the library. The complete structures were obtained through exoglycosidase sequencing. The results showed that most of the N-glycans between different antibodies are nearly the same with different abundances. The utility of this library enables one to identify structures in a rapid manner by matching LC retention times and accurate masses.

SUBMITTER: Song T

PROVIDER: S-EPMC4066919 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Rheumatoid arthritis (RA) is characterized by formation of synovial ectopic lymphoid structures (ELS) supporting B cell autoreactivity toward locally generated citrullinated (cit) antigens, including those contained in neutrophil extracellular traps (NETs). However, only a minority of RA-rmAbs from B cells isolated from ELS+ RA tissues react against NETs. Thus, alternative cellular sources of other potential autoantigens targeted by locally differentiated B cells remain undefined. RA fibroblast-like synoviocytes (FLS) have been implicated in the release of RA-associated autoantigens. In this study, we aimed to define stromal-derived autoantigens from RA-FLS targeted by RA-rmAbs. Seventy-one RA-rmAbs were screened toward RA-FLS by living-cell immunofluorescence (IF). Western blotting was used to identify potential autoantigens from RA-FLS protein extracts. Putative candidates were validated using colocalization immunofluorescence confocal microscopy, ELISA, immunoprecipitation assay, and surface plasmon resonance on unmodified/cit proteins. Serum immunoreactivity was tested in anti-citrullinated peptide/protein Abs (ACPA)+ versus ACPA- RA patients. Ten out of 71 RA-rmAbs showed clear reactivity toward RA-FLS in immunofluorescence with no binding to NETs. One stromal-reactive RA-rmAb (RA057/11.89.1) decorated a ∼58-kDa band that mass spectrometry and Western blotting with a commercial Ab identified as calreticulin (CRT). Confocal microscopy demonstrated significant cellular colocalization between anti-CRT RA057/11.89.1 in RA-FLS. RA057/11.89.1 was able to immunoprecipitate rCRT. Deimination of CRT to cit-CRT moderately increased RA057/11.89.1 immunoreactivity. cit-CRT displayed increased blocking capacity compared with unmodified CRT in competitive binding assays. Finally, anti-cit-CRT Abs were preferentially detected in ACPA+ versus ACPA- RA sera. We identified a synovial B cell-derived RA-rmAb locally differentiated within the ELS+ RA synovium reacting toward CRT, a putative novel autoantigen recently described in RA patients, suggesting that FLS-derived CRT may contribute to fuel the local autoimmune response.

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In-depth method for the characterization of glycosylation in manufactured recombinant monoclonal antibody drugs.

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