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  • Title: Characterization of recombinant monoclonal IgG2 antibodies using LC-MS and limited Lys-C digestion.
    Author: Nowak C, Patel R, Liu H.
    Journal: J Chromatogr B Analyt Technol Biomed Life Sci; 2018 Aug 15; 1092():15-18. PubMed ID: 29879591.
    Abstract:
    Recombinant monoclonal antibodies have been routinely characterized at intact, subunit and peptide levels by LC-MS. Papain and pepsin have been the enzymes commonly used to cleave IgG into various fragments to facilitate in-depth characterization. However, non- specific cleavage for both papain and pepsin and the need for a reducing reagent for papain has limited their usage. In contrast, IdeS has gained popularity due to its specificity and independence of reducing reagent. Results presented in the current study demonstrated that the readily available endoprotease Lys-C can cleave IgG2 at a specific peptide bond in CH2 domain to generate a homogeneous F(ab')2 fragment, and the Fc regions was digested to peptides under limited digestion condition. The generated F(ab)2 fragment is suitable for further analysis because of its homogeneity. The posttranslational modifications located in the Fc region including glycosylation, deamidation and C-terminal heterogeneity can be rapidly analyzed by LC-MS.
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