earticle

영어

Glycosylation of monoclonal antibody (mAb) therapeutics plays vital roles such as biological activity, stability, plasma half-life, and immunogenicity. It has been known that the glycan variations in mAbs are caused by cell-line selection and changes in culture-medium parameters. These variations can lead to changes in drug potency of therapeutic glycoproteins. Therefore, monitoring of glycosylation on protein is required to access the quality, safety, and equivalence of therapeutic antibodies. Here, in order to develop the reference data for quality by design (QbD) in therapeutic antibodies’ production, we performed the absolute quantitation and evaluation of major glycan (G0F, G1F, and G2F) on human immunoglobulin G (IgG) and five representative recombinant mAb drugs including adalimumab, bevacizumab, infliximab, rituximab, and trastuzumab using FLD response of 2-aminobenzamide (2-AB) labeled glycan standards. N-glycans on mAbs were enzymatically released and then labeled by 2-AB reagent. Followed by 2-AB labeled glycans were purified by HILIC-SPE. 2-AB labeled glycans were analyzed by UHPLC-HILIC-FLD which can provide chromatographic glycan separation and quantitation. The calibration curves of glycan standard were linear over the range of about 10 fmole to 10 amoles (3 orders of magnitude) and its linear correlation coefficients were excess of 0.999 (R2). As a result, the concentration of the glycans on commercial mAbs measured 2 to 10 times higher than that of IgG. Additionally, we confirmed that quantitative difference of glycan in each sample. This result can be directly applied to evaluate mAbs variants and/or biosimilars for both developmental and regulatory purposes.