Calibration for quantitative Fc-glycosylation analysis of therapeutic IgG1-type monoclonal antibodies by using glycopeptide standards.

Fc-glycosylation has crucial impact on the efficacy and safety of IgG-type therapeutic monoclonal antibodies (mAbs). In order to enhance the performance of MS-based bottom-up quantitation strategy, a library of glycopeptide standards containing 26 common IgG1-type Fc-glycoforms has been constructed via modified two-dimensional hydrophilic interaction liquid chromatography (HILIC) purification. Taking advantage of the acquired glycopeptide standards, calibrated quantitation strategy for Fc-glycosylation analysis of mAbs was established and evaluated on the basis of three LC-MS-based methods, including HILIC-MRM (multiple reaction monitoring), HILIC-SIM (selected ion monitor) and RPLC-SIM. Molar concentrations of eleven individual Fc-glycoforms (0.03 ± 0.001-13.77 ± 0.64 nmol mg) as well as degree of fucosylation (75.44-97.04%), galactosylation (3.39-49.47%) and mannosylation (1.12-21.22%) in six IgG1-type mAbs were achieved. In addition, Fc-glycosylation site occupancy was also determined from 98.05% to 99.83%. Compared with traditional MS-based quantitation via peak area normalization, the quantitation accuracy and precision of the calibrated strategy had been remarkably improved, especially when combining with HILIC separation. In addition, the transferability of calibrated quantitation as assessed by using MRM-based method had also been significantly enhanced on different instruments from different laboratories. This calibrated quantitation strategy using glycopeptide standards as calibrators will be useful for Fc-glycosylation analysis of IgG1-type mAbs with multiple glycosylation sites.