关键词: 单克隆抗体, N糖, PNGaseF酶

Abstract: In order to  develop and optimize a method of hydrolyzing N-glycan from monoclonal antibodies by using PNGase F, a series of experimental conditions, including buffer pH, enzyme type, instruments, degree of enzymatic hydrolysis, denaturing buffer solution and enzyme dosage were optimized by using several monoclonal antibodies. Results showed that, buffer exchanging into 1×PBS could avoid the conversion of some mAbs from G0F to G0F-GN at low pH and improve the peak shape. Rapid PNGase F and denature buffer could effectively improve the efficiency of enzymatic hydrolysis. Combination of UPLC and chromatographic column with 1.7 μm particle size could improve the resolution. Incomplete enzymatic hydrolysis could affect the results of N-glycan content. Results indicated that the optimized hydrolyzing conditions of PNGase F can quickly and effectively hydrolyze the N-glycan from the monoclonal antibody, which can provide an effective method for controlling N-glycan content in cell line screening and quality control of monoclonal antibody drugs.

Key words: monoclonal antibody, N-glycan, peptide N glycosidase F (PNGase F)