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)

摘要： 为了优化利用N糖苷酶F（PNGase F）酶解单克隆抗体中N糖的方法，应用本公司生产的单抗对PNGase F酶的酶解条件进行优化，包括缓冲液pH、酶种类、仪器、酶解程度、变性缓冲液及酶加入量等，总结酶解条件对N糖谱结果的影响。结果显示，置换缓冲液至1×PBS中可以避免某些单抗在低pH酶解时G0F转化为G0F-GN并可改善峰型；快速PNGase F和加入变性缓冲液能有效提高酶解效率；UPLC和1.7 μm粒径色谱柱能提高分离度；不完全酶解影响N糖含量结果。研究结果表明，采用优化的酶解条件可快速、有效的酶解单抗上的N糖，使N糖谱结果准确可靠，为细胞株筛选和单抗药物质量控制提供有效手段。

关键词: 单克隆抗体, N糖, PNGaseF酶