论文部分内容阅读
目的:对于直接用N端测序仪无法进行测序的N端焦谷氨酸环化封闭的抗体类药物,去除焦谷氨酸后实现对其N端序列的从头测序。方法:对抗体的重轻链分别进行N端测序无果,再通过LC-MSMS对其重轻链N端的焦谷氨酸进行确认,证明其为焦谷氨酸封闭。对抗体蛋白进行焦谷氨酸氨肽酶酶解后,再通过N端测序仪进行Edman降解法N端测序,获得抗体药物重轻链的N端氨基酸序列。结论:抗体药物的重轻链N端经过N端测序仪测序和LC-MSMS分析,确定均为焦谷氨酸封闭。再经过焦谷氨酸氨肽酶处理后进行N端测序仪从头测序证实抗体药物的N端序列与理论序列一致。
OBJECTIVE: To de novo pyroglutamic acid, de novo sequencing of the N-terminal pyroglutamic acid was performed on the N-terminal pyroglutamic acid cyclized-blocked antibody that could not be sequenced directly by the N-terminal sequencer. Methods: N-terminal sequencing of antibody heavy and light chains was unsuccessful, and then the pyroglutamic acid at the N-terminal of heavy and light chain was confirmed by LC-MSMS, which proved that it was pyroglutamic acid blocked. The antibody against pyroglutamate aminopeptidase was digested with N-terminal sequencer and sequenced by N-terminal Edman degradation to obtain the N-terminal amino acid sequence of antibody heavy chain. Conclusion: The heavy and light chain N-terminus of the antibody drug was confirmed to be pyroglutamic acid blocked by N-terminal sequencing and LC-MSMS. After pyroglutamate aminopeptidase treatment, de novo sequencing by N-terminal sequencer confirmed that the N-terminal sequence of the antibody drug is consistent with the theoretical sequence.