N端焦谷氨酸环化封闭类单克隆抗体药物的N端测序方法

Research of N Terminal Sequencing Methods for Monoclonal Antibody Pharmaceuticals Blocked by Pyroglutamic Acid

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： Some monoclonal antibody pharmaceuticals with N terminal sequence blocked by pyroglutamic acid can＇ t be sequenced directly by N terminal sequencer which is designed based on Edman degradation principle. Removal of the pyroglutamic acid to realize de novo sequencing of the antibody. Methods ： First, using N terminal sequencer to test the light and heavy chains of antibody without treated. Second, confirm the existing of pyroglumatic acid by LC-MSMS. Last, Cutting pyroglumatic acid with pyroglumate aminopeptidase, and then, sequencing the light and heavy chains of antibody with N terminal sequencer. Conclusion： Antibody＇ s light and heavy chains blocked with pyroglumatic acid can be proved by N terminal sequencer and LC-MSMS. And, N terminal sequence of the samples can be proved the same with theoretical one after removal of pyroglumatic acid.