单克隆抗体A在液体制剂中铰链区裂解位点的鉴定

Identification of cleavage site in hinge region of monoclonal antibody A in liquid formulation

目的:对治疗性单克隆抗体A在液体制剂储存中发生裂解的位点进行鉴定。方法:单克隆抗体A在pH 5.9以及pH 6.5条件下,40℃保存1个月后,利用分子排阻色谱法(SEC)对产生的碎片进行分离收集,并用电喷雾-四级杆-飞行时间质谱进行分析,确定其裂解位点。结果:未经还原处理时,利用SEC法收集到的碎片相对分子质量为100 565.30,100 726.27和100 886.57 Da,经还原处理后,测得的相对分子质量为23500.80,26623.75,26785.96,26943.65,50448.09,50609.62和50766.90 Da。上述结果显示,发生裂解的位点为重链铰链区224位赖氨酸以及225位苏氨酸。结论:单克隆抗体A在溶液中发生裂解的位点是铰链区赖氨酸以及苏氨酸之间,该降解现象可通过降低贮存溶液的pH值得到控制。

Objective： To identify the cleavage site of therapeutic monoclonal antibody A（ mAb A） during storage in liquid formulation. Methods： The mAb A was stored in liquid formulation with pH value of 5. 9 or 6. 5at 40 ℃ for 1 month. Fractions of fragmentation observed using size-exclusion chromatography（ SEC） were collected and analyzed using electronic spray ionization quadrupole time of flight mass spectrometry（ ESI-Q-TOF） to identify the cleavage site. Results： The observed molecular weights of the fragment collected using SEC-HPLC were100 565. 30 Da,100 726. 27 Da and 100 886. 57 Da without reduction,and 23 500. 80 Da,26 623. 75 Da,26 785. 96 Da,26 943. 65 Da,50 448. 09 Da,50 609. 62 Da and 50 766. 90 Da after reduction treatment. These results indicated that the cleavage site was between lysine 224 and threonine 225 on one heavy chain of mAb A in the hinge region. Conclusion： The cleavage site is lysine-threonine bond in the hinge region of mAb A when stored in liquid formulation,and the fragmentation could be controlled by lowering storage pH.