抗人TNF-α单克隆抗体液质联用肽图分析方法的建立及验证

Establishment and validation of an LC/MS peptide mapping method for the characterization of anti-TNF-α monoclonal antibody

目的:建立液相色谱-质谱联用肽图分析方法用于抗人肿瘤坏死因子α(Tumor necrosis factor alpha,TNF-α)单抗的专属性鉴别。方法:TNF-α单抗样品经盐酸胍变性、还原,释放出的游离半胱氨酸残基进行烷化,还原剂中和过量的烷化剂。超滤置换酶切缓冲液后进行胰酶酶切并终止。色谱条件:采用Waters UPLC BEH 300 C_(18)(2.1 mm×150mm,1.7μm)色谱柱,以0.1%甲酸水溶液(A)-0.1%甲酸乙腈溶液(B)为流动相,梯度洗脱(5-120 min,2%B→45%B),流速为0.2 mL·min^(-1),检测波长为214 nm;质谱条件:采用电喷雾离子源及正离子模式,数据采集范围m/z为100~1990。结果:TNF-α单抗重链及轻链的6个互补决定区(CDR)对应肽段由质谱鉴定出,HC CDR2及HC CDR3在色谱峰图中共流出;利妥昔单抗用于评估本方法的专属性,结果显示本方法专属性强,且不受基质的干扰;选定m/z 1 344(M^(+5))的色谱峰为参考峰,根据CDR的相对保留时间考察该方法的变异程度,5次重复测定CDR相对保留时间的RSD在0.57%~1.19%之间;中间精密度考察测定的相对保留时间的RSD在0.00%~1.08%之间;胰酶酶切比例在20:1~30:1,酶切时间在17~23h范围内变化时,相对保留时间的均较小,符合方法耐用性的要求;样品消化后在8℃储存24h以及-20℃储存5d的稳定性良好。结论:基于CDR相关肽段鉴别的液质联用肽图分析方法可定性鉴定出TNF-α单抗,方法学验证结果显示该方法适用于抗人TNF-α单抗的专属性鉴别,可用于其质量控制及批检验放行。

Objective： To establish a liquid chromatography-mass spectrometry peptide mapping method for specific identification of anti-human TNF- α monoclonal antibody （ TNF-α mAb ）. Methods： After being denatured by guanidine hydrochloride and reduced by DTT, the flee cysteine sulfhydryl of TNF-α mAb was alkylated and the excessive alkylating agents were neutralized by DTT. Finally, digestion buffer was replaced by ultrafiltration, and the digestion reaction was terminated by FA. A Waters UPLC column（ BEH 300 C18, 2.1 mm× 150 mm, 1.7μm ） was adopted using water （ containing 0.1% formic acid ） as the mobile phase A and acetonitrile （ containing 0.1% formic acid ）as the mobile phase B. Gradient elution of mobile phase B was started from 5 min to 120 rain followed with changing from 2% to 45%. The flow rate was 0.2 mL ·min-1 and the detection wavelength was 214 nm. Data were obtained with electrospray ionization and positive ionization, and the acquisition range was m/z 100- 1 990. Results： Six complementary CDRs of the heavy and light chains of TNF-α mAb were identified by mass spectrometry. The HC CDR2 and HC CDR3 were coeluted. Rituximab was used to assess the method specificity. Tile result showed that this method has high specificity and was not affected by matrix. A chromatographic peak with the mass of i 344 （ M＋5 ） was set as reference peak when validation projects were conducted. Extent of variation of this method was evaluated by the relative retention time （ RRT ） of CDR to reference peak ： the RSD of RRT of CDR in the range of 0.57%-1.19% after 5 times extent of variation was small and met robustness digestion time was 17-23 h. The stability of digested detection; the RSD of intermediate precision was 0.00%-1.08%; requirements when trypsin digestion ratio within 20 ： 1-30 ： 1 and sample could be maintained for 24 h at 8℃and for 5 days at -20 ℃ Conclusion： Based on CDR related peptides identification, liquid chromatography-mass spectrometry peptide method could qualitatively identify TNF-α mAb. Method validation results showed that the established method couhl be applied to specificity identification of anti-human TNF-α mAb and used for quality control and batch release test.