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

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 C₁₈（2.1 mm×150 mm，1.7 μm）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100~1 990。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17~23 h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24 h以及-20℃储存5 d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 free 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,1.7 μm)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 min 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 co-eluted.Rituximab was used to assess the method specificity.The result showed that this method has high specificity and was not affected by matrix.A chromatographic peak with the mass of 1 344(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 detection;the RSD of intermediate precision was 0.00%-1.08%;extent of variation was small and met robustness requirements when trypsin digestion ratio within 20:1-30:1 and digestion time was 17-23 h.The stability of digested 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 could be applied to specificity identification of anti-human TNF-α mAb and used for quality control and batch release test.

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