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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 method for the identification of anti-human tumor necrosis factor alpha (TNF-α) monoclonal antibody by liquid chromatography-mass spectrometry coupled with peptide mapping. Methods: The sample of TNF-α monoclonal antibody was denatured, reduced and liberated free cysteine residue was alkylated with guanidine hydrochloride, and the reducing agent neutralized excess alkylating agent. UF replacement enzyme digestion buffer followed by trypsin digestion and termination. Chromatographic conditions: The mobile phase consisted of a mobile phase of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) using a Waters UPLC BEH 300 C 18 (2.1 mm × 150 mm, 1.7 μm) -120 min, 2% B → 45% B) at a flow rate of 0.2 mL · min -1 with a detection wavelength of 214 nm. MS conditions: electrospray ionization and positive ion mode were used. Data acquisition range m / z For the 100 ~ 1990. RESULTS: The six CDRs of the heavy and light chains of the TNF-α mAb were identified by mass spectrometry. HC CDR2 and HC CDR3 were co-eluted in the chromatograms. Rituximab was used in the assessment of this The results show that the method is specific and independent of the matrix. The chromatographic peak of m / z 1 344 (M ~ (+5)) is selected as the reference peak and the relative retention time of CDR The RSD of the relative retention time of CDR was between 0.57% and 1.19% in five replicates. The relative retention time (RSD) of intermediate CDR was between 0.00% and 1.08% 20: 1 ~ 30: 1. When the digestion time varied from 17h to 23h, the relative retention time was small, which met the requirements of method durability. After digestion, the samples were stored at 8 ℃ for 24h and -20 ℃ for 5d Good stability. Conclusion: Based on the peptide-related peptide identifications, the LC-MS method can be used to qualitatively identify the TNF-α monoclonal antibody. The method validation shows that this method is suitable for the specific identification of anti-human TNF- Its quality control and batch inspection release.