目的  建立一种快速的IgG1型重组单克隆抗体（单抗）药物反相-超高效液相色谱肽图分析方法，用于IgG1型单抗专属性鉴别、质量控制和批检验放行。方法  选取4种IgG1单抗（贝伐珠单抗、阿达木单抗、曲妥珠单抗、利妥昔单抗），经变性、还原、烷基化、Lys-C蛋白酶解后，终止反应，离心取上清用反相-超高效液相色谱法检测，评价方法的专属性、重复性、耐用性，并通过Xevo G2-XS QTof质谱进行方法确认及鉴定。结果  确定流动相为0.1%三氟乙酸/水和0.1%三氟乙酸/乙腈，色谱柱为Agilent Poroshell 120SB-C18型色谱柱(2.1 mm×150 mm，2.7 μm)，检测波长214 nm，柱温40 ℃，样品室温8 ℃，梯度洗脱时间为65 min。方法学验证结果显示空白对照无干扰峰存在，专属性良好；各特征峰的6次重复性相对保留时间的相对标准偏差为0.00%~0.19%；不同仪器、不同色谱柱、不同酶量及样品稳定性研究的结果显示各特征峰相对保留时间的相对标准偏差均＜2.0%，耐用性较好。结论  建立的单抗药物肽图分析方法可用于IgG1型重组单抗的专属性鉴别及质量控制和批检验放行。

Objective  To establish a rapid peptide map analysis method by reversed phase-ultra-high performance liquid chromatography (RP-UPLC) of IgG1 type recombinant monoclonal antibodes (mAbs) for specificity identification, quality control and batch inspection release. Methods  Four IgG1 type mAbs (Bevacizumab, Adalimumab, Trastuzumab and Rituximab) were selected for test, which were then denatured, reduced, alkylated and hydrolyzed by Lys-C protease. After the reaction was terminated, the supernatant was centrifuged and detected by RP-UPLC. The method’s specificity, repeatability and durability were evaluated. The method was also confirmed and identified by Xevo G2-XS QTof mass spectrometry. Results  It was determined that the mobile phase was 0.1% trifluoroacetic acid (TFA)/water and 0.1%TFA/acetonitrile, the chromatographic column was Agilent Poroshell 120SB-C18 (2.1 mm×150 mm, 2.7 μm), the detection wavelength was 214 nm, the column temperature was 40 ℃, the sample room temperature was 8 ℃, and the gradient elution time was 65 min. The blank control had no interference peak, which showed good specificity. For each characteristic peak, the relative standard deviation (RSD) of relative retention time (RRT) of 6-time repeatability was in the range of 0.00%~0.19%. The durability results of different instruments, different chromatographic columns, different enzyme amounts and sample stability studies show that the RSD of RRT of each characteristic peak was less than 2.0%, which showed good durability. Conclusion  The established mAbs peptide map analysis method can be used for specificity identification, quality control and batch inspection release of IgG1 type recombinant mAbs.