阿达木单抗还原肽指纹图谱鉴别方法的建立与验证  

Establishment and validation of a reductive peptide mapping identification method for adalimumab

作　　者：武刚[1] 刘冰 陈全姚 倪永波 郭璐韵 梅玉婷 于传飞[1] 王兰[1] WU Gang;LIU Bing;CHEN Quanyao;NI Yongbo;GUO Luyun;MEI Yuting;YU Chuanfei;WANG Lan(Division of Monoclonal Antibodies,National Institutes for Food and Drug Control,Key Laboratory of Ministry of Health for Research on Quality and Standardization of Biotech Products,Beijing 102629,China;Division of Biological Products,Chongqing Institute for Food and Drug Control;School of Pharmacy,Yantai University)

机构地区：[1]中国食品药品检定研究院单克隆抗体产品室,卫生部生物技术产品检定及标准化重点实验室,国家药品监督管理局生物制品质量研究与评价重点实验室,北京102629 [2]重庆市食品药品检验检测研究院生物制品室 [3]烟台大学药学院

出　　处：《山西医科大学学报》2024年第2期214-227,共14页Journal of Shanxi Medical University

基　　金：国家重点研发计划蛋白类生物制品主成分及其相关成分精准定量检测技术研究(2021YFF0600804)。

摘　　要：目的 建立阿达木单抗基于高分辨质谱的肽指纹图谱表征分析方法及用于质控放行的肽指纹图谱液相鉴别方法。方法 阿达木单抗经变性、还原、烷基化和酶解后,应用高分辨质谱测定阿达木单抗氨基酸序列覆盖率,并确证互补决定区(complementarity determining region, CDR)特征肽段。建立能够有效鉴别阿达木单抗肽指纹图谱的液相色谱分析方法,并进行方法学验证和应用评价。结果 阿达木单抗经胰蛋白酶(Trypsin)和胞内蛋白酶(Lys-C)酶解后,CDR肽段均由高分辨质谱完成确证。贝伐珠单抗和利妥昔单抗用于评估方法专属性,结果表明该方法专属性强。选择Fc段保守氨基酸序列作为参比峰,以CDR特征肽段的相对保留时间(RRT)和相对峰面积(RPA)考察方法的变异程度。Trypsin酶解的样品连续5次进样各特征肽段RRT的RSD在0.008%~1.097%之间,RPA的RSD在1.212%~7.951%之间;中间精密度考察各特征肽段RRT的RSD在0.010%~0.985%之间,RPA的RSD在2.306%~10.939%之间;酶切比例为1∶35~1∶45,酶切时间为3.5~4.5 h时,RRT和RPA的RSD均符合方法耐用性要求。Lys-C酶解的样品方法学验证结果也表明其精密度、耐用性均表现良好。不同色谱柱对肽段的选择性、保留强弱、分离效果以及峰强度等具有一定差异。结论 建立的基于Trypsin/Lys-C酶切、鉴定CDR特征肽段的高效液相肽指纹图谱鉴别方法可用于阿达木单抗的质量控制及批检验放行,同时可为企业建立单抗肽指纹图谱鉴别方法提供参考。Objective To establish a peptide characterization method based on high-resolution mass spectrometry and a peptide mapping identification method for quality control and release.Methods The adalimumab was denatured,reduced,alkylated and finally digested with trypsin or Lys-C to analyze the sequence coverage and confirm CDR peptides by high-resolution mass spectrometry.The liquid chromatography method for peptide mapping identification of adalimumab was established,validated and evaluated.Results All CDR peptides of adalimumab were confirmed by high-resolution mass spectrometry after trypsin or Lys-C digestion.The method had high specificity assessed by bevacizumab and rituximab.With the conserved sequence of Fc segment as reference peaks,the method′s variability was evaluated with relative retention time and relative peak area of CDR peptides as the indexes.For CDR peptides digested by trypsin,the RSD of RRT was 0.008%-1.097%and that of RPA was 1.212%-7.951%after five consecutive injections.Interme-diate precision test showed that the RSDs of RRT and RPA were 0.010%-0.985%and 2.306%-10.939%,respectively.The RSDs of RRT and RPA met the robustness requirements when the trypsin digestion ratio was 1∶35-1∶45 and the digestion time was 3.5-4.5 h.For Lys-C digestion,the method validation results also showed that precision and robustness were good.Different columns had different selectivity,retention,separation and intensity for peptides.Conclusion Based on CDR related peptides,the established HPLC method can be used for quality control and batch release test of adalimumab and may also provide a reference for manufacturers.

关 键 词：阿达木单抗 肽指纹图谱 互补决定区 高效液相色谱法 液质联用 方法学验证 

分 类 号：R927[医药卫生—药学]