目的 对基于半胱氨酸偶联方式的抗体偶联药物(antibody-drug conjugate,ADC)抗CD79b单抗-vc-MMAE进行一级结构表征。方法 本实验采用超高效液相色谱-串联质谱(ultra-performance liquid chromatography-tandem mass spectrometry, UPLC-MS)对抗CD79b单抗-vc-MMAE的完整相对分子质量、轻重链亚基相对分子质量以及药物抗体偶联比(drug-to-antibody ratio,DAR)进行测定;采用UPLC-MS/MS对抗CD79b单抗-vc-MMAE的序列覆盖度、偶联位点及位点占有率和糖基化位点进行分析,对抗CD79b单抗进行二硫键定位。结果 抗CD79b单抗-vc-MMAE的完整相对分子质量包括:ADC药物的未加药形式的相对分子质量为147893、修饰2个小分子药物的相对分子质量为150527、修饰4个小分子药物的相对分子质量为153161以及修饰6个小分子药物的相对分子质量为155796;抗CD79b单抗-vc-MMAE的轻重链亚基相对分子质量包括:轻链的相对分子质量为23726、轻链修饰1个小分子药物的相对分子质量为25043,重链的相对分子质量为48778、重链修饰1个小分子药物的相对分子质量为50095、重链修饰2个小分子药物的相对分子质量为51412、重链修饰3个小分子药物的相对分子质量为52728;抗CD79b单抗-vc-MMAE的DAR为3.60;抗CD79b单抗-vc-MMAE的序列覆盖度为100%;抗CD79b单抗-vc-MMAE的药物偶联位点及位点占有率:轻链C218和重链C220、C226和C229的位点占有率分别为80.46%、80.39%、21.85%以及16.98%。抗CD79b单抗-vc-MMAE的糖基化位点为重链N297;抗CD79b单抗的16对二硫键全部检出。结论 表征了抗CD79b单抗-vc-MMAE的一级结构,为该类ADC药物一级结构的研究提供参考依据。
Abstract
OBJECTIVE To characterize the primary structure of antibody-drug conjugate(ADC) of anti-CD79b antibody-vc-MMAE based on cysteine conjugation. METHODS The intact molecular weight, light chain molecular weight, heavy chain molecular weight and drug to antibody ratio(DAR) of anti-CD79b antibody-vc-MMAE were determined by UPLC-MS. The sequence coverage,conjugation sites, site occupancy, and glycosylation sites of anti-CD79b antibody-vc-MMAE were analyzed using UPLC-MS/MS. The disulfide bonds of anti-CD79b antibody were assessed using UPLC-MS/MS. RESULTS The intact molecular weight of anti-CD79b antibody-vc-MMAE included the following: the molecular weight of ADC without drug was 147893, the molecular weight of ADC with two drugs was 150527, the molecular weight of ADC with four drugs was 153161, the molecular weight of ADC with six drugs was 155796. The molecular weights of light chain and heavy chain of anti-CD79b antibody-vc-MMAE were as follows:the molecular weight of light chain was 23726, the molecular weight of light chain with one drug was 25043, the molecular weight of heavy chain was 48778, the molecular weight of heavy chain with one drug was 50095, the molecular weight of heavy chain with two drugs was 51412, the molecular weight of heavy chain with three drugs was 52728, the DAR of anti-CD79b antibody-vc-MMAE was 3.60. The sequence coverage of anti-CD79b antibody-vc-MMAE was 100%. The conjugation sites and the site occupancy of anti-CD79b antibody-vc-MMAE included light chain C218 and heavy chain C220, C226 and C229, and their site occupancies were 80.46%, 80.39%, 21.85% and 16.98% respectively. The glycosylation site of anti-CD79b antibody-vc-MMAE was heavy chain N297. All 16 disulfide bonds of anti-CD79b antibody were detected. CONCLUSION In this study, it has been characterized the primary structure of anti-CD79b antibody-vc-MMAE, which may provide a reference for the study of the primary structure of this kind of ADC.
关键词
抗体偶联药物 /
药物抗体偶联比 /
药物偶联位点 /
一级结构
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Key words
antibody-drug conjugate /
drug-to-antibody ratio /
conjugation site /
primary structure
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中图分类号：
R917
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参考文献
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基金
国家药典标准提高课题资助(2022S04)
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