液质联用(LC／MS)技术在单克隆抗体(曲妥珠单抗)结构表征上的应用

Structural characterization of the therapeutic monoclonal antibody trastuzumab by LC /MS and LC/MS^E

目的:应用LC/MS(液质联用)和LC/MSE方法(液相色谱/非数据依赖二级质谱数据采集方法,等频率切换高与低碰撞能量进行质谱数据扫描)对单克隆抗体(曲妥珠单抗,人源化单克隆免疫球蛋白IgG1)的结构进行深度表征。方法:先用LC/MS对曲妥珠单抗完整蛋白及其亚基(轻链和重链)的相对分子质量进行测定;然后用多种酶(胰蛋白酶和AspN)酶解蛋白并用LC/MSE肽图分析法对曲妥珠单抗的氨基酸全序列进行解析和确定,同时对可能发生的翻译后修饰(PTMs)进行定性和定量分析。再通过PNGase F酶来释放抗体上的糖链并应用LC/FLR/MS(液相色谱荧光检测与质谱联用)方法对其糖基化修饰进行结构分析及确认。结果:对曲妥珠单抗的完整蛋白及其亚基(轻链和重链)的相对分子质量测定得到良好的质量准确度,由此可判定特定蛋白修饰在轻链和重链上的分布。通过对曲妥珠单抗酶解后的氨基酸全序列确认,可对其翻译后修饰以及可能的错误氨基酸序列进行解析。该抗体肽图分析的氨基酸序列覆盖率是100%。糖基化分析是通过对游离的N-链接寡糖进行2-AB荧光标记后再用LC/FLR/MS(液相色谱荧光检测与质谱联用)方法来进行的。联合使用这些分析方法,能够有效地对不同批号和不同工艺的抗体药物进行常规质量检测和深度表征,控制药物质量,为患者提供安全可靠的生物治疗药品。结论:结合液质联用(LC/MS和LC/MSE)技术,通过完整蛋白相对分子质量的测量,肽图分析测定氨基酸序列以及翻译后修饰的定性和定量分析,结合游离N-链接寡糖的分析表征,对单克隆抗体曲妥珠单抗进行了全面的结构表征,并建立了一套单克隆抗体结构表征的平台化方案。

Objective： To provide in-depth characterization of trastuzumab, a humanized monoclonal antibody （mAb） , by LC/MS and LC/MSE. Methods ： Molecular weights were determined through intact and reduced protein analysis ; peptide mapping was carried out by the analysis of antibody digests from multiple enzyme （trypsin and AspN） digestion using a LC/MSE method; glycosylation profiling was conducted through 2-AB labeled released glycan and LC/FLR/MS analysis. Results： Intact and reduced molecular weights of trastuzumab were measured with good mass accuracy, and glycosylation was localized to the heavy chain of the antibody. Amino acid sequence coverage of 100% for trastuzumab was obtained through peptide mapping experiments by analyzing enzymatic （tryp- sin and AspN） digest of the antibody. Post-translational modifications （PTMs） were examined at the peptide level. Glycosylation profiling was achieved through the analysis of enzymatically released N-linked glycan by LC/FLR/MS detection and quantification. The combination of these analytical methods could be very effective in routine QC testing and comprehensive characterization for antibody therapeutics, including comparability study. Conclusion： Tryptic peptide mapping with LC/MSE , combined with intact mass measurements and released glycan profiling, can provide comprehensive structure characterization to the therapeutic monoclonal antibody trastuzumab.