Upton Rosie, Migas Lukasz G, Pacholarz Kamila J, Beniston Richard G, Estdale Sian, Firth David, Barran Perdita E
Manchester Institute of Biotechnology , Michael Barber Centre for Collaborative Mass Spectrometry , University of Manchester , 131 Princess Street , Manchester , M1 7DN , UK . Email:
Covance Laboratories Ltd. , Otley Road , Harrogate , HG3 1PY , UK.
Chem Sci. 2019 Jan 17;10(9):2811-2820. doi: 10.1039/c8sc05029e. eCollection 2019 Mar 7.
To quantify the measurable variations in the structure of a biopharmaceutical product we systematically evaluate three lots of Herceptin®, two mAb standards and an intact Fc-hinge fragment. Each mAb is examined in three states; glycan intact, truncated (following endoS2 treatment) and fully deglycosylated. Despite equivalence at the intact protein level, each lot of Herceptin® gives a distinctive signature in three different mass spectrometry approaches. Ion mobility mass spectrometry (IM-MS) shows that in the API, the attached N-glycans reduce the conformational spread of each mAb by 10.5-25%. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) data support this, with lower global deuterium uptake in solution when comparing intact to the fully deglycosylated protein. HDX-MS and activated IM-MS map the influence of glycans on the mAb and reveal allosteric effects which extend far beyond the Fc domains into the Fab region. Taken together, these findings and the supplied interactive data sets establish acceptance criteria with application for MS based characterisation of biosimilars and novel therapeutic mAbs.
为了量化生物制药产品结构中可测量的变化，我们系统地评估了三批赫赛汀®、两种单克隆抗体标准品和一个完整的Fc-铰链片段。每种单克隆抗体在三种状态下进行检测：糖基完整、截短（经内切糖苷酶S2处理后）和完全去糖基化。尽管在完整蛋白质水平上具有等效性，但每批赫赛汀®在三种不同的质谱方法中都给出了独特的特征。离子淌度质谱（IM-MS）表明，在活性药物成分（API）中，附着的N-聚糖使每种单克隆抗体的构象分布减少了10.5-25%。氢/氘交换质谱（HDX-MS）数据支持这一点，将完整蛋白质与完全去糖基化蛋白质相比，溶液中的整体氘摄取较低。HDX-MS和活化IM-MS描绘了聚糖对单克隆抗体的影响，并揭示了变构效应，这种效应远远超出Fc结构域，延伸到Fab区域。综上所述，这些发现以及提供的交互式数据集确立了适用于基于质谱的生物类似药和新型治疗性单克隆抗体表征的验收标准。
