Manchester Institute of Biotechnology, Michael Barber Centre for Collaborative Mass Spectrometry, University of Manchester , 131 Princess Street, Manchester, M1 7DN, United Kingdom.
Covance Laboratories Ltd. , Otley Road, Harrogate, HG3 1PY, United Kingdom.
Anal Chem. 2016 Oct 18;88(20):10259-10265. doi: 10.1021/acs.analchem.6b02994. Epub 2016 Sep 26.
In the development of therapeutic antibodies and biosimilars, an appropriate biopharmaceutical CMC control strategy that connects critical quality attributes with mechanism of action should enable product assessment at an early stage of development in order to mitigate risk. Here we demonstrate a new analytical workflow using trastuzumab which comprises "middle-up" analysis using a combination of IdeS and the endoglycosidases EndoS and EndoS2 to comprehensively map the glycan content. Enzymatic cleavage between the two N-acetyl glucosamine residues of the chitobiose core of N-glycans significantly simplifies the oligosaccharide component enabling facile distinction of GlcNAc from GlcNAc with core fucose. This approach facilitates quantitative determination of total Fc-glycan core-afucosylation, which was in turn correlated with receptor binding affinity by surface plasmon resonance and in vitro ADCC potency with a cell based bioassay. The strategy also quantifies Fc-glycan occupancy and the relative contribution from high mannose glycans.
在治疗性抗体和生物类似药的开发中，连接关键质量属性与作用机制的适当生物制药 CMC 控制策略，应能够在开发的早期阶段对产品进行评估，以降低风险。在这里，我们展示了一种新的分析工作流程，使用曲妥珠单抗作为模型，该流程结合了 IdeS 和内切糖苷酶 EndoS 和 EndoS2 的使用，进行“中向上”分析，以全面绘制聚糖含量。内切糖苷酶在 N-糖链中壳二糖核心的两个 N-乙酰葡萄糖胺残基之间的切割，显著简化了寡糖成分，使得容易区分带有核心岩藻糖的 GlcNAc 和 GlcNAc。这种方法促进了总 Fc-糖链核心去岩藻糖基化的定量测定，反过来通过表面等离子体共振和基于细胞的生物测定法与受体结合亲和力以及体外 ADCC 效力相关联。该策略还定量测定了 Fc-糖链的占有率以及高甘露糖聚糖的相对贡献。
