LGC, Queens Road, Teddington TW11 0LY, UK.
Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
J Am Soc Mass Spectrom. 2020 Mar 4;31(3):553-564. doi: 10.1021/jasms.9b00022. Epub 2020 Jan 21.
As monoclonal antibodies (mAbs) rapidly emerge as a dominant class of therapeutics, so does the need for suitable analytical technologies to monitor for changes in protein higher order structure (HOS) of these biomolecules. Reference materials (RM) serve a key analytical purpose of benchmarking the suitability and robustness of both established and emerging analytical procedures for both drug producers and regulators. Here, two simple enzymatic protocols for generating Fc-glycan variants from the NISTmAb RM are described and both global and localized changes in HOS between the RM and these Fc-glycan variants are characterized using hydrogen deuterium exchange-mass spectrometry (HDX-MS) and ion mobility spectrometry-mass spectrometry (IMS-MS) measurements. An alternative statistical approach is described where measurement thresholds that differentiate between measurement variability and significant structural changes were established on the basis of experimental data. Measurements revealed decreases in structural stability correlating with the degree of Fc-glycan structure loss, especially at the C2/C3 domain interface. These data promote the use of this RM and these Fc-glycan variants for establishing the sensitivity of and validating analytical methods for the detection of HOS measurements of mAbs.
随着单克隆抗体（mAbs）迅速成为治疗领域的主要药物类别，因此需要合适的分析技术来监测这些生物分子的蛋白质高级结构（HOS）变化。参考物质（RM）在基准测试方面发挥着关键作用，可用于评估既有的和新兴的分析程序对于药物生产商和监管机构的适用性和稳健性。本文描述了两种从 NISTmAb RM 生成 Fc-聚糖变体的简单酶促方案，并使用氢氘交换质谱（HDX-MS）和离子淌度质谱（IMS-MS）测量来表征 RM 和这些 Fc-聚糖变体之间的 HOS 整体和局部变化。还描述了一种替代的统计方法，该方法基于实验数据建立了区分测量变异性和显著结构变化的测量阈值。测量结果表明，结构稳定性随 Fc-聚糖结构丧失程度的降低而降低，尤其是在 C2/C3 结构域界面处。这些数据促进了对该 RM 和这些 Fc-聚糖变体的使用，可用于建立分析方法检测 mAb 的 HOS 测量的灵敏度和验证。
