Division of BioAnalytical Chemistry, Vrije Universiteit Amsterdam , 1081 HV Amsterdam, The Netherlands.
Pharma Technical Development Penzberg, Roche Diagnostics GmbH , Penzberg 82377, Germany.
Anal Chem. 2017 May 16;89(10):5404-5412. doi: 10.1021/acs.analchem.7b00211. Epub 2017 Apr 26.
We report on the online coupling of FcRn affinity liquid chromatography (LC) with electrospray ionization mass spectrometry (ESI-MS) in native conditions to study the influence of modifications on the interaction of recombinant mAbs with the immobilized FcRn receptor domain. The analysis conditions were designed to fit the requirements of both affinity LC and ESI-MS. The mobile phase composition was optimized to maintain the proteins studied in native conditions and enable sharp pH changes in order to mimic properly IgGs Fc domain/FcRn receptor interaction. Mobile phase components needed to be sufficiently volatile to achieve native MS analysis. MS data demonstrated the conservation of the pseudonative form of IgGs and allowed identification of the separated variants. Native FcRn affinity LC-ESI-MS was performed on a therapeutic mAb undergoing various oxidation stress. Native MS detection was used to determine the sample oxidation level. Lower retention was observed for mAbs oxidized variants compared to their intact counterparts indicating decreased affinities for the receptor. This methodology proved to be suitable to identify and quantify post-translational modifications at native protein level in order to correlate their influence on the binding to the FcRn receptor. Native FcRn affinity LC-ESI-MS can tremendously reduce the time required to assess the biological relevance of the IgG microheterogeneities thus providing valuable information for biopharmaceutical research and development.
我们报告了 FcRn 亲和液相色谱 (LC) 与电喷雾电离质谱 (ESI-MS) 在 native 条件下的在线偶联，以研究修饰对重组 mAb 与固定化 FcRn 受体结构域相互作用的影响。分析条件的设计符合亲和 LC 和 ESI-MS 的要求。优化了流动相组成，以保持所研究的蛋白质处于 native 状态，并实现 sharp pH 变化，以正确模拟 IgG Fc 结构域/FcRn 受体相互作用。流动相成分需要具有足够的挥发性，以实现 native MS 分析。MS 数据证明了 IgG 的假 native 形式的保留，并允许鉴定分离的变体。对正在经历各种氧化应激的治疗性 mAb 进行了 native FcRn 亲和 LC-ESI-MS 分析。使用 native MS 检测来确定样品的氧化水平。与完整的对照相比，mAb 氧化变体的保留时间更短，表明与受体的亲和力降低。该方法被证明适用于在 native 蛋白水平上鉴定和定量翻译后修饰，以将其与与 FcRn 受体的结合相关联。Native FcRn 亲和 LC-ESI-MS 可以极大地减少评估 IgG 微观不均一性的生物学相关性所需的时间，从而为生物制药的研究和开发提供有价值的信息。
