Scientific Institute of Public Health (WIV-ISP), Brussels, Belgium; Laboratory of Pharmaceutical Biotechnology, Ghent University, Ghent, Belgium.
Laboratory of Pharmaceutical Biotechnology, Ghent University, Ghent, Belgium.
J Proteomics. 2017 Jan 30;152:312-320. doi: 10.1016/j.jprot.2016.11.007. Epub 2016 Nov 26.
Thromboembolic events (TEE) associated to trace amounts of plasmatic activated coagulation factor XI (FXIa) in administrated immunoglobulin (Ig) have recently raised concerns and hence there is a need for highly sensitive profiling of residual plasma source proteins. This study aims to consider LC-ESI-QTOF data-dependent acquisition in combination with sample fractionation for this purpose. Sample fractionation proved mandatory to enable identification of plasma residuals. Two approaches were compared: Ig depletion with protein G - protein A affinity chromatography and low-abundant protein enrichment with a combinatorial peptide ligand library (ProteoMiner™, Bio-Rad). The latter allowed a higher number of identifications. Highly sensitive detection of prothrombotic FXIa was assessed with confident identification of a 1ng/mg spike. Moreover, different residuals compositions were profiled for various commercial Ig products. Using a quantitative label free analysis, a TEE-positive Ig batch was distinguished from other regular Ig products, with increased levels of FXIa but also other unique proteins. This could have prevented the recently observed TEE problems with Ig. The method is a convenient tool to better characterize Ig products after any plasma pool or manufacture process change, gaining insights in the product quality profile without any prior information required.
This study characterized residual plasma proteins in Ig products, using bottom-up LC-MS/MS with conventional data-dependent acquisition, preceded by sample fractionation. Without any prior information or target-specific development, >30 proteins were identified in a commercial Ig product. Quality control relevance was demonstrated with the identification of FXIa spiked at 1ng/mg in Ig, which is below the minimal thrombotic dose of 3ng/mg observed in an in vivo model. Relative label-free quantitation highlighted significant differences in normalized abundances of residual proteins between Ig products. A TEE-positive batch was distinguished by unique profile of residual proteins, including FXIa but also various blood stream-regulator proteins (fibrinogen, angiotensinogen, antithrombin-III, complement component C8, …). Those results emphasize that MS screening is a relevant first-line test to prevent any undesired concentration of plasma impurities after a plasma pool or manufacturing process change.
最近，与给予的免疫球蛋白（Ig）中痕量的血浆激活凝血因子 XI（FXIa）相关的血栓栓塞事件（TEE）引起了关注，因此需要对残留血浆源蛋白进行高灵敏度分析。本研究旨在考虑为此目的结合使用 LC-ESI-QTOF 数据依赖性采集和样品分级。样品分级证明是必需的，以实现对血浆残留的鉴定。比较了两种方法：用蛋白 G-蛋白 A 亲和层析进行 Ig 耗尽和用组合肽配体文库（ProteoMiner™，Bio-Rad）进行低丰度蛋白富集。后者允许鉴定出更多数量的蛋白质。用高灵敏度检测促血栓形成的 FXIa，通过对 1ng/mg 浓度的 FXIa 的确认性鉴定来评估。此外，还为各种商业 Ig 产品分析了不同的残留成分。使用定量无标记分析，将 TEE 阳性 Ig 批次与其他常规 Ig 产品区分开来，后者的 FXIa 水平升高，但也存在其他独特的蛋白质。这可能防止了最近 Ig 观察到的 TEE 问题。该方法是一种方便的工具，可在任何血浆池或生产工艺发生变化后更好地描述 Ig 产品，无需任何先前的信息，即可深入了解产品质量概况。
本研究使用传统的基于 LC-MS/MS 的数据依赖性采集，通过样品分级，对 Ig 产品中的残留血浆蛋白进行了表征。无需任何先前的信息或针对特定目标的开发，在一种商业 Ig 产品中鉴定出了>30 种蛋白质。通过鉴定在 Ig 中以 1ng/mg 浓度添加的 FXIa，证明了质量控制的相关性，这低于在体内模型中观察到的 3ng/mg 的最小血栓剂量。相对无标记定量突出了不同 Ig 产品之间残留蛋白的相对丰度的显著差异。TEE 阳性批次通过残留蛋白的独特图谱来区分，包括 FXIa 以及各种血液调节剂蛋白（纤维蛋白原、血管紧张素原、抗凝血酶-III、补体成分 C8 等）。这些结果强调，MS 筛选是一种相关的一线测试，可防止在血浆池或生产工艺发生变化后，血浆杂质的浓度增加。
