Merck & Co., Inc., Merck Research Laboratories, Process Research & Development, Rahway, NJ, 07065, USA; Robert F. Smith School of Chemical and Biomolecular Engineering, Cornell University, 120 Olin Hall, Ithaca, NY, 14853, USA.
Merck & Co., Inc., Merck Research Laboratories, Process Research & Development, Rahway, NJ, 07065, USA.
Anal Chim Acta. 2019 Apr 25;1054:114-121. doi: 10.1016/j.aca.2018.12.034. Epub 2019 Jan 9.
Biological therapeutics are established as major contributors to the pharmaceutical pipeline. Many of these biological drugs are lyophilized to preserve their conformation and reduce decomposition during storage and shipping. Therefore, understanding and controlling the effects of lyophilization on protein higher order structure is critical for commercialization of biologics. Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) is a well-established technique for studying protein higher order structure. Previous publications have demonstrated a solid state HDX (ssHDX) method for labeling formulated, lyophilized proteins to assess their physical stability during, but this process still suffered from low throughput and undesired back exchange. Recently, our group described a method combining HDX-MS with MALDI to greatly reduce the time of analysis and nearly eliminate H/D back-exchange, but that method was not suited for interrogating solid samples. This work integrates the two techniques to assess and predict the stability of peptides and proteins following mixing and lyophilization with various excipient formulations. Sample mixing and handling were performed through the use of a bench-top robotics and programmed data MALDI-MS acquisition allowed for monitoring deuterium incorporation for dried peptides and protein samples following continuous labeling with DO vapor. Effects of excipients upon peptide stability were also tracked and compared to a control for a three day labeling time course. This workflow is automated and free from back-exchange. As demonstrated by deuterium retention of bradykinin, these features serve to reduce experimental error normally associated with conventional deuterium exchange experiments. The proposed union of MALDI-MS and ssHDX can be applied to study higher order structure of proteins and peptides and the effects of added excipients in an environment that closely resembles the storage and shipping conditions of biopharmaceuticals and may be beneficial in giving insights studying protein structural dynamics in solids.
生物疗法已被确立为药物研发的主要贡献者。这些生物药物中的许多都是通过冷冻干燥来保存其构象，减少在储存和运输过程中的分解。因此，了解和控制冷冻干燥对蛋白质高级结构的影响对于生物制剂的商业化至关重要。氢氘交换质谱（HDX-MS）是研究蛋白质高级结构的一种成熟技术。以前的出版物已经证明了一种用于标记配方、冷冻干燥蛋白质的固态 HDX（ssHDX）方法，以评估其在过程中的物理稳定性，但该过程仍然存在低通量和不理想的回交换问题。最近，我们小组描述了一种将 HDX-MS 与 MALDI 结合的方法，可以大大缩短分析时间，并几乎消除 H/D 回交换，但该方法不适合检测固态样品。这项工作整合了这两种技术，以评估和预测各种赋形剂配方混合和冷冻干燥后肽和蛋白质的稳定性。通过使用台式机器人进行样品混合和处理，并通过程序数据 MALDI-MS 采集来监测干燥肽和蛋白质样品在连续暴露于 DO 蒸汽后的氘掺入情况。还跟踪并比较了赋形剂对肽稳定性的影响，以进行为期三天的标记时间过程的对照。该工作流程是自动化的，没有回交换。如通过缓激肽的氘保留率证明的那样，这些特征有助于减少与传统氘交换实验相关的实验误差。MALDI-MS 和 ssHDX 的联合应用可以用于研究蛋白质和肽的高级结构以及添加赋形剂的影响，这种环境非常接近生物制药的储存和运输条件，可能有助于深入研究固体中蛋白质结构动力学。
