Long Zhen, Zhao Zixi, Fan Xingliang, Luo Xi
ThermoFisher scientific corporation, Beijing 100080, China.
National Institutes for Food and Drug Control, Institute for Medical Devices Control, Beijing 102629, China.
J Pharm Biomed Anal. 2025 Jan 1;252:116484. doi: 10.1016/j.jpba.2024.116484. Epub 2024 Sep 27.
The accurate and sensitive analysis of sub-proteomic samples, such as host cell proteins (HCPs) in recombinant products and stem cells in medical devices, is crucial for ensuring product safety and efficacy in the biopharmaceutical industry. However, current analytical techniques, such as conventional analytical-flow LC-MS/MS, face limitations in sensitivity due to the low concentrations of target proteins and the complexity of the sample matrix. In this study, a highly sensitive and repeatable micro-flow LC-MS/MS strategy was developed by replacing analytical-flow tubing with micro-flow tubing on an existing analytical-flow LC-MS system for sub-proteomic sample analysis. Method optimization and evaluation were first conducted with monoclonal antibody (mAb) digestion, focusing on enhancing sensitivity and repeatability. Over 8 days, relative standard deviations (RSDs) for retention time and mass area were less than 5 % and 10 %, respectively. Sensitivity improved by 2.91-4.14 times compared to the analytical-flow LC-MS/MS method. After confirming the reliability of the method, the micro-flow LC-MS/MS method was compared to the nano-flow LC-MS/MS method and the analytical-flow LC-MS/MS method in sub-proteomic sample analysis. For HCPs, the micro-flow LC-MS/MS method demonstrated superior qualitative and much better reproducibility than the nano-flow LC-MS/MS method, with more than 98 % of proteins showing intensity RSD values below 20 %. In the analysis of mesenchymal stem cells (MSCs), the micro-flow method demonstrated good reproducibility and better sensitivity than the analytical-flow method. Taking the analysis of the 20 generation of MSC products as an example, the sample analyzed by micro-flow LC-MS/MS resulted in the identification of 68 % and 8.5 % more peptides and proteins, respectively. Moreover, micro-flow maintained stable system pressure while analyzing umbilical cord stem cells, where nano-flow methods often encounter blockages. This micro-flow LC-MS/MS method is notable for its sensitivity, reproducibility, and straightforward operation, making it highly adaptable for diverse sub-proteomic analyses in biopharmaceutical laboratories.
准确且灵敏地分析亚蛋白质组样本，如重组产品中的宿主细胞蛋白（HCP）和医疗器械中的干细胞，对于确保生物制药行业产品的安全性和有效性至关重要。然而，当前的分析技术，如传统的分析型液相色谱-串联质谱（LC-MS/MS），由于目标蛋白浓度低和样品基质复杂，在灵敏度方面存在局限性。在本研究中，通过在现有的分析型液相色谱-质谱系统上用微流管替换分析流管，开发了一种用于亚蛋白质组样本分析的高灵敏度且可重复的微流LC-MS/MS策略。首先用单克隆抗体（mAb）消化进行方法优化和评估，重点是提高灵敏度和重复性。在8天的时间里，保留时间和质量面积的相对标准偏差（RSD）分别小于5%和10%。与分析型液相色谱-串联质谱方法相比，灵敏度提高了2.91至4.14倍。在确认该方法的可靠性后，将微流LC-MS/MS方法与纳流LC-MS/MS方法和分析型液相色谱-串联质谱方法在亚蛋白质组样本分析中进行了比较。对于HCP，微流LC-MS/MS方法在定性方面优于纳流LC-MS/MS方法，并且具有更好的重现性，超过98%的蛋白质强度RSD值低于20%。在间充质干细胞（MSC）分析中，微流方法比分析流方法具有更好的重现性和更高的灵敏度。以第20代MSC产品分析为例，用微流LC-MS/MS分析的样品分别多鉴定出68%的肽段和8.5%的蛋白质。此外，在分析脐带干细胞时，微流方法能保持系统压力稳定而纳流方法经常出现堵塞。这种微流LC-MS/MS方法以其灵敏度、重现性和操作简便而著称，使其非常适用于生物制药实验室的各种亚蛋白质组分析。
