Seidel Janik D, Condina Mark R, Klingler-Hoffmann Manuela, Young Clifford, Donnellan Leigh, Kyngdon Craig, Hoffmann Peter
Clinical and Health Sciences, University of South Australia, 5000 Adelaide, Australia.
Mass Dynamics, 3000 Melbourne, Australia.
J Proteome Res. 2025 Jan 3;24(1):234-243. doi: 10.1021/acs.jproteome.4c00637. Epub 2024 Dec 19.
Host cell proteins (HCPs) coexpressed during the production of biotherapeutics can affect the safety, efficacy, and stability of the final product. As such, monitoring HCP populations and amounts throughout the production and purification process is an essential part of the overall quality control framework. Mass spectrometry (MS) is used as an orthogonal method to enzyme-linked immunosorbent assays (ELISA) for the simultaneous identification and quantification of HCPs, particularly for the analysis of downstream processes. In this study, we present an MS-based analytical protocol with improvements in both speed and identification performance that can be implemented for routine analysis to support upstream process development. The protocol adopts a streamlined sample preparation strategy, combined with a high-throughput MS analysis pipeline. The developed method identifies and quantifies over 1000 HCPs, including 20 proteins listed as high risk in the literature, in a clarified cell culture sample with repeatability and precision shown for digest replicates. In addition, we explore the effects of varying standard spike-ins and changes to the data processing pipeline on absolute quantification estimates of the HCPs, which highlight the importance of standardization for wider use in the industry. Data are available via ProteomeXchange with the identifier PXD053035.
生物治疗药物生产过程中共同表达的宿主细胞蛋白（HCPs）会影响最终产品的安全性、有效性和稳定性。因此，在整个生产和纯化过程中监测HCP的种类和含量是整体质量控制框架的重要组成部分。质谱（MS）被用作酶联免疫吸附测定（ELISA）的正交方法，用于同时鉴定和定量HCPs，特别是用于下游工艺的分析。在本研究中，我们提出了一种基于质谱的分析方案，在速度和鉴定性能方面都有改进，可用于常规分析以支持上游工艺开发。该方案采用了简化的样品制备策略，并结合了高通量质谱分析流程。所开发的方法能够在澄清的细胞培养样品中鉴定和定量1000多种HCPs，包括文献中列为高风险的20种蛋白质，消化复制品显示出重复性和精密度。此外，我们还探讨了不同标准添加物和数据处理流程的变化对HCPs绝对定量估计的影响，这突出了标准化在行业中更广泛应用的重要性。数据可通过ProteomeXchange获得，标识符为PXD053035。
