Development of an Optimized LC-MS Workflow for Host Cell Protein Characterization to Support Upstream Process Development.

IF 3.8 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS
Journal of Proteome Research Pub Date : 2025-01-03 Epub Date: 2024-12-19 DOI:10.1021/acs.jproteome.4c00637
Janik D Seidel, Mark R Condina, Manuela Klingler-Hoffmann, Clifford Young, Leigh Donnellan, Craig Kyngdon, Peter Hoffmann
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引用次数: 0

Abstract

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.

开发优化的LC-MS工作流程用于宿主细胞蛋白表征,以支持上游工艺开发。
生物治疗药物生产过程中共表达的宿主细胞蛋白(HCP)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控 HCP 的种群和数量是整个质量控制框架的重要组成部分。质谱法(MS)是酶联免疫吸附测定法(ELISA)的一种正交方法,可用于同时鉴定和定量 HCP,特别是用于分析下游工艺。在本研究中,我们提出了一种基于 MS 的分析方案,该方案在速度和鉴定性能方面都有所提高,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 MS 分析管道。所开发的方法可鉴定和量化澄清细胞培养样本中的 1000 多种 HCPs,包括文献中列出的 20 种高风险蛋白质,并显示了消化重复样本的重复性和精确性。此外,我们还探讨了不同的标准加标和数据处理管道变化对 HCPs 绝对定量估计值的影响,这凸显了标准化对行业广泛应用的重要性。数据可通过 ProteomeXchange 获取,标识符为 PXD053035。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Journal of Proteome Research
Journal of Proteome Research 生物-生化研究方法
CiteScore
9.00
自引率
4.50%
发文量
251
审稿时长
3 months
期刊介绍: Journal of Proteome Research publishes content encompassing all aspects of global protein analysis and function, including the dynamic aspects of genomics, spatio-temporal proteomics, metabonomics and metabolomics, clinical and agricultural proteomics, as well as advances in methodology including bioinformatics. The theme and emphasis is on a multidisciplinary approach to the life sciences through the synergy between the different types of "omics".
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