Protagen Protein Services GmbH, Biotherapeutical Analytics, Dortmund, Germany.
Consultant, Analytical Chemistry, San Francisco, California.
Biotechnol Prog. 2019 May;35(3):e2788. doi: 10.1002/btpr.2788. Epub 2019 Mar 6.
For production of different monoclonal antibodies (mAbs), biopharmaceutical companies often use related upstream and downstream manufacturing processes. Such platforms are typically characterized regarding influence of upstream and downstream process (DSP) parameters on critical quality attributes (CQAs). CQAs must be monitored strictly by an adequate control strategy. One such process-related CQA is the content of host cell protein (HCP) which is typically analyzed by immunoassay methods (e.g., HCP-ELISA). The capacity of the immunoassay to detect a broad range of HCPs, relevant for the individual mAb-production process should be proven by orthogonal proteomic methods such as 2D gel electrophoresis or mass spectrometry (MS). In particular MS has become a valuable tool to identify and quantify HCP in complex mixtures. We evaluate up- and DSP parameters of four different biopharmaceutical products, two different process variants, and one mock fermentation on the HCP pattern by shotgun MS analysis and ELISA. We obtained a similar HCP pattern in different cell culture fluid harvests compared to the starting material from the downstream process. During the downstream purification process of the mAbs, the HCP level and the number of HCP species significantly decreased, accompanied by an increase in diversity of the residual HCP pattern. Based on this knowledge, we suggest a control strategy that combines multi product ELISA for in-process control and release analytics, and MS testing for orthogonal HCP characterization, to attain knowledge on the HCP level, clusters and species. This combination supports a control strategy for HCPs addressing safety and efficacy of biopharmaceutical products.
为了生产不同的单克隆抗体（mAbs），生物制药公司通常使用相关的上游和下游制造工艺。这些平台通常具有以下特点：上游和下游工艺（DSP）参数对关键质量属性（CQA）的影响。CQA 必须通过适当的控制策略进行严格监控。其中一个与工艺相关的 CQA 是宿主细胞蛋白（HCP）的含量，通常通过免疫测定方法（例如，HCP-ELISA）进行分析。免疫测定法检测与特定 mAb 生产过程相关的广泛 HCP 的能力应通过正交蛋白质组学方法（例如 2D 凝胶电泳或质谱法（MS））来证明。特别是 MS 已成为鉴定和定量复杂混合物中 HCP 的有价值工具。我们通过 shotgun MS 分析和 ELISA 评估了四个不同生物制药产品、两个不同工艺变体和一个模拟发酵的上 DSP 参数。与下游工艺的起始物料相比，我们在不同的细胞培养液收获物中获得了相似的 HCP 模式。在 mAb 的下游纯化过程中，HCP 水平和 HCP 种类显著降低，同时残留 HCP 模式的多样性增加。基于这些知识，我们建议采用一种控制策略，将多产品 ELISA 用于过程控制和放行分析，并结合 MS 测试进行正交 HCP 表征，以了解 HCP 水平、簇和种类。这种组合支持针对 HCP 的控制策略，以解决生物制药产品的安全性和疗效问题。
