Sahoo Ansuman, Tsukiadate Taku, Lin Bor-Ruei, Kotzbauer Erin, Houser Jason, Patel Misaal, Li Xuanwen, Madabhushi Sri Ranganayaki
Biologics Process Research & Development, Merck & Co., Inc., Rahway, New Jersey, USA.
Analytical Research & Development Mass Spectrometry, Merck & Co., Inc., Rahway, New Jersey, USA.
Biotechnol J. 2025 Jan;20(1):e202400567. doi: 10.1002/biot.202400567.
Chinese hamster ovary (CHO) cells are widely used to produce recombinant proteins, including monoclonal antibodies (mAbs), through various process modes. While fed-batch (FB) processes have been the standard, a shift toward high-density perfusion processes is being driven by increased productivity, flexible facility footprints, and lower costs. Ensuring the clearance of process-related impurities, such as host cell proteins (HCPs), is crucial in biologics manufacturing. Although purification processes remove most impurities, integrated strategies are being developed to enhance clearance of some high-risk HCPs. Current understanding of HCP expression dynamics in cell culture is limited. This study utilized data-independent acquisition (DIA) proteomics to compare the proteomic profiles of cell culture supernatants from 14 FB clones and three perfusion clones, all expressing the same mAb from the same host cell line. Results showed that perfusion processes enhance cell growth and productivity, exhibiting distinct proteomic profiles compared to FB processes. Perfusion processes also maintain a more comparable HCP abundance profile across clones, especially for 46 problematic HCPs monitored. Cluster analysis of FB proteomics revealed distinct abundance patterns and correlations with process parameters. Differential abundance analysis identified significant protein differences between the two processes. This is the first extensive study characterizing HCPs expressed by clones under different process modes. Further research could lead to strategies for preventing or managing problematic HCPs in biologics manufacturing.
中国仓鼠卵巢（CHO）细胞被广泛用于通过各种工艺模式生产重组蛋白，包括单克隆抗体（mAb）。虽然补料分批（FB）工艺一直是标准工艺，但由于生产率提高、设施占地面积灵活和成本降低，正推动着向高密度灌注工艺的转变。确保清除与工艺相关的杂质，如宿主细胞蛋白（HCP），在生物制品生产中至关重要。尽管纯化工艺可以去除大部分杂质，但正在开发综合策略以提高对一些高风险HCP的清除率。目前对细胞培养中HCP表达动态的了解有限。本研究利用数据非依赖采集（DIA）蛋白质组学比较了来自14个FB克隆和3个灌注克隆的细胞培养上清液的蛋白质组谱，所有克隆均来自同一宿主细胞系且表达相同的mAb。结果表明，灌注工艺可促进细胞生长和提高生产率，与FB工艺相比，其蛋白质组谱不同。灌注工艺在各克隆间还能保持更可比的HCP丰度谱，尤其是对于监测的46种有问题的HCP。FB蛋白质组学的聚类分析揭示了不同的丰度模式以及与工艺参数的相关性。差异丰度分析确定了两种工艺之间的显著蛋白质差异。这是第一项广泛研究不同工艺模式下克隆表达的HCP的特征的研究。进一步的研究可能会带来生物制品生产中预防或管理有问题的HCP的策略。
