National Institute for Bioprocessing Research and Training, Fosters Avenue, Blackrock, Co, Dublin, Ireland.
School of Chemical and Bioprocess Engineering, University College Dublin, Belfield, Dublin, Ireland.
Nat Commun. 2024 Oct 4;15(1):8605. doi: 10.1038/s41467-024-51870-0.
Chinese hamster ovary (CHO) cells are used to produce almost 90% of therapeutic monoclonal antibodies (mAbs) and antibody fusion proteins (Fc-fusion). The annotation of non-canonical translation events in these cellular factories remains incomplete, limiting our ability to study CHO cell biology and detect host cell protein (HCP) impurities in the final antibody drug product. We utilised ribosome footprint profiling (Ribo-seq) to identify novel open reading frames (ORFs) including N-terminal extensions and thousands of short ORFs (sORFs) predicted to encode microproteins. Mass spectrometry-based HCP analysis of eight commercial antibody drug products (7 mAbs and 1 Fc-fusion protein) using the extended protein sequence database revealed the presence of microprotein impurities. We present evidence that microprotein abundance varies with growth phase and can be affected by the cell culture environment. In addition, our work provides a vital resource to facilitate future studies of non-canonical translation and the regulation of protein synthesis in CHO cell lines.
中国仓鼠卵巢（CHO）细胞被用于生产近 90%的治疗性单克隆抗体（mAbs）和抗体融合蛋白（Fc 融合蛋白）。这些细胞工厂中非规范翻译事件的注释仍然不完整，限制了我们研究 CHO 细胞生物学和检测最终抗体药物产品中宿主细胞蛋白（HCP）杂质的能力。我们利用核糖体足迹分析（Ribo-seq）来鉴定新的开放阅读框（ORF），包括预测编码微小蛋白的 N 端延伸和数千个短 ORF（sORF）。使用扩展的蛋白质序列数据库，对八种商业抗体药物产品（7 种 mAbs 和 1 种 Fc 融合蛋白）进行基于质谱的 HCP 分析，发现了微小蛋白杂质的存在。我们提供的证据表明，微小蛋白的丰度随生长阶段而变化，并且可能受到细胞培养环境的影响。此外，我们的工作提供了一个重要的资源，以促进未来对 CHO 细胞系中非规范翻译和蛋白质合成调控的研究。
