Purification of recombinantly produced biopharmaceuticals involves removal of host cell material, such as host cell proteins (HCPs). For lysates of the common expression host Escherichia coli (E. coli) over 1500 unique proteins can be identified. Currently, understanding the behavior of individual HCPs for purification operations, such as preparative chromatography, is limited. Therefore, we aim to elucidate the elution behavior of individual HCPs from E. coli strain BLR(DE3) during chromatography. Understanding this complex mixture and knowing the chromatographic behavior of each individual HCP improves the ability for rational purification process design. Specifically, linear gradient experiments were performed using ion exchange (IEX) and hydrophobic interaction chromatography, coupled with mass spectrometry-based proteomics to map the retention of individual HCPs. We combined knowledge of protein location, function, and interaction available in literature to identify trends in elution behavior. Additionally, quantitative structure–property relationship models were trained relating the protein 3D structure to elution behavior during IEX. For the complete data set a model with a cross-validated R² of 0.55 was constructed, that could be improved to a R² of 0.70 by considering only monomeric proteins. Ultimately this study is a significant step toward greater process understanding.

中文翻译：

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制备色谱过程中大肠杆菌宿主细胞蛋白质组保留的实验表征和预测


重组生产的生物制药的纯化涉及去除宿主细胞材料，例如宿主细胞蛋白 （HCP）。对于常见表达宿主大肠杆菌 （E. coli） 的裂解物，可以鉴定出 1500 多种独特蛋白质。目前，对单个 HCP 在纯化操作（如制备色谱）中的行为了解有限。因此，我们的目标是阐明色谱过程中大肠杆菌菌株 BLR（DE3） 中单个 HCP 的洗脱行为。了解这种复杂的混合物并了解每种 HCP 的色谱行为可以提高合理纯化工艺设计的能力。具体来说，使用离子交换 （IEX） 和疏水相互作用色谱进行线性梯度实验，并结合基于质谱的蛋白质组学，以绘制单个 HCP 的保留情况。我们结合了文献中可用的蛋白质位置、功能和相互作用知识来确定洗脱行为的趋势。此外，还训练了定量结构-性质关系模型，将蛋白质 3D 结构与 IEX 期间的洗脱行为相关联。对于完整的数据集，构建了一个交叉验证 R² 为 0.55 的模型，通过仅考虑单体蛋白，可以将其改进为 R² 为 0.70。最终，这项研究是朝着更深入的过程理解迈出的重要一步。