Purity by design: Reducing impurities in bioproduction by stimulus-controlled global translational downregulation of non-product proteins
Purity by design: Reducing impurities in bioproduction by stimulus-controlled global translational downregulation of non-product proteins
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DOI：
10.1016/j.ymben.2018.11.007
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发表时间：
2019-03-01
影响因子：
8.4
通讯作者：
Fussenegger, Martin
中科院分区：
文献类型：
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作者：
Bojar, Daniel;Fuhrer, Tobias;Fussenegger, Martin
利用哺乳动物细胞进行复杂的翻译后修饰的能力，目前大多数蛋白质治疗剂在细胞培养系统中生产。向产物蛋白质中添加信号肽使其能够在细胞培养上清液中积累，但是从内源性分泌的蛋白质中分离产物仍然是昂贵且劳动密集的。我们认为非产物蛋白翻译的全局下调将是最小化下游加工要求的有效策略。因此，利用哺乳动物蛋白激酶R（PKR）在病毒感染时关闭大多数细胞翻译过程的能力，我们将咖啡因诱导的二聚化结构域融合到PKR的催化结构域。向该构建体中加入咖啡因导致PKR的同源二聚化和活化，有效地将快速的全局翻译下调重新连接到以剂量依赖性方式加入刺激物。然后，为了保护靶向治疗剂的翻译，我们筛选了已知或怀疑对PKR诱导的翻译应激具有抗性的病毒和细胞内部核糖体进入位点（IRES）。在选择了同类最佳的塞内卡谷病毒（SVV）IRES后，我们还筛选了IRES反式激活因子（ITAF）以及补充性小分子，以进一步提高产物蛋白在整体翻译下调条件下的生产滴度。重要的是，在最大下调下大约10%的残余总体翻译活性足以在至少五天的生产时间范围内维持细胞活力。用这种合成的生物学启发的蛋白激酶R-增强的蛋白质生产（PREPP）系统转染的标准工业上使用的贴壁以及悬浮适应的细胞系可以大量生产几种医学相关的蛋白质治疗剂，例如重磅炸弹药物利妥昔单抗，并且比以前的培养技术具有显著更高的纯度。我们相信，在生产过程中采用这种设计纯度技术将缓解未来生物制药生产中的下游加工瓶颈。
Capitalizing on the ability of mammalian cells to conduct complex post-translational modifications, most protein therapeutics are currently produced in cell culture systems. Addition of a signal peptide to the product protein enables its accumulation in the cell culture supernatant, but separation of the product from endogenously secreted proteins remains costly and labor-intensive. We considered that global downregulation of translation of non-product proteins would be an efficient strategy to minimize downstream processing requirements. Therefore, taking advantage of the ability of mammalian protein kinase R (PKR) to switch off most cellular translation processes in response to infection by viruses, we fused a caffeine-inducible dimerization domain to the catalytic domain of PKR. Addition of caffeine to this construct results in homodimerization and activation of PKR, effectively rewiring rapid global translational downregulation to the addition of the stimulus in a dose-dependent manner. Then, to protect translation of the target therapeutic, we screened viral and cellular internal ribosomal entry sites (IRESes) known or suspected to be resistant to PKR-induced translational stress. After choosing the best-in-class Seneca valley virus (SVV) IRES, we additionally screened for IRES transactivation factors (ITAFs) as well as for supplementary small molecules to further boost the production titer of the product protein under conditions of global translational downregulation. Importantly, the residual global translation activity of roughly 10% under maximal downregulation is sufficient to maintain cellular viability during a production timeframe of at least five days. Standard industrially used adherent as well as suspension-adapted cell lines transfected with this synthetic biology-inspired Protein Kinase R-Enhanced Protein Production (PREPP) system could produce several medicinally relevant protein therapeutics, such as the blockbuster drug rituximab, in substantial quantities and with significantly higher purity than previous culture technologies. We believe incorporation of such purity-by-design technology in the production process will alleviate downstream processing bottlenecks in future biopharmaceutical manufacturing.