Porosk Ly, Nebogatova Jekaterina, Härk Heleri Heike, Vunk Birgit, Arukuusk Piret, Toots Urve, Ustav Mart, Langel Ülo, Kurrikoff Kaido
Institute of Technology, University of Tartu, Nooruse 1, 50411 Tartu, Estonia.
Icosagen Cell Factory, Eerika tee 1, Õssu, 61713 Tartu, Estonia.
Pharmaceutics. 2022 Sep 14;14(9):1949. doi: 10.3390/pharmaceutics14091949.
Therapeutic proteins are currently at the apex of innovation in pharmaceutical medicine. However, their industrial production is technically challenging and improved methods for transient transfection of mammalian cell cultures are necessary. We aimed to find a fast, microliter-scale transfection assay that allows the prediction of protein expression in the transient production settings. We used an array of lipid, polymeric and cell-penetrating peptide transfection reagents, and compared their performance in various high throughput transfection assays to their performance in protein (antibody) expression in professional protein-producer cell lines. First, we show that some of the most frequently used microliter-scale transfection efficacy assays fail to predict performance in the protein production in milliliter and liter scale settings. We found that CHO suspension culture post-transfection EGFP(+) population and SEAP quantitation correlate with large-scale protein production, whereas the adhesion culture assays and transfection of pLuc are non-predictive. Second, we demonstrated that cell-penetrating peptide-based transfection achieves significantly higher protein yields compared to PEI and lipoplex methods in both CHO and HEK293 producer cell lines. In this work we demonstrate a CPP-based transient protein expression approach that significantly outperformed the current industry standard workhorse method of PEI.
治疗性蛋白质目前处于药物医学创新的前沿。然而，它们的工业化生产在技术上具有挑战性，因此需要改进哺乳动物细胞培养的瞬时转染方法。我们旨在找到一种快速的微升规模转染检测方法，以预测瞬时生产环境中的蛋白质表达。我们使用了一系列脂质、聚合物和细胞穿透肽转染试剂，并在各种高通量转染检测中比较了它们的性能，以及它们在专业蛋白质生产细胞系中蛋白质（抗体）表达方面的性能。首先，我们表明，一些最常用的微升规模转染效率检测方法无法预测毫升和升规模环境中蛋白质生产的性能。我们发现，转染后CHO悬浮培养的EGFP(+)群体和SEAP定量与大规模蛋白质生产相关，而贴壁培养检测和pLuc转染则无法预测。其次，我们证明，在CHO和HEK293生产细胞系中，基于细胞穿透肽的转染与PEI和脂质体方法相比，能显著提高蛋白质产量。在这项工作中，我们展示了一种基于细胞穿透肽的瞬时蛋白质表达方法，其性能明显优于目前行业标准的主力方法——PEI。
