Many proteins produced in CHO cells need evaluation for their clinical and commercial potential. Traditional methods based on stable clone generation are slow and unsuitable for screening larger numbers of proteins, while transient expression technologies are fast but unpredictable regarding product quality and lacking an optional path to subcloning. The STEP® vector technology introduced here combines the best properties of both methods. STEP® vectors contain a strong transcriptional cassette driving expression of a bicistronic mRNA. The gene-of-interest (GOI) is cloned upstream of a functionally impaired zeocin resistance gene (FI-Zeo) whose translation is coupled to that of the GOI through an IRES. Stable transfected cells surviving zeocin selection produce high levels of FI-Zeo and thus, high levels of the GOI-encoded protein. By using different spacers, the translational coupling efficiency and selection strength can be controlled allowing maximization of expression of any GOI. Production of laronidase and factor VII (FVII) is presented as examples of unrelated, difficult-to-express (DTE) proteins. First step is rapid generation of transfected pools with the STEP® vectors. All high expressing surviving pools showed high product quality homogeneity as did monoclonal cell lines obtained from the top pools. Up to 500 μg/mL laronidase was obtained with virtually identical glycosylation profile as reference product. For FVII, cell specific productivity of 0.45 pg/cell/day with 50 IU/μg protein matched highest reported levels of reference product even before process development. Taken together, STEP® vector technology is ideally suited for rapid, small to large-scale production of DTE proteins compared to traditional methods.

CHO 细胞中产生的许多蛋白质需要评估其临床和商业潜力。基于稳定克隆生成的传统方法速度慢，不适合筛选大量蛋白质，而瞬时表达技术速度快，但产品质量无法预测，并且缺乏亚克隆的可选途径。这里介绍的 STEP® 矢量技术结合了两种方法的最佳特性。STEP® 载体包含驱动双顺反子 mRNA 表达的强转录盒。感兴趣的基因 (GOI) 克隆在功能受损的 zeocin 抗性基因 (FI-Zeo) 的上游，该基因的翻译通过 IRES 与 GOI 的翻译耦合。在 zeocin 选择中存活的稳定转染细胞产生高水平的 FI-Zeo，因此产生高水平的 GOI 编码蛋白。通过使用不同的间隔，可以控制平移耦合效率和选择强度，从而最大化任何 GOI 的表达。laronidase 和因子 VII (FVII) 的产生是作为不相关的、难以表达 (DTE) 蛋白质的例子。第一步是使用 STEP® 载体快速生成转染池。所有高表达的存活池都显示出高质量的同质性，从顶部池中获得的单克隆细胞系也是如此。获得了高达 500 μg/mL 的 laronidase，其糖基化谱与参考产品几乎相同。对于 FVII，即使在工艺开发之前，50 IU/μg 蛋白质的细胞比生产率为 0.45 pg/细胞/天，与参考产品的最高报告水平相匹配。总之，STEP® 矢量技术非常适合快速、