Enhancing secretory pathway function to improve production of recombinant therapeutic proteins

基于蛋白质的治疗药物是工业生物技术领域具有重要战略意义和迅速发展的组成部分，通常通过生物加工生产，使用经过改造的哺乳动物宿主细胞来生产高水平的治疗蛋白质（Dumont等人，2016）。在生产一种新型生物制药的关键步骤是克隆细胞系的产生，这种细胞系稳定地表达和分泌大量的蛋白质。SAL Scientific开发的培养基补充物institugro通过提高细胞存活率和随后的增殖能力，极大地提高了DHFR- CHO细胞限制稀释克隆的效率。与SAL Scientific合作的联合主管实验室的初步结果导致了这样的假设，即institugro特别有利于产生产生“难以表达”蛋白质的细胞系。此外，使用instgro选择的品系似乎产生了更多的蛋白质。鉴于分泌途径的能力可能是生物制药蛋白生产的一个限制因素，这就提出了一种可能性，即institugro促进CHO细胞的分泌途径功能，从而增强它们处理和运输“困难”蛋白的能力。该项目将研究“容易”和“困难”的货物蛋白是如何通过一系列细胞类型的分泌途径进行运输的，包括CHO细胞系，它是生物制药行业生产蛋白质的“主力”细胞系（Dumont等人，2016）。我们将使用一种新的活细胞成像方法（Boncompain et al., 2012）结合生化分析来跟踪每种货物从内质网（ER）经高尔基体到达质膜（PM）的动力学和运输。我们将确定哪种类型的膜载体用于高尔基到pm的运输，以及哪种驱动蛋白超家族的微管马达驱动它们的运动（Ruane et al. 2016），因为两者在货物蛋白之间可能不同。将CHO细胞系与其他通常用于运输研究的细胞类型（HeLa, Vero， COS）进行比较，将揭示是否存在分泌途径的适应性，使CHO特别适合于蛋白质生产，并查明困难货物运输中的瓶颈，这些瓶颈可能是未来优化的目标。完成这一特征后，我们将确定instgro如何影响分泌途径和蛋白质生产水平，以及这些影响是否在“困难”vs中得到更大程度的增强。“简单”的蛋白质。

Protein-based therapeutics are a strategically-important and rapidly expanding component of the industrial biotechnology sector, and are typically manufactured via bioprocessing using mammalian host cells engineered to produce high levels of the therapeutic protein (Dumont et al., 2016). A critical step in the production of a novel biopharmaceuticals is the generation of a clonal cell line that stably expresses and secretes the protein in large amounts. InstiGRO, a media supplement developed by SAL Scientific, greatly improves the efficiency of limiting dilution cloning of DHFR- CHO cells by improving cell survival and subsequent proliferation. Preliminary results from the co-supervisor's laboratory in collaboration with SAL Scientific led to the hypothesis that InstiGRO is particularly beneficial for generating lines producing 'difficult-to-express' proteins. In addition, lines selected using InstiGRO appeared to produce more protein. Given that secretory pathway capacity can be a limiting factor in biopharmaceutical protein production, this raises the possibility that InstiGRO promotes secretory pathway function in CHO cells so that their ability to handle and traffic 'difficult' proteins is enhanced. This project will investigate how 'easy' and 'difficult' cargo proteins are trafficked through the secretory pathway in a range of cell types, including CHO lines that are the 'workhorse' cell line for protein production by the biopharmaceutical industry (Dumont et al., 2016). We will use a novel live cell imaging approach (Boncompain et al., 2012) combined with biochemical analysis to follow the kinetics and trafficking of each cargo from the endoplasmic reticulum (ER), via the Golgi apparatus, to the plasma membrane (PM). We will determine which type of membrane carriers are used for Golgi-to-PM transport, and which microtubule motor of the kinesin superfamily drives their movement (Ruane et al. 2016), as both can differ between cargo proteins. Comparing CHO lines with other cell types commonly used for trafficking studies (HeLa, Vero, COS) across a range of cargoes will reveal if there are adaptations of the secretory pathway that makes CHOs particularly well-suited to protein production, and pinpoint bottlenecks in the trafficking of difficult cargoes that could be targets for future optimisation. With this characterisation complete, we will then determine how InstiGRO affects the secretory pathway and protein production levels, and whether these effects are enhanced to a greater extent for 'difficult' vs. 'easy' proteins.