Development of an integrated continuous process for recombinant protein production using Pichia pastoris

该项目将建立在我们在生物加工研究工业俱乐部（BRIC 1）第1阶段的两个项目的成功基础上，以开发一种在具有商业重要性的生产生物体-酵母毕赤酵母中生长相关生产具有商业重要性的蛋白质产品的系统。蛋白质，特别是抗体，现在在人类疾病的治疗中发挥着重要作用。它们通常使用培养中的动物细胞生产，这是一个既昂贵又耗时的过程。另一种有吸引力的方法是改造酵母细胞，使其产生具有治疗价值的人类蛋白质。巴斯德毕赤酵母常用于此目的，因为它可以在高细胞密度下生长，并且具有从细胞释放蛋白质产物的有效分泌系统。目前的方法通过反复向酵母培养物中加入甲醇来诱导蛋白质的产生。我们在BRIC 1中的研究表明，这是完全错误的方法，因为细胞反复受到压力，产生了折叠不良的蛋白质，这些蛋白质具有低生物活性，并且不会分泌出细胞。我们需要一个连续的蛋白质生产和回收过程，这也是我们BRIC 2项目的目标。我们将在BRIC成员公司内部联系人的指导下研究产品蛋白质，其中包括许多用于人类治疗的蛋白质。与增长相关的生产将使生产过程能够连续进行，从而提高商业生产力。BRIC 1中开发的计算机模型将得到改进、扩展，并用于控制新工艺。此外，我们在BRIC 1中开发的快速蛋白质分离方法将允许连续回收产品，从而消除连续系统商业应用的主要障碍-连续生产和分批下游加工之间的不匹配。

This project will build on the successes of our two projects in phase 1 of the Bioprocessing Research Industry Club (BRIC1) to develop a system for the growth-associated production of commercially important protein products in a commercially-important production organism - the yeast Pichia pastoris. Proteins, particularly antibodies, are now playing a major role in the treatment of human disease. They are usually produced using animal cells in culture, a procedure that is both costly and time-consuming. An attractive alternative is to engineer a yeast cell to produce human proteins of therapeutic value. Pichia pastoris is often used for this purpose since it may be grown at high cell densities and has an efficient secretion system to release the protein product from the cells. Current processes induce protein production by repeatedly adding methanol to the yeast cultures. Our studies in BRIC1 demonstrated that this is exactly the wrong way to go about this since the cells are repeatedly stressed and produced badly folded proteins that have low biological activity and are not secreted out of the cells. What is required is a continuous process for protein production and recovery and this is what our BRIC2 project aims to achieve.The product proteins to be studied will be guided by our contacts within the BRIC member companies and include a number of proteins used in human therapies. Growth-associated production will enable continuous processes and thus increase commercial productivity. Computer models developed in BRIC1 will be improved, extended, and used to control the novel process. Moreover, the fast protein separation methods that we also developed in BRIC1, will permit the continuous retrieval of product, thus obviating the major barrier to the commercial adoption of continuous systems - the mismatch between continuous production and batchwise downstream processing.