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产业俱乐部(BRIC1)第一阶段的两个项目的成功基础上，开发一个系统，用于在具有商业重要性的生产有机体--毕赤酵母--中与生长相关地生产具有商业重要性的蛋白质产品。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。它们通常是在培养中使用动物细胞生产的，这一过程既昂贵又耗时。一个有吸引力的替代方案是设计酵母细胞来生产具有治疗价值的人类蛋白质。巴斯德毕赤酵母通常用于这一目的，因为它可以在高细胞密度下生长，并具有有效的分泌系统来从细胞中释放蛋白质产物。目前的工艺通过在酵母培养物中反复添加甲醇来诱导蛋白质生产。我们在BRIC1中的研究表明，这完全是错误的方式，因为细胞反复受到压力，产生严重折叠的蛋白质，这些蛋白质具有低生物活性，不能分泌出细胞。需要的是一个连续的蛋白质生产和回收过程，这也是我们的金砖四国项目的目标。要研究的产品蛋白质将由我们在金砖四国成员公司的联系人指导，包括一些用于人类治疗的蛋白质。与增长相关的生产将使连续过程成为可能，从而提高商业生产率。在BRIC1中开发的计算机模型将得到改进、扩展和用于控制这一新的过程。此外，我们在BRIC1中开发的快速蛋白质分离方法将允许产品的连续回收，从而消除了连续系统商业化采用的主要障碍-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.