Large-scale production and purification of biological compounds

摘要： 实验室活动的一个重要部分是生产商业来源无法获得的高质量和独特的生物制品。平均而言，该单位去年进行了大约100个过程，包括细菌、酵母、昆虫细胞和哺乳动物细胞的发酵(体积从2升到100升不等)，以及生物分子的初步回收和纯化。下面是所进行的过程的例子：细菌的生长，如大肠杆菌，(重组和天然)各种重组酵母菌株，如酿酒酵母和毕赤酵母。此外，还对HeLa、CHO、MDCK、HEK293等哺乳动物细胞，SF9、High Five等昆虫细胞进行了瞬时表达重组蛋白的培养。许多合作研究项目需要各种产品：毕赤酵母(NCI)的P-糖蛋白(膜蛋白转运体)的表达，人NPP1在HEK 293细胞中的表达(耶鲁大学医学院)，以及来自炭疽芽孢杆菌的肽聚糖(NIH临床中心)。 该实验室与NCI的外科分部共同发起了一个新项目，以改进目前用于生产过继T细胞治疗所需的伽马逆转录病毒载体的方法。现有的程序是基于固定支架上生长的贴壁包装细胞的生产，这是本研究中的一个限速步骤。我们的目标是通过测试使用搅拌槽反应器的各种方法来改进生长过程。在生物反应器中成功地进行了包装细胞生长的初步实验。

Summary: An important portion of the Lab activities is the production of high-quality and unique biologicals that are not available from commercial sources. On average, the Unit performs around 100 processes last year, which include fermentation of bacteria, yeast, insect cells, and mammalian cells (in volumes ranging from 2 to 100 liters), and initial recovery and purification of biomolecules. The following are examples of processes performed: growth of bacteria such as Escherichia coli, (recombinant and native) various recombinant yeast strains such as Saccharomyces cerevisiae and Pichia pastoris. In addition, mammalian cells such as HeLa, CHO, MDCK and HEK 293, insect cell such Sf 9, and High five for transient expression of recombinant proteins were propagated. The various products were needed for a number of collaborative research projects: Expression of P-glycoprotein (membrane protein transporter) from pichia pastoris (NCI), expression of human NPP1 in HEK 293 cells (Yale university medical school), and peptidoglycan from Bacillus anthraces (NIH clinical center). The lab initiated a new project together with the surgery branch of the NCI for improvement of the method currently used for production of gammaretroviral vectors needed for adoptive T cell therapy. The existing procedure is based on production from adherent packaging cells growing on stationary support which is a rate limiting step in this research. Our aim is to improve the growth procedure by testing various methodologies using stirred tank reactor. Initial experiments were successfully conducted for the growth of the packaging cells in bioreactors