Large-scale production and purification of biological compounds

生物化合物的大规模生产和纯化

• 批准号: 9356265
• 负责人: Joseph Shiloach
• 金额: $ 57.8万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9356265
• 关键词: Bacillus (bacterium); Bacteria; Biocompatible Materials; Biological; Bioreactors; Biotechnology; Cell Count; Cell Density; Cells; Clinical; Development; Escherichia coli; Fermentation; Goals; Growth; Human; Insecta; Laboratories; Laboratory Research; Lentivirus Vector; Maintenance; Mammalian Cell; Membrane Proteins; Methodology; Methods; National Institute of Diabetes and Digestive and Kidney Diseases; Operative Surgical Procedures; P-Glycoprotein; Peptidoglycan; Perfusion; Pichia; Plants; Polysaccharides; Procedures; Process; Production; Recombinant Proteins; Recombinants; Recovery; Research; Research Project Grants; Source; Suspension substance; Suspensions; System; T cell therapy; Tissues; Transfection; United States National Institutes of Health; Yeasts; base; drug development; improved; instrumentation; large scale production; microorganism; operation; particle; structural biology; vaccine development; vector

Summary: An essential part 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 50 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 Pichia pastoris. In addition, mammalian cells such as CHO, MDCK and HEK 293, insect cell such as Sf 9, for transient expression of recombinant proteins were propagated. The various products were needed for a number of collaborative research projects such as expression of P-glycoprotein (membrane protein transporter) from pichia pastoris (NCI), peptidoglycan from Bacillus anthraces (NIH clinical center) polysaccharides from E. coli (FDA) and production of human OLFM4 from HEK 293 cells. The lab continues its new project (that was initiated last year) together with the surgery branch of the NCI for improving the current method for the 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 modified process is based on growing the adherent cells on microcarriers in a stirred tank bioreactor. In this methodology we were able to produce infective particles in higher yield. We are now optimizing this process and in addition setting up perfusion system which will allow us to increase the cell number and the production yield. Another approach that we are initializing is an alternative production approach based on lentiviral vector from cells growing in suspension.

概括： 实验室活动的重要组成部分是商业来源无法获得的高质量和独特生物学作品。平均而言，该单元去年执行约50个过程，其中包括细菌，酵母，昆虫细胞和哺乳动物细胞的发酵（在2至100升的体积中）以及生物分子的初始恢复和纯化。以下是进行过程的示例：细菌的生长，例如大肠杆菌（重组和天然）各种重组酵母菌菌株，例如Pichia Pastoris。此外，将哺乳动物细胞（例如CHO，MDCK和HEK 293，例如SF 9）用于重组蛋白的瞬时表达。需要进行许多协作研究项目，例如来自Pichia Pastoris（NCI）（NCI）的P-糖蛋白（膜蛋白转运蛋白）的表达，来自芽孢杆菌（NIH临床中心）多糖的肽聚糖（FDA）和来自E. coli（FDA）的肽聚糖的表达以及来自Hek 293 Hek 293细胞的OlfM4的多糖含量。该实验室继续其新项目（去年启动）以及NCI的手术分支，用于改善当前生产胶体逆转录病毒载体的方法。现有的过程基于从固定支持上生长的粘附包装单元的生产，这是这项研究的速率限制步骤。我们的修改过程是基于在搅拌罐生物反应器中的微载体上生长的粘附细胞。在这种方法中，我们能够以更高的产量产生感染性颗粒。现在，我们正在优化此过程，此外，还设置了灌注系统，这将使我们能够增加细胞数量和生产产量。我们正在初始化的另一种方法是一种基于慢病毒载体的替代生产方法。

项目成果

ENPP1-Fc prevents mortality and vascular calcifications in rodent model of generalized arterial calcification of infancy.

• DOI: 10.1038/ncomms10006
• 发表时间: 2015-12-01
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Albright RA;Stabach P;Cao W;Kavanagh D;Mullen I;Braddock AA;Covo MS;Tehan M;Yang G;Cheng Z;Bouchard K;Yu ZX;Thorn S;Wang X;Folta-Stogniew EJ;Negrete A;Sinusas AJ;Shiloach J;Zubal G;Madri JA;De La Cruz EM;Braddock DT
• 通讯作者: Braddock DT