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、独特的生物制品。在过去的一年里，该实验室进行了几个过程，包括细菌、酵母、昆虫细胞和哺乳动物细胞的繁殖(体积从2升到50升不等)，并进行了生物分子的初步回收和纯化。下面是所执行的过程的例子：细菌的生长，如大肠杆菌、非致病性炭疽芽孢杆菌和各种重组毕赤酵母，一种甲基营养酵母菌株。此外，还对CHO、PG13、HEK293等哺乳动物细胞，SF9、Hi 5等昆虫细胞进行了瞬时表达重组蛋白的培养。不同的合作研究项目需要不同的产品，例如毕赤酵母(NCI)的P-糖蛋白(膜蛋白转运体)的表达，炭疽芽孢杆菌(NIH临床中心)的肽聚糖的表达，以及大肠杆菌(FDA)的多糖的表达。为了生产潜在的疟疾疫苗，进行了几次大规模的巴斯德毕赤酵母试验性生长(NIAID)。该实验室完成了与国家癌症研究所外科分院的项目，以改进过继T细胞治疗所需的伽马逆转录病毒载体的生产。现有的程序是基于固定载体上生长的贴壁包装细胞的生产，这是获得适当载体的速率限制步骤。改进的工艺是基于在搅拌槽式生物反应器中将贴壁细胞生长在微载体上。使用这种方法，我们可以更高产量地生产具有感染性的颗粒。对工艺进行了优化，并加入了灌流系统，使我们可以增加细胞数量和产量。我们还继续致力于开发一种替代的生产方法，该方法基于悬浮生长的细胞中的慢病毒载体。该实验室生产的另一种蛋白质是色素上皮衍生因子(PEDF)，它是从HEK Ebna细胞表达的，国家眼科研究所需要进行结构研究。我们还与疫苗研究中心一起进行了埃博拉研究抗体生产过程的早期开发。对不同类型的培养基和转染剂在不同的生物反应器结构下进行了试验。今年下半年，该实验室与约翰霍普金斯大学一起，启动了从哺乳动物细胞中生产尖峰蛋白的工艺开发，用于柯萨奇病毒19的研究。这包括放大不同哺乳动物细胞系的蛋白质表达，重点是下游的回收和纯化。另一个与COVID19有关的项目是NCI正在开发的从大肠杆菌中回收噬菌体展示的SARS-CoV-2表位的初步过程。

Summary: An essential part of the Lab activities is the production of high-quality, unique biologicals that are not available from commercial sources. During the last year the Lab performed several processes, which include propagation of bacteria, yeast, insect cells, and mammalian cells (in volumes ranging from 2 to 50 liters) and executed initial recovery and purification of biomolecules. The following are examples of processes performed: growth of bacteria such as Escherichia coli, nonpathogenic Bacillus anthracis, and various recombinant Pichia pastoris, a methylotrophic yeast strains. In addition, mammalian cells such as CHO, PG13 and HEK 293, insect cell such as Sf 9 and Hi five, for transient expression of recombinant proteins were propagated. The various products were needed for different 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). Several large-scale experimental growth of Pichia pastoris for production of potential malaria vaccine were performed (NIAID). The lab completed its project with the surgery branch of the National Cancer Institute for improving the production of gammaretroviral vectors needed for adoptive T cell therapy. The existing procedure was based on production from adherent packaging cells growing on stationary support which is a rate limiting step for obtaining the proper vector. The modified process is based on growing the adherent cells on microcarriers in a stirred tank bioreactor. Using this methodology, we could produce infective particles in higher yield. The process was optimized and perfusion system which allow us to increase the cell number and the production yield was incorporated. We also continued to work on the development of an alternative production approach based on lentiviral vector from cells growing in suspension. Another protein that the lab produced is Pigment epithelium-derived factor (PEDF) which was expressed from HEK Ebna cells and was needed for structural studies by the National Eye Institute. We also conducted earlier development of antibodies production process for Ebola research with the Vaccine Research center. Different types of media and transfection agents in different bioreactor configuration were tested. In the second half of the year the lab, together with John Hopkins university, initiated process development for production of Spike protein for COVID19 research from mammalian cells. This involved scaling up of the protein expression from different mammalian cell line with emphasis on downstream recovery and purification. Another project related to the COVID 19 was the initial development of recovery process of phage-displayed SARS-Cov-2 epitopes from E. coli which is being developed by the NCI.