Production and purification of biological compounds

简介:

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.