Large-scale production and purification of biological compounds

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

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.