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 production of unique biologicals that are not available from commercial sources at a suitable quality, for basic and clinical research. During the last year the Lab performed several processes, which include propagation of bacteria, and mammalian cells (in shake flasks and different size bioreactors) and executed initial recovery and purification of biomolecules. The following are examples of processes performed: growth of bacteria such as Escherichia coli and nonpathogenic Bacillus anthracis, yeast such as Pichia pastoris mammalian cells such as CHO, PG13 and HEK 293, for expression of recombinant proteins. 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 anthracis (NIH clinical center) and polysaccharides from E. coli (FDA). The Lab was heavily involved, together with Professor Andrew Pekosz, Department of Molecular Microbiology & Immunology Johns Hopkins University Bloomberg School of Public Health, in the production and initial purification of mg quantities of different Covid 19 proteins; especially different spike proteins and different receptor binding domain (RBD) which were needed for clinical evaluations. The production was done by expressing the covid proteins from HEK 293 cells growing in defined media. The process was associated with scaling up the protein expression with an emphasis on downstream recovery by using tangential flow filtration process. 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.