Metabolic selection is now the industry’s preferred approach during cell line development, avoiding the high cost associated with antibiotic selection (maintaining cells in antibiotics) and the need to remove the antibiotics during downstream production.
Glutamine Synthetase catalyzes the conversion of glutamate to the amino acid glutamine and is the only mechanism for cells to generate their own glutamine. If the expression of Glutamine Synthetase is reduced through chemical or genetic means, then the cells are not viable unless they are either cultured in media containing additional glutamine or have an alternative Glutamine Synthetase exogenously expressed.
Over the years this mechanism has developed into a metabolic selection system that links the expression of an exogenous GS gene to the expression of a protein of interest, such as a monoclonal antibody. As a result, when the cassette stably integrates into the genome, expression of the monoclonal antibody is proportional to the amount of Glutamine Synthetase expressed. Cells can then be placed into media that lacks glutamine, where those expressing insufficient GS (and by extension a low level of monoclonal antibody) are unable to survive.
Originally, the mouse cell line NS0 exploited this mechanism as it is naturally deficient in Glutamine Synthetase. To adapt this system for use in CHO cell expression, GS was inhibited by the chemical inhibitor Methionine Sulphoximine (MSX). However, this led to high levels of background due to the cell line increasing the expression of its endogenous GS gene, and MSX needed to be included in the production culture to maintain selection. As a highly toxic compound, MSX needs to be removed from the production media during downstream processing, leading to increased costs and time.
More recently, CHO K1 cells have been engineered to be null for Glutamine Synthetase. Horizon Discovery has engineered a GS null CHO cell line using proprietary rAAV technology, creating a system that, after transfection with a vector expressing the biotherapeutic, is becoming an industry standard for selection of highly expressing clones.
Please contact us to discuss your bioproduction/biomanufacturing cell line needs and to find out how we can meet them.