Cell line name CHO-GS

Synonyms GS-CHO

Species of origin Cricetulus griseus(Chinese hamster) (Cricetulus barabensis griseus) (NCBI Taxonomy: 10029)

Hierarchy Parent: (CHO-K1)

Sex of cell Female

Category Spontaneously immortalized cellline

Chinese Hamster Ovary (CHO) Cells

Biological therapeutics such as monoclonalantibodies are an increasingly prominent part of many drug developmentpipelines.

Horizon Discovery believes that to maintainpace with industry needs in this area, access to Chinese Hamster Ovary or CHOcells must be improved to make them available to companies of all sizes.

High entry costs and restrictive licensingconditions are prohibitive for access to CHO cells for the majority ofbiomanufacturing groups.

CHO cells have become the expression systemof choice for the manufacturing of biological therapeutics. They have beenshown to have the capacity to express a variety of proteins such as therapeuticenzymes or monoclonal antibodies at multi-gram per litre titres.

As expression technologies have developed,focus on increasing titre has mainly been achieved through improvements tomedia and feed, while the ability to identify high productivity clones has beenstreamlined through the use of different selection systems.

Antibiotic selection has been used for a numberof years, but the requirement to maintain cells in antibiotics is costly andrequires removal of the antibiotic from the production media during downstreamsteps, which means that metabolic selection has become the preferred industrymethod.

Metabolic selection can broadly be splitinto those that use Glutamine Synthetase (GS) or Dihydrofolate reductase (DHFR)systems.

Glutamine Synthetase (GS) Null

Glutamine Synthetase is an enzyme thatcatalyzes the conversion of glutamate to the amino acid glutamine and is theonly mechanism for cells to generate their own glutamine. If the expression ofGlutamine Synthetase is reduced through chemical or genetic means, then thecells are not viable unless they are either cultured in media containingadditional glutamine or have an alternative Glutamine Synthetase exogenouslyexpressed. This mechanism has been exploited for over ten years to generate ametabolic selection system that links the expression of an exogenous GS gene tothe expression of a protein of interest (for example a monoclonal antibody).This means that when the cassette stably integrates into the genome, expressionof the monoclonal antibody is proportional to the amount of GlutamineSynthetase expressed.

Cells can then be placed into media thatlacks glutamine, and those expressing insufficient GS (and by extension a lowlevel of monoclonal antibody) are unable to survive. Originally, the mouse cellline NS0 exploited this mechanism as it is naturally deficient in GlutamineSynthetase. To adapt this system for use in CHO cell expression, GS wasinhibited by the chemical inhibitor Methionine Sulphoximine (MSX). However,this led to high levels of background due to the cell line increasing theexpression of its endogenous GS gene, and MSX needed to be included inproduction culture to maintain the selection. As a highly toxic compound, thisneeds to be removed from the production media during downstream processing,leading to increased costs and time. More recently, CHO K1 cells have beenengineered to be null for Glutamine Synthetase. Horizon Discovery hasengineered a GS null CHO cell line using its proprietary rAAV technology, whileLonza used Meganucleases and Sigma Aldrich used Zinc Fingers (ZFNs). This GSnull CHO K1 selection system is now considered to be the industry standardmethod of selecting high expressing clones following transfection with a vectorexpressing the biotherapeutic.

An alternative metabolic selection systemexists that utilizes the DHFR gene. DHFR reduces dihydrofolic acid to tetrahydrofolicacid and in its absence, cells require supplementation of the media withglycine, hypoxanthine, and thymidine for viability. To reduce the level of DHFRin the cells, increasing levels of the chemical inhibitor Methotrexate (MTX)was used, or there are also cells lines (such as DG44 cells) that are null forthe DHFR gene.

Similar to the GS system, vectors have beendesigned that express the protein of interest (for example a monoclonalantibody) as well as a separate expression of an exogenous DHFR gene. However,due to decreased timelines associated with the GS system, this has become thesystem of choice for most companies.

Furthermore, metabolic selection using MSXor MTX is often performed using a number of amplification steps to increase copynumber and expression levels of clones, which has led to some concerns over thelong-term stability of the final producer clone. Together with the increaseddownstream processing burden, null cell lines are a preferable technology forutilizing the metabolic selection through DHFR or GS.

Publications

DOI=10.1016/j.enzmictec.2004.08.027

Omasa T., Yamanaka M., Tanimura N.,Katakura Y., Kishimoto M., Suga K.-I., Enosawa S.

Expression and amplification of glutaminesynthetase gene endows HepG2 cells with ammonia-metabolizing activity forbioartificial liver support system.

Enzyme Microb. Technol. 35:519-524(2004)

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