High-Level Expression of a Recombinant Antibody from Myeloma Cells Using a Glutamine Synthetase Gene as an Amplifiable Selectable Marker

Abstract

We report a method for introducing a glutamine synthetase (GS) selectable marker into myeloma cells in which transfectants are selected by growth in a glutamine-free medium. Vector amplification can subsequently be selected using the specific inhibitor of GS, methionine sulphoximine (MSX). Using this system, DNA sequences encoding a chimeric B72.3 IgG4 antibody were expressed from hCMV-MIE promoters in NSO myeloma cells. A cell line was isolated after a single round of selection for vector amplification which contains approximately 4 copies of the vector, secretes 10-15 pg/cell/day cB72.3 antibody during exponential growth and can accumulate 560 mg/l antibody in a fed-batch air-lift fermentation system. Productivity is stable in the absence of MSX selection.

An engineered anti-CA19-9 cys-diabody for positron emission tomography imaging of pancreatic cancer and targeting of polymerized liposomal nanoparticles

“…This was accomplished using standard PCR components (Invitrogen, Carlsbad, CA) with a 48 bp primer to insert the GGCGG amino acid sequence (5 0 -GAATTCTCAATGATGATGATGATGATGA CCCCCACACCCACCTGCAGA-3 0 ; IDT Integrated DNA Technologies, San Diego, CA). This construct was confirmed by DNA sequencing, excised from the pUC18 vector, and ligated into the pEE12 mammalian expression vector (Lonza Biologics, Slough, United Kingdom) containing the glutamine synthetase gene for selection and the hCMV promoter for high expression [14]. …”

Using cell engineering and omic tools for the improvement of cell culture processes

“…The most common host cell line used for the production of therapeutics is Chinese Hamster Ovary (CHO) (Cockett et al 1990, Milbrandt et al 1983) but apart from this cell line, others that are commonly used for the production of recombinant proteins are: mouse myeloma derived (NS0) (Barnes et al 2001, Bebbington et al 1992, human embryonic kidney (HEK-293) (Baldi et al 2005), baby hamster kidney (BHK) (Wurm 2004) and more recently, the human retina derived (PerC6) (Jones et al 2003) cell lines. For the purpose of process homogeneity, reproducibility and safety, chemically defined protein free medium has become a necessity.…”

“…Selection occurs in the absence of the metabolites hypoxanthine and thymidine and gene amplification is accomplished by exposing the cell line to MTX concentrations, which inhibit DHFR enzyme activity. CHO GS (Glutamine Synthetase) (Cockett et al 1990) and NS0 GS (Bebbington et al 1992) are another standardized selection system, where cells are transfected with a vector containing the gene of interest in addition to the glutamine synthetase (GS) gene. GS is an enzyme that allows the cell to synthesize its own intracellular glutamine from glutamate and the ammonia group provided from asparagines (Barnes et al 2000).…”

Development of Animal-free, Protein-Free and Chemically-Defined Media for NS0 Cell Culture

“…The NS0 wildtype cell line (NS0-WT) was obtained from the European Collection of Cell Culture (ECACC 85110503). NS0 lacks endogenous glutamine synthetase which catalyzes the formation of glutamine from glutamate and ammonia, enabling the selection and the culture of GS-NS0 recombinants in glutamine-free media (Bebbington et al 1992). GS-NS0 Cell Line I secreted a model IgG1 MAb protein, while Cell Line II (licensed from Lonza Biologics) and III both expressed a model IgG4 MAb protein.…”

“…Due to their hardy, suspension growth characteristics and their well-equipped machinery for producing and secreting immunoglobulin proteins, NS0-derived recombinant constructs are commonly employed for MAb expression (Birch and Froud 1994;Barnes et al 2000). In addition, NS0 cells are well-suited for use of the glutamine synthetase (GS) expression system because they lack endogenous GS activity, thus a plasmid containing both GS and the antibody gene of interest can be constructed to allow easy isolation of MAb-secreting transfectants in glutamine-free medium (Bebbington et al 1992). Through process optimization, MAb production from the GS-NS0 system has reached over 2.5 g/l in fed-batch reactor cultures (Zhou et al 1997;Varma et al 2003;Schmelzer et al 2004).…”