Cell Line Development for High-Level Expression of Recombinant Proteins
Texcell’s Cell Culture team has nearly 25 years of experience using cell-based systems for protein expression. In this capacity, a wide range of cell types (e.g. CHO, hybridomas, and various animal/human cell lines) have been used to produce monoclonal antibodies and other recombinant proteins using a wide variety of expression systems.
A key factor in controlling production costs of biopharmaceutical proteins is the rapid development of recombinant cell lines that produce high levels of expressed protein products.
To help facilitate achieving this goal, our team offers the following important core services to expedite the development process:
- Cloning or sub-cloning your gene-of-interest (GOI) into an expression vector of your choice
- Site-directed mutagenesis and/or codon optimization
- Sequence verification of the recombinant expression construct
- DNA preparation and quantification
- Transfection of recombinant DNA into cell lines of your choice
- Stable cell selection using appropriate selective agents (G418, hygromycin, puromycin, etc.)
- Gene amplification using DHFR/MTX or GS/MSX amplification systems to increase recombinant protein expression levels
- Single-cell cloning by limiting dilution and/or FACS sorting
- Cell clone validation by ELISA and/or Western analysis
- Cryopreservation of the high-level expresser cell line clones for future use
- Preparation of Master Cell Banks and Working Cell Banks from selected clonal cell lines
- Testing the MCB and WCB for Mycoplasma and Sterility prior to shipping
Using the above approaches, an effective selection strategy can be designed to identify cell lines that should express high levels of recombinant proteins in culture; these cells will then be ideally suited for large-scale production of proteins in standard fed-batch fermentation processes.
The entire cell line development process typically requires 5 to 12 months to complete, depending on the complexity of the gene cloning and cell line characterization requirements. Throughout this effort, the Texcell team will communicate frequently with you and keep you well-informed as various milestones in the development process are completed successfully.