Raising the bar on mammalian cell line engineering
Gene Product Expression Technology (GPEx®) sets a new standard in mammalian cell line engineering. Our proprietary offering is faster, more reliable, and more flexible that traditional methods of gene insertion. GPEx® technology can ensure that genetically stable cell lines are produced 100% of the time.
It is the ideal mammalian cell line technology when you want to:
- Quickly choose the best clinical candidate from a group of potential molecules
- Get to a stable Master Cell Bank (MCB) rapidly
- Generate protein for clinical trials as soon as possible
To date, Catalent has produced over 200 different antibodies and antibody fusions and over 50 different recombinant proteins using the GPEx® technology system.
The advanced mammalian cell line technology in GPEx® can substantially impact your outcomes:
- Faster timelines
- From cDNA to milligram quantities in 2 months
- From cDNA to master cell bank candidate in 4.5 months
- More reliability
- Consistent genetic stability and high production levels with no need for antibiotic selection
- A cell line and antibody production process yielding 2.5 - 5.0 g/L six months after start of a project
- Over 250 different proteins successfully expressed to date, with established worldwide regulatory path
- Increased yield
- Produces mammalian cell lines that deliver higher initial yields of proteins than any conventional system based on transfection or electroporation
- Targets high-expressing “open” regions in the target cell genome, leading to markedly higher expression of the gene in transduced cells compared with other systems
- Optimization of GPEx® cell lines employs an iterative insertion process that drives up the inserted gene copy number and proportionally increases protein expression levels – resulting in a dozen or more copies of the desired gene, all stably inserted and expressing the target protein
- Consistently produces cell lines with productivities greater than 50 p/c/d
- Superior cell stability to any other method
- Stability is inherent to the technology, hence stability studies do not need to be on the critical path for cell line development
- Rock-solid genetic stability
- Consistent mRNA expression from gene inserts
- Better flexibility and unmatched versatility
- Technology works on any mammalian cell line with consistent results
- Demonstrated success with a wide variety of protein types
- Monoclonal antibody heavy- and light-chain coexpression
- Receptors, coexpressed with a secreted surrogate
- Inactive proteins, coexpressed with associated processing enzymes
- Sequential transduction used to introduce required genes without antibiotic selection
- Expressions enhanced by clonal selection or additional rounds of transduction without antibiotic selection
- Risk reduction
- Established worldwide regulatory path with excellent track record
- Cell lines developed in serum-free culture media
- No antibiotic selection requirement
- Great value
- Maximized value compared to other cell line development methods with faster timelines, higher expression levels, better stability, and favorable intellectual property
How It Works
The GPEx® method utilizes replication defective retroviral vectors to stably insert single copies of genes at multiple genomic locations into dividing cells. Retrovectors deliver genes coded as RNA that, after entering the cell, are reverse transcribed to DNA and integrated stably into the genome of the host cell. Two enzymes, reverse transcriptase and integrase, provided transiently in the vector particle, perform this function. These integrated genes are maintained through subsequent cell divisions as if they were endogenous cellular genes. By controlling the number of retrovector particles accessing the cell, multiple gene insertion (desirable for high-yielding cell cultures) is achieved without any of the traditional amplification steps.
- GPEx® technology can be used on a wide variety of cell lines.
- GPEx® technology utilizes a specific envelope on the retrovector particles. This envelope protein allows the retrovectors to insert genes into all mammalian cells, in addition to numerous other cell types, due to its ability to bind to various membrane phospholipids and glycolipids. On some occasions, a protein you are making might need specific post-translational modifications that are not normally performed in CHO cells or may have toxic effects on CHO cells. In these instances a different cell type may allow efficient post-translational modification and secretion of the protein of interest.
- Each copy of the transgene is inserted at a different genomic location.
- Retrovector gene insertions occur at unique locations in the cell genome, with a single copy of the gene being inserted at each independent site. Unlike most other methods of transgene insertion that are undefined “passive” processes, each insertion by a retrovector is an “active” process that is modulated by the integrase enzyme. This unique insertion process eliminates the occurrence of “head-to-tail” multiple-single-loci transgene inserts in these cell lines. GPEx® technology genetic insertion is extremely stable, and cell lines generated by this method do not require detailed testing of stability before the production of the master cell bank.
- GPEx® transgene inserts target “open” or “active” regions of the cell genome.
- The GPEx® retrovectors have been shown to preferentially insert into or around the transcription start point of genes. This preference for transcriptionally “active” regions of the genome allows for higher, more consistent levels of expression per copy of the gene inserted as compared to other methods of gene insertion. Protein producing cell clones generated by this method are extremely consistent, and only a few hundred clones are screened to identify high-expressing master cell bank candidate clones.
- GPEx® technology does not require antibiotic selection or use of toxic compounds for gene amplification.
- Due to the extremely high gene-insertion efficiency of the GPEx® technology process, no selectable markers (e.g., neomycin, blasticidin, hygromycin, or puromycin resistance genes) are needed for cell line generation. This has a number of advantages over other cell line development methods including reduced costs for culturing cells, no additional taxing of the cells due to production of the selectable marker, and reduced time to clonal cell line selection. The high transduction efficiency and the ability to do repeat cell transductions generate high copy number cell lines using this process, eliminating the need to amplify gene copy number by adding toxic compounds such as methotrexate. Re-transduction of cells yields clonal lines with copy numbers ranging from 25 - 250.
- GPEx® technology allows straightforward addition of extra genes to cell pools or previously developed cell lines.
- The high transduction efficiency, coupled with no antibiotic selection requirement, allows easy addition of one or more genes to newly developed or already established cell lines. For antibodies, heavy and light chains are easily titrated to the correct gene ratio to yield maximum antibody production and efficient antibody formation. Gene ratio titering can be accomplished through specific screening during clonal selection or an individual transduction of a specific chain, if required. In addition, protein processing enzymes can easily be added to cell lines already producing protein that may not be fully processed.
- GPEx® technology generates high-expressing cell population prior to clonal selection
- After the initial transductions are completed, cell pools are available for small to large scale production. These cell pools have been expanded up to 100L scale for production. Typical antibody producing cell pools will secrete antibody at levels of 500 mg/L to 1,500 mg/L at this early stage in the development process (prior to selection of a high-expressing clone). Milligrams to multi-gram quantities of antibody are produced from these cell pools.
- Cell line development
- Initial protein product in 2.5 months and MCB candidate in 4.5 months
- Process development
- Cell line optimization and gram-scale production and purification from small bioreactors
- Consistent processes yielding 2.5 – 5.0 g/L for antibodies 6 months after start of cell line development
- Robust purification processes developed in parallel with upstream process development
- cGMP manufacturing
- Phase I/Phase II cGMP production of clinical trial material
- Development program planning
- Customized program designed including:
- Milestone-based feasibility study reflecting your development plan
- Production of a stable high-expressing mammalian cell line and gram quantities of protein for your analysis and preclinical needs
- Licensing cell line for production
- At the conclusion of the feasibility study there are several options:
- Cell line for production available to be out-licensed at the facility of your choice
- cGMP product for Phase I/Phase II clinical trials manufactured at our Madison, Wisconsin facility
- Monoclonal antibody production
- GPEx® technology expresses any type of IgG: full-length antibodies, Fab fragments, chimeric antibodies, antibody fusion proteins, and single-chain antibodies.
- Antibody variable regions are capable of being inserted into our backbone retrovectors for cell line development and protein production of your desired antibody.
- Backbone vectors containing the constant region for human IgGs 1, 2, 3, and 4 and light-chain kappa and lambda regions are maintained by Catalent.
- DNA sequences encoding IgG variable regions capable of being fused to any constant region in the GPEx® retrovector system.
- Any desired IgG molecule produced using this system.
- Variant analysis
- GPEx® technology ideal for protein variant analysis and screening
- Milligram amounts of several protein variants delivered quickly from a stable pool
- A selected clone from this pool is then used for a fast path to clinical manufacturing