ProteoGenix strives to overcome the economic issues related to stable cell line generation by offering the most productive IP free cell lines on the market. With a one-time fee and strong yield guarantees, our stable cell line development service, based on our proprietary cell line, aims at offering high productivity at competitive price and FTO without royalties! ProteoGenix also offers all other most common cell lines to perfectly meet your project requirements!

ProteoGenix strives to offer best-in-class services from target selection to bioproduction. Our unique knowledge of the whole process, combined with our expertise in monoclonal antibody development and protein production, is your best guarantee to obtain a high producing stable cell line.

As a major player in the therapeutic antibody development field, bringing your biotherapeutic from the bench to the clinic is our challenge. Let us support your stable cell line development project and pave the way to success!

Discover our stable cell line development service content

Expression vector construction

  • Gene synthesis optimized for the target expression system.
  • Gene subcloning in a high performing expression vector.

Host cell transfection and generation of stable pools

  • Transfection of the host cells with the recombinant plasmid.
  • Generation and characterization of several stable transfected pools.

Characterization of the best stable pools

  • Selection and amplification of the most producing stable pools for further clone selection

Isolation and selection of the best monoclones

  • Isolation of monoclones by limiting dilution.
  • Screening by ELISA, WB, FACS, IP or Octet Red96.
  • Selection and expansion of the most producing clones.

Characterization of the best monoclones

  • Productivity evaluation in batch and/or fed-batch culture
  • Screening by ELISA, WB, FACS, IP or Octet Red96.
  • RCB preparation and stability study

Upstream process optimization

  • Test of several culture medium and several feed strategy (media/culture optimization).

Stable cell line and protocol transfer



Step Content Timeline Deliverables
Gene synthesis
  • Gene design including codon optimization
  • Gene synthesis
  • Subcloning in expression vector
3 to 4 weeks
  • Gene optimized for the required expression system
  • Gene cloned in pUC57 plasmid
Stable pool generation
  • Stable transfection
  • Pool selection and amplification
9 to 10 weeks
  • 50mL of the supernatant from stable pool
  • Stable pool vials
  • Detailed report
Single cell clones screening and subcloning
  • Subcloning of the top producing clones
  • Characterization of clones
  • Stability study
  • RCB preparation
~4 months
  • Research cell bank -tested for Mycoplasma
  • Certificate of Analysis
  • Detailed report
Process optimization
  • Test of several media/feed conditions
4 to 5 weeks
  • Detailed report
  • Optimized protocol transfer


  • PoC based on transient expression evaluation before to start
  • Additional characterization (KD determination, thermostability, aggregation…)
  • RCB stability study
  • Master and working cell bank development
  • Scale up production / Bioreactor

Available cell lines

Proprietary CHO

  • Freedom to operate (one-time fee payment)
  • MTX/DHFR-mediated amplification and selection


  • Methionine Sulfoximine (MSX)/GS-mediated amplification and selection
  • Rapid and easy transfer for cGMP production


  • No Licence fee before moving towards commercial use
  • MTX/DHFR-mediated amplification and selection


  • Methionine Sulfoximine (MSX)/GS-mediated amplification and selection


  • No Licence fee before moving towards commercial use
  • MTX/DHFR-mediated amplification and selection
  • Used for the bioproduction of FDA-approved biotherapeutics

Other cell lines available

  • HEK293, 293F, 293E
  • Customer cell line

As each stable cell line generation project is unique, our service is tailored to meet your requirements and to ensure a safe investment. Send your requirements to our PhD account managers who will put together a customized offer. Our stable cell line development service is composed of several milestones (Go/ No Go steps) that are accompanied by a detailed report and discussed with our expert team. By building your your project in this way, you can be guaranteed of support throughout the development process giving you unique flexibility.

Have you not successfully achieved a high antibody transient expression yield? Then carry out a transient expression evaluation on our best-in-class high producing proprietary CHO cell line XtenCHO™.

Our XtenCHO™ cell line together with our in-house Xten Protocol is your best chance at overcoming your difficult-to-express protein challenge. This is usually the first step before moving to stable cell line development in order to be able to ensure that all antibody features meet the requirements in CHO based productions.

We maximize your chance to get a high producing stable cell line at competitive price

As a contract research organization (CRO) having successfully produced more than 1500 recombinant proteins and 300 monoclonal antibodies, ProteoGenix constantly strives to offer innovative tools to satisfy and even exceed its customer’s requirements. Proposing a high performance cell line at competitive price is one of our daily challenges. For this reason, a team of dedicated R&D experts is currently working on the development of one of the most productive GS deficient cell line on the market.

Do you need to develop a highly productive stable cell line? Try our customized stable cell line development service.

Stable Cell line generation at ProteoGenix: FAQ

Stable Cell line generation

Metabolic selection and amplification for stable cell line generation

Selection of highly productive stable cell clones is of primary importance when it comes to stable cell line development. For several decades now, metabolic selection has become the preferred method to select the most productive clones for bioproduction. This field is characterized by intense research activity due to the important economic issues related to the production of biotherapeutics. Overcoming this challenge means reducing production costs by increasing the productivity of stable cell lines, while reducing their development timelines. Metabolic selection is based on the disruption of a vital metabolic pathway. In the biopharmaceutical industry, the enzymes glutamine synthetase (GS) and dihydrofolate reductase (DHFR) are the most commonly used selectable markers for the disruption of metabolic pathways. GS and DHFR are involved in the synthesis of the vital metabolites glutamine and purines, respectively.

When using a metabolic selection strategy, the stable clone selection process is based on cell culture in a medium lacking the vital metabolite. Thus, only the cells able to endogenously produce the target metabolite will be able to survive in the culture media. In expression systems lacking DHFR or GS activity, endogenous production of the vital metabolite is only possible for cells stably transfected with a plasmid containing the GS or DHFR gene. This selection process can further be amplified by the addition of DHFR or GS inhibitors such as MTX or MSX, respectively. Gradually increasing inhibitor concentration leads to gene amplification in order to overexpress GS or DHFR. Therefore, since the marker gene is linked to the recombinant protein gene to express (both genes on the same plasmid), recombinant protein production is amplified in the same proportion as the marker production.
The GS system has been gaining ground over other systems due to its simplicity. For this reason, although GS-NS0 has for long been considered the gold standard of GS-based selection, multiple CHO cell lines lacking GS are currently being developed.
As a protein production expert, ProteoGenix masters all the steps of the process leading to a highly productive and stable CHO cell line. Together with our FTO cell line and unrivalled guarantees, our expertise ensures you a safe investment and a strong ROI.

Other selection markers used for stable cell line generation

Alternative resistance markers can be used to select stable cell lines. The most commonly used for mammalian cell line selection include Blasticidin, G418/Geneticin, Hygromycin B, Puromycin, Zeocin, etc. Unlike metabolic-based selection, resistance-based selection does not require gene knockout or inhibition, only the identification of a “kill curve” (growing cells in different concentrations of antibiotic).

Monoclonal antibody production in stable mammalian cell lines

How long does it take to generate stable mammalian cell lines for monoclonal antibody production?

When starting from an optimized expression vector, stable cell line generation starts at 4-6 months. First, antibody genes need to be optimized (i.e. codon optimization) and cloned into a high-expression vector. The vector is then transfected or co-transfected in parallel with another vector containing the selective markers (i.e. DHFR or GS are the two most widely used in CHO-based production) into competent mammalian cells. We carry out vector linearization to increase stable integration efficiency.

Successful transfection can be achieved by chemical (e.g. PEG or other reagent to increase membrane permeability) or physical methods (e.g. electroporation) in non-selective medium and optimal conditions. The process of transfection takes about 24-48 hours.

After transfection, the process of selection starts; cells are amplified in selective medium (for several weeks) and positive clones subsequently isolated either by:

1. Limiting dilution (most conventional method)
2. Verified In-Situ Plate Seeding (VIPS™)

Independently of the method used for single clone selection, the process is laborious and complex and starts at 4 months. Subsequent steps comprise assessing the production yield, cell line stability, and antibody properties in several promising single clones to select the best clone for further development.
Further development includes process optimization (4-5 weeks), cell bank preparation and stability studies (variable), and production scale-up.

Main mammalian cell lines used for stable monoclonal antibody production

Stable production of monoclonal antibodies for commercialization is mainly achieved in mammalian systems. These cells are known to perform proper protein folding and complex post-translational modifications (including human-like glycosylation), vital to ensure optimal antibody reactivity and therapeutic effectiveness.
For the past decades, Chinese hamster ovary (CHO) and mouse myeloma (NS0 and Sp2/0) cells have been considered the industry’s gold standard for therapeutic antibody production. These cells are well-adapted to growth in suspension in serum-free medium and many are compatible with well-established systems of selection – a crucial step in stable cell line generation for monoclonal antibody production.
The majority of currently licensed antibody therapeutics are produced by either CHO or NS0 cell lines, and only a minority of therapeutic antibodies are expressed by bacteria (Escherichia coli) or yeast (Pichia pastoris) (antibodies approved until 2020).
Of these, all antibodies produced in E. coli approved for clinical use consist of antibody fragments: brolucizumab (humanized scFv), caplacizumab (humanized dsFv), moxetumomab pasudotox (murine dsFv), and certolizumab pegol (humanized Fab). Moreover, the sole antibody produced in P. pastoris approved for clinical use is eptinezumab (Vyepti™), approved only in 2020 for migraine prevention.

Why are CHO cells favored for therapeutic antibody production?

The most common CHO cell lines used for the production of licensed antibody pharmaceuticals include CHO-S, CHO-K1, and CHO-DG44. Besides the advantages listed above, many methods for recombinant production (particularly large-scale stable expression) have been optimized for the unique genetic background and nutritional requirements of CHO cells. This expression system is also extremely tolerant to changes in oxygen concentrations, pH, temperature, and pressure – crucial properties for industrial production processes.

Additionally, CHO cells can secrete proteins with complex post-translational modifications very similar to those produced by human cells. Plus, there are several well-established systems for gene amplification in CHO cells leading to higher titers of recombinant proteins.

CHO cells are constantly being engineered and optimized for protein production and they are typically selected using metabolic markers such as:

• Dihydrofolate Reductase (DHFR) system
• Glutamine Synthetase (GS) system

The choice of selection system heavily depends on the genetic background of the cell line being used. For instance, CHO-DG44 cells were engineered from CHO-K1 cells by gamma radiation in the 1960s to eliminate both alleles of DHFR. For this reason, when antibody genes are co-transfected with exogenous DHFR, positive clones are easily selected by eliminating glycine, hypoxanthine, and thymidine (GHT) from the growth medium. Alternatively, methotrexate (MTX) can be used to inhibit endogenous DHFR in DHFR+ CHO cell lines.

CHO cell lines deficient in GS have only been recently developed. For this reason, GS-based selection is still recurrently made using methionine sulphoximine (MSX) able to inhibit endogenous GS.

Despite all the advantages of CHO cell lines for monoclonal antibody production, these cells still produce slightly different glycans to those produced by human cells (α-gal and NGNA glycans which may cause adverse reactions in humans), plus, they lack α[2-6] sialyltransferase α[1-3/4] fucosyltransferases (human enzyme). These limitations continue driving the search for alternative production systems.

Alternative stable production systems for monoclonal antibodies

The regulatory process for licensing new therapeutic antibodies can take up to 10-12 years. For this reason, the cell lines that currently dominate the market aren’t necessarily the ones being used by developers with monoclonal antibodies on the clinical development pipeline.

In recent years, alternative expression systems have been tested for the production of monoclonal antibodies including:

• Baby hamster kidney (BKH21) cells
• HEK including HEK293T
• C6 cells
• C6 cells

Each of these cell lines has a complex genetic background and thus requires very specific protocols for successful stable integration and stable antibody production. Notably, human cell lines like HEK are particularly attractive alternatives given their ability to synthesize all human glycans, thus reducing the risks of eliciting adverse reactions (immunogenicity).
However, the greatest disadvantage of these cells is the lack of species barrier. Human cell lines may harbor and potentially contribute to the transmission of human viruses. Although viral charges can be inactivated or eliminated by processes such as nanofiltration, these additional steps increase production costs. Plus, HEK cell lines pose a moderate health hazard to production plant workers and thus require tighter containment and security measures.

For this reason, plant and yeast cells represent a desirable alternative and we may soon witness the rise of licensed antibodies produced by these systems.