The choice of expression system used in different stages of antibody drug discovery is dependent on a number of factors, including the number of antibody variants, the quantity of material required, the quality of material required and the turnaround timeline.

  • If both Transient and Stable cell lines can be used for Recombinant antibody production, how do I pick the right one?

    Good question! Unfortunately, there is no right answer.

    It all depends on what your experimental needs are. Both these transfection methods involve getting the foreign (target) gene into the cells. In transiently transfected cells, the foreign DNA does not integrate into the host genome and as such it does not replicate and is eventually lost through cycles of cell division over several days. Transient transfection is good if you have relatively small requirements for rAb and if you need it fast. Transient gene expression results in short term recombinant Ab production, typically 6-10 days from the point of DNA transfection. HEK293 cells normally achieve higher transfection rates and higher yields with PEI which is inexpensive and resulting in lower overall production costs.

    Stable transfection also begins transiently but through a process of careful selection and amplification, stable clones are generated. In stably transfected cells the foreign gene becomes part of the host genome and is therefore replicated. Descendants of these transfected cells, also express the foreign gene, resulting in a stably transfected cell line. Because the stable transfection of cells is a longer and more arduous process, it is practical for rAb production on larger scales. During initial selection process when research quantities of rAbs are sufficient (10-1000 mg), rapid methods of rAb production are required and so large scale transient expression is routinely used. For large scale production of therapeutic antibodies, stable gene expression is the preferred option as it allows for greater process consistency and control of the final product quality.

How Are rAbs Produced?

Recombinant antibodies are constructed in vitro using recombinant DNA technology. Antibody genes are isolated from the appropriate source or synthesized based on sequence information. They are then cloned into expression vectors for expression in a suitable expression system. Full length rAbs are produced in mammalian expression system owing to its superior post-translational modification capabilities. Another advantage of mammalian system is the low risk of immunogenicity that could arise from altered, non-human glycosylation patterns. rAbs are produced using Transient or Stable expression technology.

Transient Expression

Transient Expression

Figure 1: A typical transient expression workflow for rAb production would involve gene synthesis and/or subcloning the light chain and heavy chain genes into the appropriate expression vector followed by transient co-transfection into suspension (usually HEK293 cells). Once the cells start secreting Antibody, the cell culture broth is centrifuged and supernatant filtered (usually 5-6 days post-transfection). Filtered supernatant is then loaded onto protein A resin for purification by affinity chromatography.

  • Stable Expression

    Two expression vector systems dominate antibody production in mammalian cell culture, one based on dihydrofolate reductase (DHFR) genes1 and the other on glutamine synthetase genes2.

    DHFR Principle: In a DHFR expression system, the folate analog methotrexate (MTX) is used to inhibit the function of DHFR, an essential metabolic enzyme for purine and pyrimidine synthesis. Transfection with an expression vector containing the DHFR gene prevents MTX from poisoning transfected cells, whereas the cells without DHFR are selected against in the presence of MTX. Frequently, an antibiotic resistance gene is used in DHFR expression vectors to act as the selectable marker, turning the primary function of the DHFR gene to facilitating vector amplification. DHFR system is effective and has been used in conjunction with other aspects of cell line development to achieve mAb expression levels of multiple grams per liter.

    Glutamine Synthetase Principle: Glutamine Synthetase synthesizes glutamine from glutamate and ammonium. Since glutamine is an essential amino acid, transfection of cells lacking endogenous Glutamine Synthetase, such as NS0, with the Glutamine Synthetase vector confers the ability to grow in glutamine-free media. Selection occurs in the absence of glutamine. Even for cells that do express functional levels of Glutamine Synthetase (CHO), including Glutamine Synthetase inhibitor methionine sulfoximine (MSX), enables use of the Glutamine Synthetase expression vectors. To learn more about the Glutamine Synthetase technology principle and view case studies, click here.

  • Key attributes

    Key attributes of GenScript's CHO-Glutamine Synthetase technology

    • CHO K1 suspension cell line adapted to growth in serum-free media
    • CHO-Glutamine Synthetase expression vector [double gene vector for mAbs]
    • Guaranteed >g/L rAb expression

    Transient Expression

    Comparison of workflow between DHFR and GenScript Glutamine Synthetase Technologies shown above. Glutamine Synthetase Expression Technology guarantees g/L rAb expression in ~4 months from the point of transfection.

    Transient Expression

    Figure 2: Schematic of GenScript Glutamine Synthetase Technology workflow.

    The previous section in this series is "Antibody Expression in Eukaryotes". To review, click here.

GenScript Antibody Drug Discovery Services

Antibody Discovery

GenScript's Antibody Engineering group can build antibody library with up to 1010 individual clones, to speed up your antibody discovery efforts.

Antibody Sequencing

GenScript's advanced Antibody Sequencing technology offers fast and professional sequencing services for your monoclonal antibodies.


GenScript has developed several cell-based ADCC/CDC functional assays to profile the efficacy and potency of your therapeutic antibodies using proprietary recombinant effector cells.

Antibody Engineering

GenScript scientists' extensive experience in antibody engineering can provide superior services such as antibody humanization, affinity maturation and more.

Antibody Production

With solid expertise in recombinant antibody (rAb) production techniques, GenScript provides a comprehensive rAb service portfolio that deliver microgram to gram quantities of pure rAb for each stage of your Ab drug discovery program.

PK/PD Study

GenScript offers over 120 tumor and inflammation models for evaluation of in vivo efficacy, PK/PD, biomarker and bioanalysis studies. GenScript Anti-idiotype Antibody services are also a powerful tool for antibody drug PK/PD and immunogenicity studies.

You can also view our Recombinant Antibody Service Selection Guide to identify services that are the best match for your application.


  1. Schimke, R. T., Roos, D. S. & Brown, P. C. Amplification of genes in somatic mammalian cells. Methods Enzymol 151, 85-104 (1987).
  2. Bebbington, C. R. et al. High-level expression of a recombinant antibody from myeloma cells using a glutamine synthetase gene as an amplifiable selectable marker. Biotechnology (N Y) 10, 169-175 (1992).

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