Transient transfection is an effective way of inserting foreign genetic material into eukaryotic cells. Due to its rapid production rates, it is often used in studies focusing on the short-term expression of genes whilst being easily adapted to small and large-scale production, such as in recombinant antibody production.
The Chinese Hamster Ovary (CHO) cell line is the industry standard for protein production and manufacturing in multiple fields across life sciences, from research to gene therapy. The CHO mammalian cell line can produce high protein yields with high efficiency through transient transfection, allowing for optimizations in production processes as well as time and resource savings.
This article will provide an overview of transient transfection, describe the differences with stable transfection, and examine its crucial role in the production of antibodies using CHO cells.
Transfection — an overview
Földes and Thomas Trautner originally coined the term transfection to mean the infection of cells with viral nucleic acid from a bacteriophage or virus, which resulted in the production of a complete virus. The term has become more generalized and is now used to describe the introduction of foreign nucleic acid (e.g. DNA, RNA, miRNA, siRNA, and other small non-coding RNAs) delivered to a cell to modify the cell’s genetic makeup and properties. Two main types of transfection exist — transient and stable transfection.
Stable transfection
Stable transfection refers to the integration of foreign DNA into the host nuclear genome or the maintenance of an episomal vector in the host nucleus as an extra-chromosomal element1. This results in the transcription of target genes in an ongoing manner, even with the replication of stably transfected cells.
Transient transfection
Genetic material in the form of plasmids or oligonucleotides is transfected into the host cell. As transgene expression does not rely on the integration of nucleic acids into the host cell genome, the expression exists for a limited time and is lost once the host cells replicate via cell division.
Benefits of transient transfection?
The main purpose of transient transfection experiments is to study the effects of short-term expression of genes and gene products. The advantages of transient transfection are the rapid speed of the process and low cost compared with stable transfection. While stable transfection supports gene expression over a long period of time through the integration of nucleic acid into the host genome, it is more laborious and requires more effort and resources.
Particularly in bioprocessing, transient transfection allows the fast development of recombinant antibody production and generates milligram quantities of material in the space of days. This circumvents the need to screen hundreds of clones in search of high-producing stable cell lines for antibody development.
One of the main flaws of transient transfection in classical CHO cells is the generally lower titer compared with stable pool generation due to the expression being episomal and the lack of selection for plasmid transfection. However, optimization of specific host cells, like HEK293 and CHO cells, has made it possible to use transient transfection protocols to achieve high-level transient expression, producing milligrams to grams of recombinant protein per liter2.
How does transient cell transfection work?
To achieve transient transfection, the nucleic acid must be introduced through the cell membrane without damaging the cell to result in protein synthesis. Different types of transfection methods exist, including chemical and physical, but also the use of expression vectors like viral vectors for gene transfer.
The challenge of transfection is to deliver negatively charged nucleic acid molecules through the negatively charged cell membrane of the host cell. Chemical methods employ chemicals that impart a neutral or overall positive charge to the nucleic acid molecule. Examples of chemical transfection reagents include calcium phosphate and diethylaminoethyl (DEAE)‐dextran.
Another type of chemical transfection reagent that is commonly employed is liposomal-based transfection reagents. Positively charged — cationic lipid structures — bind electrostatically with negatively charged residues of nucleic acid and cell membrane. The formation of these lipid-nucleic acid complexes can merge with the host cell’s phospholipid bilayer to facilitate endocytic entry and subsequent release into the cytoplasm3.
Physical transfection methods for gene delivery include electroporation, gene microinjection, and laser irradiation. Electroporation is a commonly employed method that exposes cells to an electric voltage to increase the cell membrane permeability and allow the entry of foreign nucleic acid.
Process steps for transient transfection
The stable cationic polymer — polyethylenimine (PEI) — is commonly utilized to introduce plasmid and oligonucleotides1. The different steps include:
- Cell culture of host cells and ex vivo preparation of high-quality nucleic acid (e.g. in incubators)
- In vitro preparation of complex nucleic acid and transfection reagent (e.g. PEI) complexes
- Transfection: nucleic acid transfection reagent mix added to cultured cells to initiate cellular uptake of the gene of interest and DNA transfection
- Harvest cells and assess transfection efficiency: 48–72 h post-transfection (or at later time points of the cell culture process), the mRNA expression level or protein expression level by using GFP as a reporter could be utilized with characterization assays by microscopy or flow cytometry. Alternatively, protein expression can be analyzed using protein separation and Western blotting/mass spectrometry.
Optimization of cell lines for transient transfection
CHO and Human Embryonic kidney cells (HEK293 cells) are the main host cell lines for producing antibodies. In most cases, transient transfection is performed in suspension culture rather than in adherent cells. Both cell types are readily adaptable not only to suspension culture but also to serum-free media (SFM) and chemically defined (CD) media, which are often employed in antibody production.
SFM and CD media enable easier purification and downstream processing due to their reproducible formulations that lack animal products. The lower the risk of contamination from bacteria, fungi, or viruses is a further benefit of SFM and CD media. The proteins produced through transient transfection are fully post-translationally modified and active mammalian proteins. The secreted proteins can be easily purified from serum-free cell culture medium, with methods like affinity chromatography and protein polishing techniques like size exclusion or ion exchange chromatography as offered by evitria.
Read more: Benefits of transient transfection
HEK293 cells
Derived from human embryonic kidney cells and transformed with adenoviral 5 DNA4, transient protein expression in HEK293 cells has in the past been preferred due to the higher protein titers compared with CHO cells. HEK293 offer the benefit of human post-translational modifications (PTMs).
- The Expi293 cell line has been developed to produce higher expression yield over a quicker period compared with classical HEK293 cells. Expi293 are able to grow to higher densities than HEK293 cells and require less handling and less medium — instead of needing splitting every 2–3 days, Expi293 cells only require splitting every 4–5 days5. In addition, Expi293 cells express proteins better than HEK293 cells and are compatible with PE1 transfection, a cost-effective, cationic polymer commonly used for both HEK293 and CHO cells.
The main drawback of HEK293 cells, however, is due to their human origin; they are at a higher risk of being contaminated with human-specific viruses. This can lead to immunogenicity or allergic reactions to the final product.
CHO cells
CHO cells are the most popular cell culture lines in biopharmaceutical production. Their advantages are their ability to produce human-like glycosylation patterns and secrete exogenous proteins into the culture medium. Classical CHO cell lines demonstrate lower transfection rates compared to HEK cell lines. However, the development of cell lines and transfection reagents has improved the production of high-level transient recombinant protein in CHO cell lines.
- High-level transient recombinant protein production in ExpiCHO cell line: ExpiCHO cells were selected for their high transfection efficiency, production, and growth properties. Made commercially available in 2015 and in combination with a specialised transfection reagent and transfection enhancers, recombinant protein production in ExpiCHO can exceed transient HEK293-based systems. Not only does ExpiCHO generate similar or greater titers 5–7 days post-transfection compared to the Expi293 system, but it also continues to produce proteins for up to 14 days, resulting in significantly higher overall protein titers6.
The optimization of CHO cell lines to produce higher protein titers means that CHO cells remain more widely employed than HEK293 cells. In addition, toxicity risks are lower when using CHO cells because they are less susceptible to infection by human viruses7.
Conclusion — why we choose transient CHO cell transfection
Optimisation of CHO-based transient transfection for antibody production has resulted in the CHO cell line achieving high transfection rates and corresponding high yields, as well as maintaining quick biotherapeutic production times. This makes them the ideal host cell line for enabling the recombinant monoclonal antibody production at evitria.
Please get in touch with us for more information on our recombinant antibody production service.
References
- Lufino, M. M. P., Edser, P. A. H. & Wade-Martins, R. Advances in high-capacity extrachromosomal vector technology: Episomal maintenance, vector delivery, and transgene expression. Molecular Therapy vol. 16 Preprint at https://doi.org/10.1038/mt.2008.156 (2008). ↩︎
- Jain, N. K. et al. A high density CHO-S transient transfection system: Comparison of ExpiCHO and Expi293. Protein Expr Purif 134, (2017). ↩︎
- Kim, T. K. & Eberwine, J. H. Mammalian cell transfection: The present and the future. Anal Bioanal Chem 397, (2010). ↩︎
- Graham, F. L., Smiley, J., Russell, W. C. & Nairn, R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. Journal of General Virology 36, (1977). ↩︎
- Fang, X. T., Sehlin, D., Lannfelt, L., Syvänen, S. & Hultqvist, G. Efficient and inexpensive transient expression of multispecific multivalent antibodies in Expi293 cells. Biol Proced Online 19, (2017). ↩︎
- Liu, C. Y., Spencer, V., Kumar, S., Lui, J. & Chiou, H. Attaining High Transient Titers in CHO Cells: Case Study Involving the Use of the ExpiCHOTM Mammalian Transient Expression System. Genetic Engineering & Biotechnology News 35, (2015). ↩︎
- Wiebe, M. et al. A multifaceted approach to assure that recombinant tPA is free of adventitious virus. Dev Biol Stand. 70, 147–151 (1989). ↩︎