Application of HEK293 Cell Line in the Production of Recombinant Proteins and Viral Vectors

HEK293 Cells and Their Derivatives

The HEK293 cell line was established by transforming human embryonic kidney cells with sheared adenovirus type 5 DNA. Since then, many subtypes and derivatives have been established, with HEK293, HEK293-T, and HEK293-F frequently used in the production of biopharmaceuticals. HEK293-T is a derivative of the HEK293 cell line, established by the expression of a temperature-sensitive SV40 T-antigen mutant. HEK293-F cells are cloned from HEK293 and adapted to suspension culture in serum-free media. Other notable HEK293 derivatives commonly used in recombinant protein production include HEK293-E and HEK293-6E.

Recombinant Protein Production in HEK293

Eukaryotic expression systems are used to produce complex recombinant protein with complex PTMs for proper protein function. The use of the HEK293 host cell eliminates issues of potential immunogenicity due to the presence of non-human PTMs. Its ease of transfectability and relatively high protein productivity makes it a popular choice for small scale production of recombinant proteins for scientific studies and its capability to adapt to a suspension free culture facilitates its use in large scale biotherapeutics production.

Stable producer cell lines are preferred for the large-scale production of recombinant proteins. Because of its ease of transfection, HEK293 is widely used for transient gene expression (TGE). Recombinant proteins can be produced quickly by transfecting HEK293 with readily available reagents such as calcium phosphate or polyethylenimine (PEI). Such transfection processes have been demonstrated in stirred-tank bioreactors and have proved to be a good method for the rapid production of recombinant proteins for lab to pre-clinical use.

Fig. 1 HEK293 provides improved secreted titers of difficult to express proteins compared to CHO in two different expression systems.

Viral Vector Production in HEK293

HEK293 is commonly used in the production of viral vectors. Recent advancements in transient transfection technologies, cell line and media development have demonstrated that viral vector production can be performed using serum-free suspension-based transfection process in chemically defined cell culture medium. In addition, stable viral vector producer cell lines have been established. Together, these developments have enabled the scalable production of viral vectors in HEK293 cells.

Retroviral vector production

Retroviral vectors are enveloped, single-stranded RNA viruses. In this family of viruses, γ-retroviruses (γRV) based on the Murine Leukemia Virus and lentiviruses (LV) based on the Human Immunodeficiency Virus type 1, are commonly used in gene therapies.

Retroviral vectors are one of the best tools for gene transfer due to its ability to transduce wide range of cell types. Retroviral vectors are typically produced by the transient transfection of adherent HEK293T cells. To improve process scalability, several serum-free suspension-based production methods have been developed. The suspension-based production methods demonstrated comparable volumetric productivities compared to adherent-based production methods.

Adeno-associated virus production in HEK

Adeno-associated viruses (AAV) are non-enveloped, single-stranded DNA viruses which have shown safety and efficacy as gene therapy vectors. Recombinant AAVs (rAAVs) are typically produced via the transient-transfection of adherent HEK293 cells with three plasmids containing: 1) Adenoviral helper factors, E4, E2a, and VARNA; 2) Adeno-associated virus rep and cap genes; and 3) Cargo gene flanked by AAV ITR sequences. HEK293 cells are most frequently used for rAAV production due to the expression of adenoviral E1a/b genes which are essential for AAV production. Although the HeLa cell line was used for rAAV production in early studies, HEK293 based cell lines are typically favored due to the expression of the adenoviral viral helper genes, E1A/B, which improves rAAV titers during production.

Fig. 2 Mechanistic model for production of recombinant adeno-associated virus via triple transfection of HEK293 cells.

Production of adenoviral-vectored vaccines and oncolytic adenoviruses

Adenoviruses (AdV) are non-enveloped, double-stranded DNA viruses. It was first used as a gene therapy vector as they possess high genomic capacity, high transduction efficiency and were non-integrating with high epichromosomal persistence. The COVID-19 pandemic drove the rapid development of adenoviral vectored vaccines and their eventual emergency use. Of the three adenoviral vectored vaccines approved for emergency use by the World Health Organisation (as of August 2021), Ad5-nCOV and ChAdOX1-nCoV are produced in HEK293 cells, while Ad26.

Future Prospective

The human cell line, HEK293, have emerged to be the main production platform for therapeutic viral vectors. Advancements in cell line developments would see more HEK293 clones or derivatives developed for growth in a serum-free suspension medium for high-titer viral vector production.

References:

Tan, E.; et al. HEK293 cell line as a platform to produce recombinant proteins and viral vectors. Frontiers in bioengineering and biotechnology. 2021: 1288.
Nguyen, T. N. T.; et al. Mechanistic model for production of recombinant adeno-associated virus via triple transfection of HEK293 cells. Molecular Therapy-Methods & Clinical Development. 2021, 21: 642-655.
Malm, M.; et al. Harnessing secretory pathway differences between HEK293 and CHO to rescue production of difficult to express proteins. Metabolic engineering. 2022, 72: 171-187.

* For research use only. Not intended for any clinical use.

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Application of HEK293 Cell Line in the Production of Recombinant Proteins and Viral Vectors

HEK293 Cells and Their Derivatives

Recombinant Protein Production in HEK293

Viral Vector Production in HEK293

Future Prospective

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