HEK-293 cells in CGTs: Key challenges, accelerating production, and strategic innovation

Human Embryonic Kidney or HEK-293 cell line was isolated in the past century, gaining popularity with the rise of novel treatments and reliance on viral vectors. Similar to numerous cell lines, seed train expansion of HEK-293 is not perfected and prompts innovative solutions

HEK-293 cells represent adherent cells that naturally grow as a monolayer (illustration partially generated with the assistance of OpenAI). © Green Elephant Biotech

The HEK-293 adherent cell line was originally established by transforming human embryonic kidney (HEK) cells with a modified adenovirus type 5 DNA to immortalize the former. This widely used human cell line and its derivatives are central to biomanufacturing, particularly for the production of complex biomolecules such as recombinant proteins (e.g., antibodies, growth factors, clotting factors) and viral vectors.

The use of human-derived cell lines has become increasingly popular in biopharmaceutical production, as the resulting therapeutics often display more human-like post-translational modifications (PTMs). These PTMs, such as glycosylation, are critical for ensuring proper protein folding, biological function, reduced immunogenicity, and product potency. For instance, certain biologics and gene therapy agents can only be manufactured in HEK-293 cells, as their PTM requirements cannot be met by other mammalian expression systems, such as Chinese hamster ovary (CHO) cells (1). Within the cell and gene therapy (CGT) landscape, examples of approved biologics that use HEK-293 for the respective viral vector production include Luxturna, the first FDA-approved gene therapy in 2017, and Abecma, a CAR-T cell therapy approved in 2021.

Despite these breakthroughs, around 95% of rare diseases still lack approved treatments. With an estimated 300 million people worldwide living with rare genetic disorders (2), there is a pressing demand to accelerate HEK-293 seed train expansion and upstream bioprocess optimization. Here, we discuss the HEK-293 applications and introduce innovative strategies, such as CellScrew®, designed to help manufacturers maximize the cell density and cost-time ratio of this critical cell line platform.

HEK-293 has built-in strategies for expansion

Although nearly 70% of recombinant biologics are produced in CHO cells due to their high productivity and viability, the HEK-293 cell line plays a critical role in developing many experimental recombinant protein therapies. Unlike non-human cell lines, which often generate incomplete PTMs and potentially immunogenic glycosylation patterns, HEK-293 cells provide more human-like PTMs, resulting in safer and more effective therapeutic proteins. For example, tyrosine sulfation and glutamic acid carboxylation are PTMs where HEK-293 demonstrates clear advantages over other mammalian expression systems (3).

To further improve recombinant protein titers, researchers frequently apply cell engineering strategies to enhance characteristics such as cell proliferation, apoptosis resistance, protein folding capacity, and central carbon metabolism. One example is the upregulated expression of eukaryotic initiation factor 3 (eIF3i) in HEK-293, which in turn activates the transcription factor c-Myc. This modification leads to significantly improved proliferation and an approximately 1.5-fold increase in protein synthesis capacity, as confirmed by luciferase reporter assays. Interestingly, this enhancement was not reproducible in CHO cells, possibly due to an already high baseline protein expression (4).

Key challenges of these strategies are largerly associated with the tools used for execution and potential off-target effects. These opportunities for genetic manipulation and the broad adaptability of the HEK-293 platform make the cell line particularly attractive for biopharmaceutical applications.

There are as many suspension transition protocols as there are scientists

Thanks to their high viability and adaptability, HEK-293 cells are considered among the most easily transfectable mammalian cell lines, comparable to HeLa cancer cells, enabling both stable and transient gene expression (5). Over the years, diverse transfection methods have been developed, and the HEK-293 platform has become a milestone for the production of lentiviruses (LVs), adenoviruses, and adeno-associated viruses (AAVs) used in CGTs.

A well-documented step toward establishing a scalable seed train for viral vector manufacturing is the transition from adherent to suspension culture. Although experimental protocols vary, the adaptation of HEK-293 starter cultures typically begins with gradually weaning cells from serum-containing adherent media to a chemically defined suspension. This process simultaneously exposes cells to new bioprocessing conditions, such as shear stress, mixing, and an altered oxygen transfer rate, crucial for growth in stirred-tank bioreactors. Once adaptation is achieved, transfection optimization focuses on balancing the amount of transfection cocktail (i.e., reagents, nucleic acids, and enhancer agents) with process efficiency (6).

Additional strategies, such as implementing a fed-batch regimen, have been shown to increase HEK-293 cell density and improve viral vector titers (7). Nevertheless, these approaches must be approached critically, as the field still faces limitations related to inconsistent methodologies, variable target viable cell densities, and concerns about viral vector quality across extended passages.

Seed train expansion might no longer be a challenge

While genetic modifications or adaptation to suspension culture remain conventional approaches for scaling up HEK-293 cell growth in industrial biomanufacturing, more efficient and streamlined alternatives are emerging. Some of these strategies place a heavy workload on researchers, while others rely heavily on automation at the cost of reduced process control. In contrast, CellScrew® technology overcomes these limitations by combining both efficiency and flexibility. Offering approximately 33% reduction in labor compared to cell culture flasks and roller bottles while retaining precise control over culture parameters, CellScrew® has been extensively validated to support HEK-293 seed train expansion.

Thanks to its versatile product formats, CellScrew® mini, 6K, and 10K, the system provides a substantially larger growth surface area than conventional cell culture platforms, while maintaining optimal in vitro growth conditions. In trials with HEK-293 cultures, CellScrew® 6K achieved growth kinetics of 200,000 cells/cm² within 96 hours, accelerating workflows for seed train scale-up, viral vector production, and cell banking. Additionally, susceptibility to contamination, a frequent challenge with HEK-293 cells, is significantly reduced due to minimized handling steps (5). Beyond productivity, CellScrew® also contributes to sustainable bioprocessing by lowering environmental impact and reducing carbon emissions by up to 90%, thereby alleviating the industrial burden often associated with large-scale bioprocessing.

As therapy development continues to advance, certain cell lines will remain a cornerstone of CGT manufacturing. HEK-293 cells exemplify the balance between economic advantages, including rapid growth and methodological simplicity, and biological relevance at the molecular level. Absence of native PTMs and HEK-293-specific genetic changes, observed in the CHO cells, can raise concerns around immunogenicity and therapeutic effectiveness. The pursuit of greater efficiency has driven the development of techniques, such as genetic engineering and suspension adaptation, each contributing to the scale-up of this platform. While process optimization and intensification are significant, novel manufacturing solutions should also be embraced.

At Green Elephant Biotech, we believe that efficiency and excellence must not come at the cost of reproducibility, reliability, or sustainability. Our products and goals define this principle and we work to support the researchers that share this vision. If you want to learn more about our trials with HEK-293 using CellScrew®, we got you! Register for Green Elephant newsletter to stay updated with our latest product releases and inspiring insights.

References:

1. Tan, E, Hui Chin, CS, Sherman Lim, ZF, Kong Ng, S, HEK293 Cell Line as a Platform to Produce Recombinant Proteins and Viral Vectors (2021), Front Bioeng Biotechnol, 9; 796991: 1-9. doi: 10.3389/fbioe.2021.796991.
2. A Historic Moment for the First-Ever WHA Resolution on Rare Diseases (2025). Rare Diseases International. Available at https://www.rarediseasesinternational.org (Accessed 16 September 2025).
3. Abaandou, L, Quan, D, Shiloach, J, Affecting HEK293 Cell Growth and Production Performance by Modifying the Expression of Specific Genes (2021), Cells, 10; 7: 1-21. doi: 10.3390/cells10071667.
4. Roobol, A, Roobol, J, Smith, ME, Carden, MJ, Hershey, JWB, Willis, AE, Smales, CM, Engineered transient and stable overexpression of translation factors eIF3i and eIF3c in CHOK1 and HEK293 cells gives enhanced cell growth associated with increased c-Myc expression and increased recombinant protein synthesis (2020), Metab Eng, 59: 98-105. doi: 10.1016/j.ymben.2020.02.001.
5. HEK293 cells vs. CHO cells (2022). evitria AG. Available at https://www.evitria.com/cho-cells/hek293-cells-vs-cho-cells/ (Accessed 17 September 2025).
6. Grieger, JC, Soltys, SM, Samulski RJ, Production of Recombinant Adeno-associated Virus Vectors Using Suspension HEK293 Cells and Continuous Harvest of Vector From the Culture Media for GMP FIX and FLT1 Clinical Vector (2015), Mol Ther, 3; 24: 287-297. doi: 10.1038/mt.2015.187.
7. Rodenbrock, A, Manceur, A, Broussau, S, Transfiguracion, J, Gilbert, R, Shen, CF, Loignon, M, Development of a Scalable Fed-Batch Bioreactor Process for High-Titer Production of Lentiviral Vector Using an Inducible HEK293 Producer Cell Line (2024), BioProcess J, 23: 1-10. doi: 10.12665/J23OA.Manceur.