Optimizing Transient Transfection for Viral Vector Production

With over 1,000 gene therapies and gene-modified cell therapies in clinical trials, many of which target prevalent diseases, the demand for the viral vectors needed to deliver the genetic material is expected to increase 100- to 1,000-fold. While capacity additions are planned, they will be insufficient to meet the dramatically higher demand. Improvements in the efficiency of transient transfection processes are urgently needed to boost productivity and to reduce cost.

Why Transient Transfection Predominates for Viral Vector Production

Transfection involves the introduction of foreign DNA/RNA into eukaryotic cells to modify their genetic makeup and generally cause the expression of certain proteins.1 When the foreign nucleic acids are integrated into the host nuclear genome, and long-term transgene expression results, even after cell division, the transfection is considered to be stable. If the foreign plasmids or oligonucleotides are not integrated, then transient or short-term transgene expression occurs. The former is often preferred for lengthy genetic studies, while the latter has been widely adopted for protein expression, including the production of viral vectors.

For viral vector production, including adeno-associated viral (AAV) and lentiviral (LV) vectors used for direct gene therapy and gene-modified cell therapy, transient transfection, typically into HEK293 or HEK293T cells, is generally the preferred method because it is both flexible and robust.2 In addition, the HEK293 cell line is widely used in pharma manufacturing, and changing the gene of interest (GOI) is relatively easy. For AAV, transient transfection also enables rapid switching from one serotype to another.

In addition, the short duration of the transient transfection process also allows for faster process optimization and quicker access to products for assessment of therapeutic potential.3 Furthermore, transient transfection enables rapid manufacture of viral vectors in sufficient quantities to support clinical development and commercial manufacturing, at least for gene and gene-modified cell therapies targeting rare diseases. For AAV vectors, transient transfection also makes rapid switching from one serotype to another feasible.

Alternatives to transient transfection include infection of Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression vector system (BEVS) and/or Herpes simplex virus cells, but there are issues with these methods, including lengthy development times, limited AAV serotype applicability, and quality concerns.4 Longer-term efforts are focused on developing stable packaging/producer cells lines that provide high virus titers while avoiding cell toxicity. One challenge with this approach is the need to invest significant time and money up front, which can be difficult given the short development timelines for gene and gene-modified cell therapies.

Efficient Vector Production at Scale is Essential to the Future Success of Gene Therapies

The few gene therapies on the market today were developed to treat rare diseases with limited populations and generally involve local administration to a small area, such as the eye. The number of gene and gene-modified cell therapies expected to receive approval in the next few years could surpass 50, most of which will leverage viral vector delivery vehicles.5 In addition, close to 1,250 gene therapies were in preclinical development and an additional 520 in clinical trials in 2021. Importantly, it is estimated that nearly 60% of those clinical trials for gene and gene-modified cell therapies target more prevalent diseases that are administered systemically and thus require larger doses.6 Many could serve as first-line therapies rather than as last hopes.7

To meet future demand, therefore, much larger quantities of AAV and LV vectors will need to be produced. However, scaling transient transfection processes is not a simple matter. Unlike recombinant proteins and antibodies, for which platform processes have been developed, the variability from one viral vector to another — and even one serotype to another — makes standardization a challenge. Transient transfection is also a more complex process that involves the formation of transfection reagent–plasmid complexes that are unstable and shear-sensitive, creating mixing and transfer challenges, as well as process volume issues, upon scaling.

In addition, because three or four plasmids must be transfected, transcribed, and translated to generate AAV or LV vector capsids, respectively, the transfection process tends to be less efficient than protein expression.8 Titers can also be limited by the short duration of the transient transfection process. Further complicating the situation for AAV vectors is the potential for the generated capsids to be missing the GOI or to contain only part of it or even host-cell DNA.

With the increase in demand for viral vectors anticipated to be 100- to 1,000-fold, increasing the yield of high-purity vector products is essential. In addition to boosting the transfection efficiency, it will be necessary to achieve higher transgene expression and better posttranslational packaging.8 This need is particularly strong given the limited capacity for viral vector production among both contract manufacturers and gene therapy developers.

Some steps have been taken. There has been significant collaboration among gene therapy developers, equipment manufacturers, and raw material suppliers to develop cost-effective, platformizable solutions to enable industrialization of transient transfection processes for viral vector production.9 For instance, solutions have been and continue to be developed to enable scaling of transient transfection processes in bioreactors rather than flatware, for both suspension and adherent cell culture. Use of design-of-experiment (DoE) studies for process development is allowing optimization of multiple process parameters and is leading to better performance. Tailor-made plasmids and fit-for-purpose transfection reagents specifically designed for the production of viral vectors are also enabling higher yields of functional vectors.

Manufacturing Challenges are Still Significant

One of the main challenges to the development of cost-effective, scalable viral vector manufacturing processes is the very short timelines allotted for process optimization.4 Receiving regulatory approval for monoclonal antibody products typically takes 10 years; for cell and gene therapies, that time is reduced by more than half to just 3–4 years.10

Consequently, compromises are often made on cost of goods and scalability –– for instance, lack of investment in the development of robust cell lines11 –– that would not be accepted for traditional recombinant protein and antibody products. Complicating this issue is the complexity of the multi-step transient transfection process. Biologics license applications for gene therapies can be tens of thousands of pages long with the bulk related to manufacturing data,12 and the greatest portion of regulatory agency review time is focused on manufacturing and quality issues.13 Other factors include the lack of easy platformization and the reliance at early development stages on small-scale adherent cell culture processes performed in plasticware and requiring numerous manual interventions — an approach that is not practically industrializable.

Other challenges relate to the nature of transient transfection, which inherently becomes less efficient at larger scales.4 With AAV vectors, there is also significant variation in the yield of functional viral particles with changes in serotype and transgene. Rapid, sensitive, and robust analytical techniques to support process development through commercial GMP production, including comparison of vectors produced using different processes and product release, are also lacking.

Time must be invested in understanding the specific requirements for a viral vector product so that suitable, robust cell lines can be developed.11 A quality-by-design approach to process development is also needed to enable identification of optimal process parameters (e.g., cell density at the time of transfection, choice of media and supplements, reagent-to-DNA ratio, plasmid ratios, total DNA amount, choice of transfection reagent, complexation conditions and time, rate of addition of complexation mix, timing of transfection, and so on8).14–16

DNA plasmids must be properly sequenced and free of impurities, ideally using next-generation sequencing technology to confirm quality and purity.11 Transfection reagents must be selected that provide high titers of functional vectors at all scales while minimizing raw material needs and processing volumes. Manufacturing processes should also be sufficiently flexible to allow for response to changes in demand. Overall, the best strategy is to adopt a right-first-time approach to the development of transient transfection processes that provide high-quality product and are scalable and practical to implement under cGMP conditions.3,11

Suspension and Adherent Cell Culture Solutions

To overcome the limitations of transient transfection using adherent cell culture in plasticware, two different strategies have been pursued. Fixed-bed bioreactors have been developed that allow the scaling –– at least to some degree –– of adherent processes. Separately, HEK293 cells have been modified to support suspension cell culture in traditional stirred-tank bioreactors.

Fixed-bed bioreactors provide a three-dimensional surface for adherent cell growth within a bioreactor and are designed to enable scalability beyond what is possible with flatware. The bioreactor design provides optimal process conditions to encourage high cell viability, growth, and vector expression. Products are on the market from Pall Corporation (iCELLis®),17 UTECH (scale-X™),18 and Corning Life Sciences (Ascent™)19 that support scaling from lab to pilot to production scale (500 liters).

Suspension-based transient transfection is attractive because it avoids the need for detachment of the cells from whatever type of support is used. It also enables the use of existing equipment, including sensor and automation technology. While the same can be said for fixed-bed bioreactors, the bioreactor technology is designed specifically for adherent processes. Both approaches, therefore, reduce variability and simplify process development.

However, there are only limited modified cell lines commercially available for suspension-based transient transfection, and high-performing proprietary cell lines can take as much as a year to develop. In addition, HEK293-derived cells are known to clump at the higher cell densities typically associated with suspension cell culture.

Much effort is being directed at overcoming these issues, however, and progress is being made. In general, though, the choice of suspension versus adherent cell culture should be made, taking into consideration the nature of the target vector and the desired quality attributes.20 For products only requiring small vector volumes, adherent production in flatware or fixed-bed bioreactors may be sufficient and even preferable, as it is a proven technology. For gene therapies intended to treat large patient populations that will require large quantities of vector, suspension-based processes will likely be more appropriate. It is thus anticipated that, as producer cell lines are improved and become more widely used, suspension-based processes will become predominant for large-scale viral vector production.

Leveraging Design-of-Experiment Studies for More Effective Process Development

The traditional approach to transient transfection process development has been to use a one-factor-at-a-time (OFAT) approach to investigating process parameters.16 With the limited development timelines available for most gene and gene-modified cell therapies, such an approach precludes full investigation of the design space, owing to the inability to explore all the important process parameters or how they interact with one another.

Numerous recent studies have shown that performing even limited studies using a DoE approach that allows simultaneous exploration of multiple process parameters yields significantly more optimized transient transfection production processes.

In one example, the transgene, packaging, and helper plasmid ratios, the total DNA concentration, and the cell density were simultaneously varied, revealing an unexpected combination of process parameters that provided significantly improved yields of functional viral genomes.16 There is a growing recognition that implementing a targeted DoE approach using scale-down models at during early development can provide measurable increases in process efficiency and productivity, impacting yield, quality, and cost.20 This type of DoE strategy can be further maximized by evaluating not only process parameters that impact quality but those that influence scalability.21

Tailored Plasmids

Viral vectors used as gene therapies and in the production of gene-modified cell therapies are manufactured from plasmids, small, typically circular double-stranded DNA units found in many bacterial species. Plasmid quality is a crucial determining factor for transient transfection efficiency.22 Certain properties of plasmids must be carefully evaluated when selecting plasmids for viral vector manufacturing, including their origin of replication, which determines the vector copy number, antibiotic resistance genes, and many others. In addition, plasmids used for viral vector production must consist largely (at least 80% and preferably 90–95% or higher) of the supercoiled DNA (vs. relaxed, nicked, nicked circular, etc.) isoform.23

In general, the trend is toward smaller plasmids free of any unnecessary genetic material, including antibiotic resistance genes, to facilitate increased transfection efficiency. PlasmidFactory, for instance, offers customized mini-circle plasmids that almost exclusively contain the GOI and its regulating sequence motifs.24 Nature Technology claims that its Nanoplasmid DNA offers higher levels of gene expression than traditional plasmids and mini-circles, as well as greater potency for a longer duration.25 Other examples include the Antibiotic-free Maintenance System from Cobra Biologics26, doggybone DNA (dbDNA™) from TouchLight Genetics,27 and tailor-made plasmids from eZyvec (now part of Polyplus) constructed in one step from DNA templates (“bricks”) using proprietary software and four standardized assembly protocols.28

Even gene therapy developers are focusing on the development of optimized plasmids. One such company is LogicBio Therapeutics, Inc., a clinical-stage company developing gene-editing and gene-delivery platforms, which has created plasmids for transient transfection of HEK293 cells that maximize the production of AAV capsids while preventing generation of replication-competent AAVs (rcAAVs).2

Given the critical importance of plasmid quality, regulatory authorities have placed increasing emphasis on the use of GMP-grade material (rather than research-grade plasmids) for the production of viral vectors used in late-stage clinical and commercial gene therapy products. High-quality, non-GMP plasmids can be used in early-stage clinical trials, however. Many plasmid manufacturers therefore offer research, clinical, and GMP grades of plasmids for use at different development stages.

Fit-for-Purpose Transfection Reagents

Greater understanding of transient transfection processes has revealed the importance of the transfection agent in determining transfection efficiency and productivity. Common first-generation transfection reagents have included linear polymer polyethyleneimine (PEI) and lipid-based lipofectamine reagents, most of which were not initially developed for this purpose and therefore do not enable optimum process performance.

Polyplus and Mirus Bio are two companies focused on the supply of transfection reagents for many different applications. Both have developed novel transfection reagents designed specifically to support transient transfection.

Polyplus first offered PEIPro®, a PEI-based reagent designed for large-scale viral vector production via transient transfection. More recently, the company developed FectoVIR®-AAV for suspension-based production of AAV vectors of different serotypes.29 The company has shown that this fit-for-purpose reagent provides at least twice the yield of AAV vectors than PEIPro and up to 10 times that of other conventional transfection reagents. It also allows the use of 40–60% less pDNA, 20–30% less cells and media, and 10–20% less transfection agent, reduced complexation volumes (by 50% or more), and greater complex stability for practical transfer of complexation mixes at large scale. FectoVIR®-AAV is available in research and GMP grades, and the scalability of reactions with it are being explored through numerous partnerships.2,21,30,31

Mirus Bio pursues a rational approach to transient transfection agent design, taking into consideration the properties needed to optimize interaction with plasmids and cell membranes for enhanced uptake and delivery with minimal cytotoxicity.8 Its TransIT-VirusGEN® Transfection Reagent comprises lipid and polymer components suitable for both suspension and adherent processes. Performance is enhanced for specific vectors by the VirusGEN® AAV Complex Formation Solution and Enhancer and the VirusGEN® LV Complex Formation Solution and VirusGEN® LV Enhancer. They are available in Research Use Only, SELECT, and GMP configurations and result in increased in functional titers, genome copies (GCs), total capsid counts, and GC/capsid ratios.

Many Positive Impacts of Increased Efficiency and Productivity

Higher yields of functional vectors contribute to greater efficiency and productivity and ultimately reduced cost. If more vector is produced per batch, more doses can be obtained. For instance, assuming all other aspects of the process (e.g., bioreactor size, downstream processing, ratio of consumables) remained the same, a two-fold increase in titer would lead to twice the number of doses being manufactured at the same cost level. Therefore, the cost per dose would be half, yet twice as many patients could be treated.32

Alternatively, fewer batches would be needed to produce the same quantities, or the same number of batches could be produced in smaller equipment.8 In all cases, reduction of the cost of goods combined with time savings would result. The ultimate impact, therefore, would be acceleration of gene therapy development and greater patient access to these life-changing and lifesaving treatments.



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  13. Remarks by Commissioner Gottlieb to the Alliance for Regenerative Medicine’s Annual Board Meeting. Speech by Scott Gottlieb. U.S. Food and Drug Administration. 22 May. 2018. https://www.fda.gov/NewsEvents/Speeches/ucm608445.htm.
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  5. Leinonen, Hannah M. et al. “Benchmarking of Scale-X Bioreactor System in Lentiviral and Adenoviral Vector Production.” Gene. Ther. 31: 376–384 (2020).
  6. Upton, Todd and Vasily Goral.How fixed bed bioreactors are changing the game for adherent cell culture-based vector production.” Cell & Gene Therapy Insights. 7: 337–343 (2021).
  7. Kole, Denis, et al. “Round table discussion: Successful suspension-based viral vector manufacturing scale-up, from process development to clinics.” Cell & Gene Therapy Insights Webinar. 7 Sep. 2022.
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  9. El-Hajjami, Nargisse and Laurens Vergauwen.Plasmid DNA Production for Cell and Gene Therapy.” Cell Culture Dish. 30 Nov. 2020.
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  11. Customized Minicircle DNA.” PlasmidFactory. n.d.
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  13. Antibiotic-free Maintenance System (ORT®).” Cobra Biologics n.d.
  14. Technology.” Touchlight Genetics. n.d.
  15. Tailor-Made DNA Plasmids for Viral-Vector Manufacturing.” n.d.
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  17. Porte, Mathieu, Claire Wartel, and Bhargavi Kondragunta.Optimization of AAV Production for High-yielding and Scalable GMP Processes with Catalent.” Polyplus. 23 May 2022.
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Cynthia A. Challener, Ph.D.

Dr. Challener is an established industry editor and technical writing expert in the areas of chemistry and pharmaceuticals. She writes for various corporations and associations, as well as marketing agencies and research organizations, including That’s Nice and Nice Insight.