There are several strategies you can take to improve viral vector production, though some will require more sizeable changes to your existing process than others. In this post, we will briefly discuss variables you can tweak to increase viral titer and to potentially modulate percentage of full AAV capsids. These variables are discussed in order of least to most cumbersome to change:
- Transfection Reagent
- Cell Line and Cell Culture Media
- Transfection Procedure
- DNA Constructs
- Cell Culture Platform
Transfection Reagent
Trying a different transfection reagent is perhaps the least involved way to achieve higher viral titers. Among the variety of reagents on the market for transfection, a subset exists that is specifically designed for high-titer virus production. Using one of these specialized transfection reagents can greatly improve results with minimal modification to your existing process (see section 'Transfection Procedure' below).
For example, the TransIT-VirusGEN® Transfection Reagent can be used in a diversity of adherent and suspension cell culture platforms, such as multi-well or -layered plates and fixed bed or stirred-tank bioreactors. As shown below for AAV of different serotypes, the titer and percentage of full AAV was greatly impacted by the reagent(s) used in the transfection.
Transfection with TransIT-VirusGEN® Transfection Reagent yielded significantly more full AAV vectors than PEI-based Competitor A1 and A3 reagents, and further gains to yield were observed when the TransIT-VirusGEN® Transfection Reagent was used in conjunction with the VirusGEN® AAV Complex Formation Solution and Enhancer, i.e. VirusGEN® AAV Transfection Kit. Notably, no commercial license is required for use of VirusGEN® transfection reagents in further manufacturing. Thus, swapping the transfection reagent in your virus production workflow is relatively painless and may be worthwhile for improvements to titer and/or percentage of full AAV capsids.
Cell Line and Cell Culture Media
Another relatively simple way to improve vector yield is through use of highly productive cell lines grown in supportive cell culture media. As with transfection reagents, specialized HEK 293 cell lines and production media that are geared toward maximizing viral vector productivity are commercially available. One obstacle to adopting a new cell line or media into your process may be licensing requirements surrounding their commercial use. Below is data from an experiment where functional lentivirus titer was evaluated for identical transfections in four different commercially available cell culture media.
In this example (using the VirusGEN® LV Transfection Kit and Expi293™ cells), high lentivirus titer was obtained in all cases, but was even higher for cells cultured in certain media. When testing new media or cell lines, it is important to ensure cell growth and viability are normal before beginning transfection experiments. Cells may require adaptation to new media or a recovery period post-thaw for best results.
Transfection Procedure
While gains in viral production can immediately be had by a simple swap of reagents, further improvements may be possible through finetuning of the transfection procedure. It is advised to follow the protocol supplied by the transfection reagent manufacturer as a starting point and to examine the following parameters for further optimization:
- Cell confluency or density at transfection
- Nucleic acid mass
- Transfection reagent volume
- Incubation time of transfection reagent and DNA
- Addition of 'Enhancer' solutions
- Time of harvest
Interestingly, the optimal value of each parameter for producing the highest viral titer and percentage of full AAV may vary by vector and cell culture platform. Thus, these parameters should ideally be evaluated anew for different vectors (e.g. AAV versus LV) and cell culture platforms (e.g. adherent versus suspension cells). Please contact Mirus Bio Technical Support if you would like to discuss strategies for efficiently optimizing your transfection.
DNA Constructs
If cloning is in your wheelhouse, then you may be interested in exploring different packaging system and transfer plasmid options to increase viral vector production. Examine the y-axis in the graphs below which show lentivirus titer from transfections using different packaging and transfer plasmids. A difference in lentivirus titer of over an order of magnitude was observed that was dependent on the packaging and transfer plasmid system used in the transfection (compare A and B).
Higher titers can be achieved through vector design, also known as 'vectorology,' though applying new plasmids to an existing process may require some time for cloning and testing. Fortunately, many plasmids are commercially available; most require a commercial license to modify or use in further manufacturing.
Cell Culture Platform
Lastly, the most dramatic change one can take in efforts to increase viral titer would be to consider a change in cell culture platform. The sheer increase in cell culture volume alone when moving from small to large scale systems can increase overall viral vector yield--assuming per cell productivity remains the same. The VirusGEN® reagents and kits have proven to be compatible with diverse cell culture platforms, which allows for high titer virus production when scaling up from flatware to multi-layered flasks or bioreactors.
Are any of these modifications feasible for your existing viral vector production workflow? Do you have more tips or tricks for improving viral titer? Please leave a comment below or contact Mirus Bio Tech Support to continue the discussion!