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How can I increase my viral titers and improve the percentage of full AAVs?

A graphic of stylized AAV showing one full capsid among a sea of empty.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.

Graphs showing AAV titer and % full can be affected by choice of transfection reagent.

TransIT-VirusGEN® Reagent and VirusGEN® AAV Kit Generate High Quality Virus across AAV Serotypes. Viral Production Cells 2.0 (Thermo Fisher) grown in Viral Production Medium (Thermo Fisher) were seeded immediately prior to transfection (2 ml, non-treated 6-well plate); AAV2 and AAV9 were produced by transfecting cells at 2×106 cells/ml, and AAV5 was produced by transfecting cells at 3×106 cells/ml. Transfections were performed according to manufacturer-recommended protocols with the indicated reagents and reagent-to-DNA ratio (vol:wt). AAV was harvested at 72 hours post-transfection. Genome copies were determined by dPCR using primers and a probe targeting the CMV promoter. Serotype-specific ELISA kits (Progen) were used to quantitate capsids. The % full capsids was calculated by dividing genome titer by capsid titer. The error bars represent the standard deviation of duplicate wells.

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.

Graph showing lentivirus titer can depend on the type of cell culture medium used.

High Functional Lentivirus Titers Are Achieved Using VirusGEN® LV Kit in Multiple Media Formulations. Lentivirus was produced by transfecting Expi293™ cells grown in either Expi293™ Expression Media (Thermo Fisher), FreeStyle™ F17 Media (Thermo Fisher), LV-MAX™ Media (Thermo Fisher) or BalanCD HEK 293 Media (Irvine Scientific). Cells were seeded at 4×106 cells/ml (2 ml, non-treated 6-well plate) and transfected with the TransIT-VirusGEN® Transfection Reagent (3:1 reagent-to-DNA ratio by wt:vol) and pALD-Lenti-EGFP-A transfer plasmid and pALD-VSV-G-A, pALD-Rev-A, pALD-GagPol-A packaging plasmids (Aldeveron, 3:0.5:0.5:2 ratio by wt; 1 µg total DNA/ml culture volume), according to the recommended protocol. The VirusGEN® LV Enhancer was added 18 hours post-transfection. Functional titer from filtered, virus-containing supernatant was measured after 72 hours post-transduction of 293T/17 cells using Guava® easyCyte™ 5HT Flow Cytometer. Functional titers were measured from virus dilutions with less than 20% GFP positive cells. The error bars represent the standard deviation of triplicate wells.

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, e.g. RevIT™ AAV Enhancer
  • 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).

Graphs showing lentivirus titer depends on transfection reagent and viral vector plasmid constructs used during production.

TransIT-VirusGEN® Reagent and the VirusGEN® LV Transfection Kit Outperform PEI-based Reagents with Multiple Lentivirus Packaging Systems. Lentivirus production using TransIT-VirusGEN® Transfection Reagent +/- the VirusGEN® LV Enhancer was compared to Competitor A3 (2:1 reagent-to-DNA ratio) and Competitor C1 (4:1 reagent-to-DNA ratio) using their recommended protocol. Lentivirus was produced by transfecting Expi293™ cells grown in Expi293™ Expression Media (Thermo Fisher Scientific) at 4 x 106 cells/ml with two different LV vector mixtures (1 μg/ml, 2μg/ml) including: (A) 3rd generation vectors pALD-LentiEGFP-A transfer vector and pALD-VSV-G-A, pALD-Rev-A, pALD-GagPol-A packaging vectors (3:0.5:0.5:2 DNA ratio, Aldevron) or (B) 3rd generation vectors pLKO.1-puro-CMV-TurboGFP™ transfer vector (MilliporeSigma) and ViraSafe™ Pantropic Packaging mix (pRSV-Rev, pCMV-VSV-G, pCgpV, CellBio Labs) at a 3:0.5:0.5:2 DNA ratio. The VirusGEN® LV Enhancer was added to the TransIT-VirusGEN® condition at 18 hours post-transfection. Virus containing supernatant was used to transduce 293T/17 cells and GFP expression was measured at 72 hours post-transduction using Guava® easyCyte™ 5HT Flow Cytometer. Lentivirus functional titers were measured from virus dilutions with less than 20% GFP positive cells. The error bars represent the range of duplicate wells.

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 contact Mirus Bio Tech Support to continue the discussion!

Explore Related Info & Links

  • Watch the webinar ‘How to increase AAV production with a novel enhancer designed for use with any transfection reagent in any suspension HEK cell’ [31:11]
  • Learn about transfection reagents for cell and gene therapy here
  • Check out TransIT-VirusGEN® Transfection Reagent
  • Transfection 101 – Understand Viral Titer and Quality

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