Conclusions
- The addition of Recombinant Insulin to a chemically-defined cell culture media led to a 1.5-fold increase in AAV production without increasing viable cell density.
- LV titers were 2-fold higher following addition of Recombinant Insulin to the cell culture media, associated with an increase in HEK293 cell density.
- Supplementing Recombinant Insulin into chemically defined cell culture media, 2 hours before transfection of HEK293 cells, resulted in increased AAV and LV viral titers, potentially leading to substantial cost savings in the manufacturing process of viral vectors for gene therapy.
Background
Viruses naturally possess the ability to deliver genetic material into cells while evading genetic modification, rendering them valuable for cell and gene therapies. Viral vector-based gene therapy remains the most widely utilized form of gene therapy.
As the potential to scale up production processes emerges, the successful identification of optimized cell culture media with specific components becomes crucial to enhance productivity. This can be accomplished through the use of highly defined cell culture media with selected ingredients that boost production. The role of insulin in promoting cell growth and productivity in serum-free media is well known within biomanufacturing.
This study shows the impact of optimizing a chemically-defined cell culture media with Recombinant Insulin to boost the production of Adenovirus-associated virus (AAV) and Lentivirus (LV) in HEK293 cells.
Study description
To establish a HEK293 cell culture with high-yield performance, a range of insulin concentrations were added at different addition times to maximize cell growth and productivity. Suspension-adapted HEK293 cells were cultivated using an in-house developed chemically defined media. Insulin (0, 5, 10, and 20 mg/L) was added at specific time-points (before, during, or after transfection).
The production of LV and three common AAV serotypes (AAV-2, AAV-5, and AAV-8) was evaluated. For AAV production, a three-plasmid system was transfected into HEK293 cells. After 72 hours, AAV particles were collected, the content of ITR gene was detected by qPCR, and the titre (VG/mL) of AAV was calculated.
For LV production, a four-plasmid system containing GFP tags was transfected into HEK293 cells. LV particles were harvested 48 hour after transfection. LV particles were used to infect HT1080 cells. 72 hours post-infection GFP-positive cells were determined using flow cytometry and the transduction units per ml (TU/ml) were calculated.
Results
While insulin did not significantly impact cell proliferation in AAV-producing HEK293 cells, (Fig. 1A, 1C, 1E), irrespective of addition time and concentration, it notably increased viral titers. Specifically, adding 10 mg/L Recombinant Insulin 2 hours before transfection increased the AAV-2 titer by 1.5-fold, and both the AAV-5 and AAV-8 titers by 1.4-fold (Fig. 1B, 1D, 1F).
Furthermore, insulin supplementation led to enhanced cell proliferation in LV-producing HEK293 cells (Fig. 1G), and resulted in a significant 2-fold increase in LV titer, when 20 mg/L insulin were added to the culture 2 hours before transfection (Fig. 1H).
Figure 1: Addition of insulin to HEK293 cell culture media boosts AAV and LV production. Addition of insulin (2 hours before transfection) does not affect cell density of HEK293 cells expressing AAV-2 (A), AAV-5 (C), and AAV-8 (E), but increases cell proliferation of HEK293 cells expressing LV (G). Addition of different insulin concentrations at different times increases AAV-2 (B), AAV-5 (D), AAV-8 (F) and LV (H) titers.
The highest fold-change increase in the viral titer is showed by the dashed blue box. The horizontal dashed line represents the baseline for the untreated control (0 mg/L insulin). * P < 0.05, ** P < 0.01; statistical analysis was performed using an unpaired t-test.
