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Chemically Defined iCELLis® Bioreactor-Based Lentiviral Vector Mass Production
Published on 18 August 2021
A Powerful Tool to Measure Cell Density and Optimize Processes
InVitria: Atherly Pennybaker, Media Formulation and Product Applications Specialist, Randy Alfano, PhD, VP Product Development, Sofia Pezoa, PhD, Cell Culture Scientist
Pall: Todd Lundeen, Andrew Laskowski
Efficient Lentiviral Vector Production Scalable to the iCELLis® Bioreactor Technology
Abstract
Retroviral vectors are a promising candidate for the treatment of rare, monogenic diseases. Lentivirus — a type of retrovirus based on HIV — is currently being clinically evaluated in stage 3 trials for the treatment of rare blood disorders in addition to the genetic modification of human T cells in oncology applications. While the efficacy looks promising in the clinic, numerous questions surrounding the feasibility of large-scale manufacturing of lentivirus remain. Traditionally, production of these retroviral vectors has been performed using adherent platforms that rely on the use of fetal bovine serum for the adherence and growth of HEK cells used to produce lentivirus vectors. At scale, fetal bovine serum presents numerous problems including but not limited to lot-to-lot variation, constraints on the global supply chain, and increasing cost due to global demand. To overcome these limitations, we have developed OptiPEAK® HEK serum-free, chemically defined cell culture medium that is free from any blood-derived proteins and supports adherent HEK cells in 2D and 3D formats. With OptiPEAK HEK cell culture medium, we can achieve equivalent growth kinetics and viral titer compared to medium supplemented with serum. We also demonstrate that OptiPEAK HEK medium can be readily scaled up to an iCELLis Nano bioreactor, achieving high viral titers without the addition of serum. The presented data here demonstrate that high titer, retroviral vectors can be manufactured without the constrictions brought on by the inclusion of serum in cell culture medium.
High Titer Lentivirus Production in a Serum-Free Medium: Optimization of parameters for lentivirus production in serum-free medium in flatware
To determine if a serum-free, blood-free, chemically defined medium could be used for lentivirus (LV) production at scale with adherent HEK293T cells, production parameters were first optimized in flatware. Second generation pseudotyped LV was produced in OptiPEAK® HEK293t medium and compared to DMEM+10% fetal bovine serum (FBS), which is traditionally used for adherent HEK293T.
- FBS inclusion at scale presents numerous problems, including cost, variability, and lack of chemical definition
- OptiPEAK HEK293t is a serum-free chemically defined medium that supports adherent HEK293T cells and is completely free of blood-derived proteins
- We hypothesized that OptiPEAK HEK293t medium could achieve equivalent titer compared to serum-containing media

Flatware lentivirus production in a chemically defined, blood-free and serum-free medium
Pseudo typed VSV-g lentivirus vectors were produced in flatware in OptiPEAK HEK293t and DMEM + 10% FBS. HEK293T cells for LV production were seeded at 75,000 cells/cm2 18 hours before transfection. Cells were transfected for 2 hours, followed by a complete media change. LV production in flatware was carried out for 48 hours. After 48 hours, Conditioned culture media containing LV virus particles was harvested and titered by functional transduction with HT-1080 cells. Transduced HT-1080 cells were analyzed by flow cytometry 48-72 hours post transduction. N=3 separate experiments, error bars represent standard deviation. Y-axis shows estimated infectious units (IFU) per mL.
We achieved equivalent or greater functional titer in OptiPEAK HEK293t compared to DMEM + 10% FBS. These results suggest that serum-free media blood-free, chemically defined media has equal to or greater performance compared to FBS-supplemented media, thereby eliminating the problems faced with serum supplementation at scale.
HEK293t growth in the ICELLis Nano Bioreactor is highly similar between OPTIPEAK HEK293t Medium and DMEM+FBS
- We hypothesized that the same parameters for cell growth and LV production in flatware would be scalable to the iCELLis Nano bioreactor in OptiPEAK HEK293t medium
- The iCELLis Nano bioreactor is a scalable bioreactor technology for adherent cells suitable for commercial production of viral vectors
- 0.53 m2 iCELLis Nano bioreactors were inoculated with HEK293T cells at an initial density of 10,000 cells/cm2
- Glucose and lactate were monitored offline by daily sampling
- Growth kinetics were monitored by the Aber Futura biomass probe
- Target cell density for transfection was 70,000 cells/cm2 (based on 2D experiments)

iCELLis Nano bioreactor glucose, lactate, and growth kinetics of HEK293T in OptiPEAK HEK293t and DMEM+10% FBS
Representative glucose, lactate, and growth kinetics of a production run in OptiPEAK HEK293t and DMEM + 10% FBS in 0.53 m2 iCELLis Nano bioreactor.
(A) Glucose consumption was monitored by daily sampling of the iCELLis Nano bioreactor in OptiPEAK HEK293t and DMEM + 10% FBS.
(B) Lactate production was monitored by daily sampling of the iCELLis Nano bioreactor in OptiPEAK HEK293t and DMEM + 10% FBS.
(C) Growth kinetics of HEK293T were monitored in the iCELLis Nano bioreactor in OptiPEAK HEK293t and DMEM + 10% FBS, and the cell density in cells/cm2 was calculated from the raw capacitance of the Aber biomass probe [1].
(D) Table 1. Optimized set point parameters determined for OptiPEAK HEK293t in the iCELLis Nano bioreactor.
In the iCELLis Nano bioreactor, we achieved equivalent growth kinetics of HEK293T in OptiPEAK HEK293t compared to DMEM + 10% FBS. Glucose and lactate measurement trends were also similar between the two media. However, DMEM + 10% FBS initially started at higher levels for both glucose and lactate. By the end of the production run, glucose levels did not drop below 1 g/L for both media. Lactate, however, did rise above 12 mM in DMEM + 10% FBS. Both media conditions were transfected when the cell density was 70,000 cells/cm2 as determined by the Aber biomass probe. These results demonstrate that OptiPEAK HEK293t is scalable to the iCELLis bioreactor and achieves equivalent growth performance as DMEM + 10% FBS.
OPTIPEAK HEK293t medium produces high titer lentivirus in the ICELLis Nano Bioreactor: High lentivirus titer was achieved with OptiPEAK HEK293t medium in the iCELLis Nano bioreactor after optimization
- We achieved equivalent growth kinetics in OptiPEAK HEK293t compared to DMEM + 10% FBS, demonstrating that serum-free, blood-free, chemically defined media supports adherent HEK293T at scale
- We hypothesized that we would achieve equivalent functional titer in OptiPEAK HEK293t compared to DMEM + 10% FBS
- We demonstrate that high titer LV production can be scaled from flatware to the iCELLis Nano bioreactor in OptiPEAK HEK293t with overall high viral yield.
Four representative LV production runs in 0.53 m2 iCELLis Nano bioreactors in either OptiPEAK HEK293t or DMEM + 10% FBS are shown in Figure 3: two pre-optimization runs (A: Before Optimization) and two runs after optimization of OptiPEAK HEK293t conditions (B: After Optimization). Functional titer was determined by HT-1080 transduction assay. Transduced cells were analyzed by flow cytometry. For each bioreactor run, 900 mL of virus-containing supernatant was collected.

Functional LV titer produced in OptiPEAK HEK293t medium and DMEM+10% FBS in the iCELLis Nano bioreactor
High LV functional titer was achieved in the iCELLis Nano bioreactor in OptiPEAK HEK293t serum-free medium. Optimization of production parameters (transfection cell density and the addition of InVitria Optiferrin® transferrin) resulted in an 4X increase in viral titer in OptiPEAK HEK293t compared to DMEM + FBS.
Conclusion
- In flatware, OptiPEAK HEK293t serum-free medium produces equivalent LV titer as DMEM + FBS
- In the iCELLis Nano bioreactor, growth and metabolite profiles are highly similar between HEK293T cells grown in OptiPEAK HEK293t and DMEM + FBS
- In the iCELLis Nano bioreactor using InVitria’s cell culture protocol, OptiPEAK HEK293t medium produces functional titers approximately four times higher than DMEM + FBS
Footnotes
InVitria: +1 800 916 8311 (USA) • Email us at: https://invitria.com/contact-us/ or info@invitria.com • Web: www.invitria.com
Pall Corporation: +1 800 717 7255 (USA) • +41 (0)26 350 53 00 (Europe) • +65 6389 6500 (Asia/Pacific) • Email us at: www.pall.com/contact • Web: www.pall.com/biotech
Alfano, R. et al. (2020), Implementation of Aber’s Futura Biomass Probe in Pall’s iCELLis Nano Bioreactor Provides a Robust and Reproducible Method to Assess Cell Density [Poster], ASGCT, < https://www.pall.com/en/biotech/posters-presentations/reproducible-method-assess-cell-density.html>