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Published on: September 12, 2019
Perfusion process with tangential flow filtration for oncolytic VSV-GP production
Orsolya Hamusics1, Anja Wittmann2, Katrin Hasler1
1Viral Therapeutics Center, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany.
Abstract:
The oncolytic vesicular stomatitis (VSV)-GP virus is a promising therapeutic against cancer. To ensure clinical efficacy, doses with high titers are required, which poses a challenge for the manufacturing process. Perfusion cultivation processes with high cell densities have attracted great interest to improve the production titer. This work aimed to enhance the titer of the VSV-GP production process with suspension human embryonic kidney 293 (HEK293) cells by using perfusion with tangential flow filtration (TFF) and virus retention. For this purpose, six potential critical process parameters were evaluated using I-optimal design of experiments (DoE). The study showed that several input parameters and their interactions have significant impact on the infectious titer. Increasing the seeding cell density significantly improved the infectious titer, allowing infection at up to 46.6 × 106 cells mL-1 without decrease in the cell-specific virus yield. Keeping the perfusion pause after infection at minimum (1.1-1.3 h) and subsequently start the perfusion with a higher exchange rate (0.045-0.051 nL cell-1 d-1) was shown to be beneficial. The process was sensitive to shear stress and thus, the optimal crossflow rate was between 44 and 55 mL min-1, which corresponds to 950-1150 s-1 shear rate. By optimizing the perfusion process, the titer reached up to 5.1 × 1010 TCID50 mL-1, which is 17-fold higher than in batch cultivation. Overall, this work presents perfusion cultivation as an efficient technology to improve the VSV-GP titer with virus retention.
Insights
High-titer production of vesicular stomatitis virus (VSV)-GP for cancer therapy was achieved using optimized perfusion cultivation. This method significantly increased virus yield by enhancing cell density and controlling perfusion parameters for improved manufacturing.
Area of Science:
- Biotechnology
- Virology
- Chemical Engineering
Background:
- Oncolytic vesicular stomatitis virus (VSV)-GP shows therapeutic potential for cancer treatment.
- Achieving high virus titers is crucial for clinical efficacy but challenging in manufacturing.
- Perfusion cultivation at high cell densities is a promising strategy to boost production titers.
Purpose of the Study:
- To enhance the production titer of VSV-GP using suspension human embryonic kidney 293 (HEK293) cells.
- To optimize a perfusion cultivation process incorporating tangential flow filtration (TFF) and virus retention.
- To identify and evaluate critical process parameters influencing VSV-GP yield.
Main Methods:
- Utilized I-optimal design of experiments (DoE) to assess six critical process parameters.
- Employed suspension HEK293 cells in a perfusion system with TFF and virus retention.
- Investigated the impact of seeding cell density, perfusion pause duration, exchange rate, and shear stress.
Main Results:
- Increasing seeding cell density improved infectious titer up to 46.6 × 10^6 cells mL^-1.
- Minimizing perfusion pause (1.1-1.3 h) and using a higher exchange rate (0.045-0.051 nL cell^-1 d^-1) were beneficial.
- Optimal crossflow rate (44-55 mL min^-1) minimized shear stress sensitivity.
- Achieved a 17-fold increase in titer (up to 5.1 × 10^10 TCID50 mL^-1) compared to batch cultivation.
Conclusions:
- Optimized perfusion cultivation significantly enhances VSV-GP production titers.
- High cell densities and controlled perfusion parameters are key to maximizing virus yield.
- Perfusion cultivation with virus retention is an effective technology for improving VSV-GP manufacturing.
