Aeration and Shear Stress Are Critical Process Parameters for the Production of Oncolytic Measles Virus.

Tanja A Grein,Hauke Dieken,Daniel Loewe,Peter Czermak,Tobias Weidner,Denise Salzig

doi:10.3389/fbioe.2019.00078

Abstract

Oncolytic Measles virus is a promising candidate for cancer treatment, but clinical studies have shown that extremely high doses (up to 1011 TCID50 per dose) are required to effect a cure. Very high titers of the virus must therefore be achieved during production to ensure an adequate supply. We have previously shown that Measles virus can be produced in Vero cells growing on a Cytodex 1 microcarrier in serum-containing medium using a stirred-tank reactor (STR). However, process optimization and further process transfer or scale up requires the identification of critical process parameters, particularly because the use of STRs increases the risk of cell damage and lower product yields due to shear stress. Using a small-scale STR (0.5 L working volume) we found that Measles virus titers are sensitive to agitator-dependent shear, with shear stress ≥0.25 N m−2 reducing the titer by more than four orders of magnitude. This effect was observed in both serum-containing and serum-free medium. At this scale, virus of titers up to 1010 TCID50 mL−1 could be achieved with an average shear stress of 0.1 N m−2. We also found that the aeration method affected the virus titer. Aeration was necessary to ensure a sufficient oxygen supply to the Vero cells, and CO2 was also needed to regulate the pH of the sodium bicarbonate buffer system. Continuous gassing with air and CO2 reduced the virus titer by four orders of magnitude compared to head-space aeration. The manufacture of oncolytic Measles virus in a STR can therefore be defined as a shear-sensitive process, but high titers can nevertheless be achieved by keeping shear stress levels below 0.25 N m−2 and by avoiding extensive gassing of the medium.

Highlights

Measles virus has natural oncolytic properties and an excellent safety profile as an attenuated vaccine (WHO, 1998)
We recently described an adapted process that can achieve titers of 1010 TCID50 mL−1, in which Vero host cells attached to Cytodex 1 microcarriers are cultivated in a small-scale stirredtank reactor (STR) using serum-containing medium (Grein et al, 2018)
Infection with Measles virus increased the oxygen consumption rate by 10-fold, to −1.3 ± 0.7 mmol h−1 per consumption rate or the specific lactate production rate. These results indicated that the active aeration of Vero cells in a STR should be avoided during Measles virus production

Summary

Introduction

Measles virus has natural oncolytic properties and an excellent safety profile as an attenuated vaccine (WHO, 1998). Engineered oncolytic Measles viruses selectively kill cancer cells and induce a systemic anti-tumor immune response, making this virus an attractive choice for the treatment of patients suffering from incurable cancer (Rammensee, 2014; Russell et al, 2014; Galanis et al, 2015). Clinical studies have shown that high doses of oncolytic Measles virus (108-1011 TCID50 per dose) are required for successful treatment (Russell et al, 2014; Galanis et al, 2015). Current production methods for the measles vaccine do not yield enough virus for oncolytic therapy, with maximum titers of 106 TCID50 mL−1 reported for mammalian cells growing on microcarriers (Trabelsi et al, 2014). The high titers were realized by harvesting the thermosensitive virus at the optimal time, determined online by dielectric spectroscopy (Grein et al, 2018)

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Discussion

Conclusion