Application Note - Integrating Dielectric Spectroscopy for High-Titer Oncolytic Measles Virus Production

Production of oncolytic viruses  as anti-cancer agents. Oncolytic viruses, including the measles virus (MeV), exhibit a preferential killing of cancer cells, rendering them promising candidates for novel tumor therapies. As part of the viral infection cycle, an oncolytic virus infects a cancer cell. It initiates its destruction from the inside by rewiring the cell‘s synthetic machinery to synthesize viral components. Viruses are synthesized until the cell finally bursts, resulting in the death of  the host cell and the release of new viral particles, which then reinitiate infection cycle. Viruses released into the culture medium gradually lose their infectivity over time. Consequently, a critical challenge in the biomanufacturing of infectious viral particles for therapeutic use is the identification of the optimal time of harvest (TOH). In the context of biotechnological processes focused on the production of oncolytic viruses for therapeutic application, it is essential to achieve a balance between maintaining viable host cells for continued virus production and harvesting the culture at the peak of viral infectivity. 

Aim: Determine the best time  of harvest (TOH) to maximize the yield of infectious virus particles

However, the commonly used methods for determining viral infectivity, such as plaque assay and TCID50 (median tissue culture infectious dose) assay, are time-consuming to set up, require incubation periods of several days and are susceptible to infection due to the repetitive and manual nature of setting up the experiments. Moreover, these methods are not suitable for r eal-time in-process analysis during virus production in a timely manner to determine the optimal TOH. 

Challenge: traditional off-line methods are time-consuming and subjective. 

The working group “Cell Culture” of the Institute of Bioprocess Engineering and Pharmaceutical Technology (IBPT) at the University of Applied Sciences Mittelhessen (THM) uses process analytical technology (PAT) for the development of novel processes in mammalian cell culture. Dielectric spectroscopy (DS) is one PAT tool currently used in several studies. 

DS, a non-invasive analytical technique, offers a promising approach for monitoring cell culture processes. By employing DS, the cell’s permittivity can be measured, which reflects its ability to get polarized. This provides valuable insights into cell viability, growth, and alterations in cellular composition and morphology –important factors during cell culture for virus production. Permittivity’s reliance on the cell’s membrane for polarization ensures the selective measurement of whole, intact cells, thus distinguishing them from non-living components such as dead cells, debris, and scaffolds (which may be used during cell culture). Overall, DS can provide valuable information for optimizing cell culture conditions and improving virus production yields. 

Solution: identify an in-line PAT  tool that can deliver objective,  real-time data with the option for integrating data analysis software and automated control loops. 

Production of measles virus MeV were produced in Vero cells on Cytodex 1 microcarriers (3 g L-1) in a 1 L stirred tank reactor (STR; with siliconized glass vessel) with 0.5 L working volume. The temperature was set to 32°C, the pH was regulated to 7.4 ± 0.1 (using 1 M NaOH or CO2 gassing) and the stirrer speed was set to 70 rpm. The dissolved oxygen was maintained between 50-70 % by discontinuous aeration (0.01 vvm), as previous studies had shown continuous aeration had negative effects on virus titer1. The concentration of viable cells was monitored in-line by DS (Incyte and ArcView, both Hamilton) via measuring permittivity at 1 MHz with a F-scan of 0.3-10 MHz. In addition, the total cell concentration was determined off-line by dissolving the cells (bound on microcarriers) using crystal violet solution (1 g L-1 in 0.1 % [w/v] citric acid) and counting cell nuclei using a Neubauer counting chamber. Subsequent determination of  MeV infectivity in daily frozen reactor samples was performed by TCID50 assay.

Advantages

• Dielectric spectroscopy (DS) is a suitable PAT tool for the in-line-measurement of viable cell density in uninfected Vero cells. 

• The maximum virus titer in the supernatant is directly related to the global permittivity maximum with a time off-set. 

• DS can be used to determine the time frame from viral infection to cell lysis, making it possible to predict the optimal TOH.

Results

In uninfected Vero cells, the permittivity signal correlates with the total cell concentration determined off-line from the period of the cell adhesion process (approximately 4-7 h post inoculum) to the end of the exponential cell growth phase (approximately 50 h  post inoculum). In contrast, in Vero cells infected with MeV, there was no correlation observed between permittivity signal and cell concentration determined off-line. In case of a high multiplicity of infection (MOI) with 30 TCID50 /cell, in, the permittivity signal increases during the period  0-48 h post infection, while the total cell concentration determined off-line remains almost constant (Figure 3). Subsequently, the permittivity signal remains relatively constant for a short period of time until it starts to decrease steadily (Figure 3). This time course of the permittivity signal is consistent with microscopic observations of Grein et al. 2 regarding morphological changes in MeV-infected Vero cells with high MOI. These observations revealed the formation of syncytia within the first 48 h after infection, which persisted for up to 24 h. Subsequently, the infected Vero cells detached from the microcarrier surface until cell lysis f inally occurred.

Conclusions

Although DS cannot be used to determine cell concentration in the case of MeV-infected Vero cells, it is possible to determine morphological changes of the cell and thereby the time frame for syncytia formation and cell lysis process using DS. This allows for the prediction of the optimal TOH. Following an evaluation of the permittivity data of 16 independent MeV productions in the STR and the corresponding virus titers of daily reactor samples (TCID50 assay), it was demonstrated that the maximum virus titers in the supernatant occurred 39.6 ± 7 h after reaching the global permittivity maximum 2. Further experiments and shorter sampling intervals may allow a more precise prediction of the optimal TOH.