pH, pCO₂, and Temperature Effect on R‐Adenovirus Production

Abstract

The effects of pH, carbon dioxide vapor pressure, pCO(2), and temperature on E1 and E3 deleted recombinant adenovirus vector (rAV) production with HEK293S cells have been studied in the ranges of pH = 6.7-7.7, pCO(2) = 0.05-0.20 atm, and T = 32-39 degrees C, respectively. The experiments were performed in four 500-mL bioreactors in parallel, which make possible the reduction of inter-run variability. Cell concentration and viability, relative oxygen uptake rate (OUR), fluorescence, and viral titer were measured. It was found that, although pH and pCO(2) did not affect significantly cell viability in the range studied, they had an important effect on virus titer. pCO(2) allowed the maximum production of rAV at 0.05 atm, and pH showed a very sharp optimum at 7.2. Temperature had an effect on both cell metabolism and virus titer. Low temperature prolonged cell viability and high OUR. Most of all, a 3-fold increase in virus yield was found at 35 degrees C compared to that at 37 degrees C, while 32 degrees C was not as beneficial (1.5-fold increase). This finding could have an important impact on large-scale production. This phenomenon was modeled using a simple 3-parameter synthesis-decay model. This model shows how the optimum gain in virus production at 35 degrees C is due to a balance between the production and decay processes at that temperature.

Cell Culture Processes for the Production of Viral Vectors for Gene Therapy Purposes

“…Higher titers were obtained at 35 and 32°C, with maximum titers being reached at 40 and 74 hpi, respectively. The highest titers were produced at 35°C, which were 3.4-fold higher than at 37°C (Jardon and Garnier 2003). This finding was confirmed by Cortin et al (2004) who showed a 2.4-fold increase in viral titer when 293S cells were cultured at 35°C compared to 37°C.…”

“…The virus infection phase is prolonged with increasing pCO 2 , which also decreases the virus yield. The maximum titer at 0.2 atm was 2-fold lower than the maximum titer at 0.05 atm (Jardon and Garnier 2003).…”

“…The same authors reported an optimal pH of 7.3 for virus productivity which decreased by 50% at pH of 6.7 and 7.7 (Xie et al 2002). Jardon and Garnier (2003) reported a 4-fold increase in virus titer at pH 7.2 compared to pH 6.7 and 7.7 using 293 cells. Infectivity of the virus was not deemed to be responsible for this difference as the increase of infected cells was similar at all pH.…”

Comparisons of optical pH and dissolved oxygen sensors with traditional electrochemical probes during mammalian cell culture

“…Shifts of 0.15 units from the optimal growth and production pHs resulted in reductions in specific growth and production rates of 13 and 16%, respectively. Larger changes in pH during culture of hybridoma and other cell lines has resulted in more severe reductions in growth and protein production (Hayter et al, 1992;Jardon and Garnier, 2003), impacted protein glycosylation (Borys et al, 1993; Muthing et al, 2003), and endogenous virus-like particle production (Brorson et al, 2002). As demonstrated in the pH sensitivity plot (Fig.…”

Histone deacetylase inhibitor trichostatin A enhances the antitumor effect of the oncolytic adenovirus H101 on esophageal squamous cell carcinoma in vitro and in vivo

“…This leads to an inconsistency between the efficacy obtained from cell culture experiments in vitro and in clinical trials. Therefore, their efficacy of viral oncolysis need to be improved (42)(43)(44)(45). The present study examined whether the enhanced in vitro tumor cytotoxicity, mediated by TSA and H101 in combination, was also exhibited during EC1 cell tumor growth in vivo.…”