High-Titer Hepatitis C Virus Production in a Scalable Single-Use High Cell Density Bioreactor

Abstract

Hepatitis C virus (HCV) infections pose a major public health burden due to high chronicity rates and associated morbidity and mortality. A vaccine protecting against chronic infection is not available but would be important for global control of HCV infections. In this study, cell culture-based HCV production was established in a packed-bed bioreactor (CelCradle™) aiming to further the development of an inactivated whole virus vaccine and to facilitate virological and immunological studies requiring large quantities of virus particles. HCV was produced in human hepatoma-derived Huh7.5 cells maintained in serum-free medium on days of virus harvesting. Highest virus yields were obtained when the culture was maintained with two medium exchanges per day. However, increasing the total number of cells in the culture vessel negatively impacted infectivity titers. Peak infectivity titers of up to 7.2 log10 focus forming units (FFU)/mL, accumulated virus yields of up to 5.9 × 1010 FFU, and a cell specific virus yield of up to 41 FFU/cell were obtained from one CelCradle™. CelCradle™-derived and T flask-derived virus had similar characteristics regarding neutralization sensitivity and buoyant density. This packed-bed tide-motion system is available with larger vessels and may thus be a promising platform for large-scale HCV production.

Inactivated genotype 1a, 2a and 3a HCV vaccine candidates induced broadly neutralising antibodies in mice

“…Applying this evolutionary approach until no further increase in viral infectivity titres is observed, and until a homogeneous viral population with no obvious evidence for ongoing selection of additional putative adaptive substitutions is recorded is expected to result in selection of highly fit and genetically stable virus populations 31. Thus, the developed genetically stable, high-yield HCV recombinants can in the future be used to initiate virus vaccine antigen production with sequence confirmed early viral passage seed stocks 44 45. Based on results from this study using full-length recombinants, as well as on results from the previous study using a JFH1-based recombinant,31 the upper limit for HCV infectivity titres in monolayer Huh7.5 cell cultures is between 6 and 7 log10 FFU/mL, which might be due to limited availability of required host cell factors.…”

“… 31 Thus, the developed genetically stable, high-yield HCV recombinants can in the future be used to initiate virus vaccine antigen production with sequence confirmed early viral passage seed stocks. 44 45 Based on results from this study using full-length recombinants, as well as on results from the previous study using a JFH1-based recombinant, 31 the upper limit for HCV infectivity titres in monolayer Huh7.5 cell cultures is between 6 and 7 log10 FFU/mL, which might be due to limited availability of required host cell factors. Future studies should focus on investigation of the effect of acquired viral substitutions on the viral life cycle.…”

“…Further preclinical and clinical development requires optimisation of vaccine production and processing conditions to ensure compatibility with vaccine manufacturing 36 44. Moreover, further research should define the most powerful of the developed vaccine candidates, based on performance in an optimised bioprocess and detailed immunogenicity studies of resulting antigens, as well as dose finding studies.…”

“… 36 44 Moreover, further research should define the most powerful of the developed vaccine candidates, based on performance in an optimised bioprocess and detailed immunogenicity studies of resulting antigens, as well as dose finding studies. In initial upstream bioprocess studies employing a scalable bioreactor 44 57 and virus seed stocks generated from early viral passages following transfection, genotype 2a and 3a HI-viruses and high-yield genotype 5a HCV 35 yielded considerably higher infectivity titres than the genotype 1a HI-virus, signifying an advantage for these three candidates in the production process. No immunocompetent HCV in vivo challenge model is available.…”