A serum-free Vero production platform for a chimeric virus vaccine candidate

Abstract

MedImmune Vaccines has engineered a live, attenuated chimeric virus that could prevent infections caused by parainfluenza virus type 3 (PIV3) and respiratory syncytial virus (RSV), causative agents of acute respiratory diseases in infants and young children. The work here details the development of a serum-free Vero cell culture production platform for this virus vaccine candidate. Efforts to identify critical process parameters and optimize culture conditions increased infectious virus titers by approximately 2 log(10) TCID(50)/ml over the original serum-free process. In particular, the addition of a chemically defined lipid concentrate to the pre-infection medium along with the shift to a lower post-infection cultivation temperature increased virus titers by almost 100-fold. This improved serum-free process achieved comparable virus titers to the serum-supplemented process, and demonstrated consistent results upon scale-up: Vero cultures in roller bottles, spinner flasks and bioreactors reproducibly generated maximum infectious virus titers of 8 log(10) TCID(50)/ml.

Fig. 1

Virus production profiles at different MOIs. Duplicate T-75 flasks of serum-free Vero cells were infected 3 days post-seeding with MEDI-534 at MOI of 0.1, 0.01, 0.001, 0.0001, or 0.00001. Cultures were incubated at 37°C pre- and post-infection

Fig. 2

Effects of time of infection and post-infection temperature on virus titers. Serum-free Vero cultures were infected with MEDI-534 at MOI 0.01 either (a) 3 days post-seeding (0.6 × 10⁷ cells/flask) or (b) 5 days post-seeding (1.7 × 10⁷ cells/flask). Duplicate T-75 flasks were incubated at either 33°C or 37°C post-infection

Fig. 3

Virus production profiles in RB cultures titrated with FBS pre-infection. Vero cells were seeded in one of the following media in triplicate RBs: OptiPRO SFM, OptiPRO SFM + 0.5% (v/v) FBS, and OptiPRO + 2% (v/v) FBS. Three days post-seeding, one RB in each condition was trypsinized for cell counting: OptiPRO SFM (1.9 × 10⁷ cells/flask), OptiPRO SFM + 0.5% (v/v) FBS (9.3 × 10⁷ cells/flask), and OptiPRO + 2% (v/v) FBS (10.4 × 10⁷ cells/flask). The remaining 2 × 3 RBs were infected with MEDI-534 at MOI 0.001

Fig. 4

Comparison of (a) cell growth and (b) virus production in RBs using different pre-infection media. Vero cells were seeded in either OptiPRO + 0.5% (v/v) FBS or VP-SFM + 1% (v/v) CDLC. To generate the growth curves (a), duplicate RBs in each condition were counted daily. To generate the virus production profiles (b), duplicate RB cultures in the two different pre-infection media were infected with MEDI-534 3 days post-seeding. The infected cultures were sampled daily from 2 to 7 days post-infection

Fig. 5

Comparison of (a) cell growth and (b) virus production in microcarrier cultures using different pre-infection media. Vero cells were seeded in spinner flasks containing 2 g/l Cytodex 1 in either OptiPRO + 0.5% (v/v) FBS or VP-SFM + 1% (v/v) CDLC. To generate the growth curves (a), samples were taken daily from uninfected duplicate flasks for nuclei counts. To generate the virus production profiles (b), duplicate flasks in each pre-infection media were infected 5 days post-seeding with MEDI-534