Improved poliovirus D-antigen yields by application of different Vero cell cultivation methods

Abstract

Vero cells were grown adherent to microcarriers (Cytodex 1; 3 g L(-1)) using animal component free media in stirred-tank type bioreactors. Different strategies for media refreshment, daily media replacement (semi-batch), continuous media replacement (perfusion) and recirculation of media, were compared with batch cultivation. Cell densities increased using a feed strategy from 1×10(6) cells mL(-1) during batch cultivation to 1.8, 2.7 and 5.0×10(6) cells mL(-1) during semi-batch, perfusion and recirculation, respectively. The effects of these different cell culture strategies on subsequent poliovirus production were investigated. Increased cell densities allowed up to 3 times higher D-antigen levels when compared with that obtained from batch-wise Vero cell culture. However, the cell specific D-antigen production was lower when cells were infected at higher cell densities. This cell density effect is in good agreement with observations for different cell lines and virus types. From the evaluated alternative culture methods, application of a semi-batch mode of operations allowed the highest cell specific D-antigen production. The increased product yields that can easily be reached using these higher cell density cultivation methods, showed the possibility for better use of bioreactor capacity for the manufacturing of polio vaccines to ultimately reduce vaccine cost per dose. Further, the use of animal-component-free cell- and virus culture media shows opportunities for modernization of human viral vaccine manufacturing.

Keywords: Adherent; Batch; Feed; Microcarriers; Perfusion; Recirculation.

Fig. 1

Growth curves of Vero cells using different cultivation modes batch (B), semi-batch (S), perfusion (P) and recirculation (R). Different batches are indicated with different grey shades and symbols, where squares, circles and triangles represent cultures that were infected with poliovirus type 1, 2 and 3 respectively. Infection of cultures was done at the end of the shown growth curve.

Fig. 2

Photographs of Vero cells on microcarriers at the time of virus inoculation. Presence of multilayers at higher cell concentrations (1.0, 1.8, 2.7 and 5.0 × 10⁶ cells mL⁻¹ for respectively batch, semi-batch, perfusion and recirculation) is clearly visible.

Fig. 3

Glucose (squares) and glutamine (diamonds) concentrations present in Vero cell cultivations using batch (B), semi-batch (S), perfusion (P) or recirculation (R). Figures are averaged numbers. Daily media exchange was started at day 2 (S), media feed at day 3 (P) and circulation at day 1 (R).

Fig. 4

Lactate (squares) and NH₄⁺ (diamonds) concentrations present in Vero cell cultivations using batch (B), semi-batch (S), perfusion (P) or recirculation (R). Figures are averaged numbers. Daily media exchange was started at day 2 (S), media feed at day 3 (P) and circulation at day 1 (R).

Fig. 5

Cell specific d-antigen yields (poliovirus type 1, black; type 2, grey; type 3, white) after different cell culture methods. Similar virus culture conditions were applied.