Sabin inactivated polio vaccine upstream process development using fixed-bed bioreactor technology

Figures

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  Fig. 1. Overview of the scale-X fixed-bed bioreactor and NevoLine Polio prototype. A) The scale-X bioreactor technology uses a structured fixed-bed matrix composed of two immobilization layers, alternated with a single spacer layer for flow distribution. The fixed-bed is enclosed in a bioreactor vessel with integrated process control and recirculation using an impeller. B) The NevoLine prototype developed for the sIPV process, composed of 3 isolators respectively containing upstream, downstream and inactivation process steps.

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  Fig. 2. Cell densities and cell growth in fixed-bed bioreactors. (A) Comparison of the cell density of WHO Vero 10–87 cell line in three 2.4 m2 structured fixed-bed bioreactors (from 2 separate laboratories in different locations belonging to different companies) and two 4 m2 unstructured fixed-bed bioreactors over time. The cell density was determined from picked fibers of the fixed bed. The target cell density of 1.5 × 105 cells/cm2 is indicated with a dotted line. (B) The structured fixed-beds of two 2.4 m2 bioreactors were dissected to analyze the distribution of cell growth across the fixed-bed surface with a radial dissection (0, 8, 17 mm) and across the height of the 10 cm fixed-bed (top, middle, bottom). Data are represented as mean ± standard deviation. (C) Vero cell density over time was assessed using fiber picking for 36 independent runs on 2.4m2 fixed-bed bioreactors. The target cell density of 1.5 × 105 cells/cm2 is indicated with a dotted line. (D) Glucose (blue) and lactate (green) concentrations over time of Vero cells on the structured fixed-bed bioreactors. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version…

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  Fig. 3. Implementation of TFF for footprint reduction of the sIPV process (n = 4 biological replicates per virus serotype with n = 3 technical replicates for fiber picking each). (A) Vero cell densities were determined through fiber picking before media replacement and infection with PV strains on day 5. The target cell density of 1.5 × 105 cells/cm2 is indicated with a dotted line. (B) Population doubling times (PDTs) of Vero cells cultured in the 2.4 m2 fixed-bed bioreactor or T-flasks. Data is represented with min-to-max floating bars with the mean. To reach the target of 1.5 × 105 cells/cm2 on day 5 from 6000 cells/cm2 seed a PDT of ≤25.8 h is required and depicted with a dotted line. (C & D) Glucose and lactate levels in the culture medium over time. The time of the media replacement and viral infection (day 5) is indicated with a dotted line. (E) Scheme describing the process of media replacement in perfusion mode after viral infection (infection medium), and subsequent continuous viral harvest using without (left panel) and with TFF (right panel, red box) for manufacturing footprint reduction. During the TFF, the (easily decontaminated) permeate is discharged and the harvest is…

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  Fig. 4. Scale-up of the TFF sPV process to 30 m2. (A) Vero cell density over time at 2.4 m2 and 30 m2 fixed bed measured with fiber picking (2.4 m2) or modeled based on the biomass glucose yield from glucose levels in the culture medium (30 m2). The target cell density of 1.5 × 105 cells/cm2 is indicated with a dotted line. (B) Vero cell growth over time in 30 m2 and 150 m2 fixed-bed bioreactors was measured by fiber-picking and calculated based on medium glucose levels. Data obtained in laboratory A. The target cell density of 1.5 × 105 cells/cm2 is indicated with a dotted line. (C) Comparison of Vero cell density over time at 150 m2 fixed bed, data from laboratory A vs B.

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  Fig. 5. D-antigen productivity and infectious virus titers in a 30 m2 fixed-bed bioreactor (FBR) for three Sabin poliovirus types and proof-of-principle for scaled-up production of sPV-3 using a 150 m2 FBR in a containment isolator. (A) D-antigen productivity per surface area in DU/cm2 in the bioreactor harvest of the fixed-bed bioreactor for sPV-1 (at 30 m2 scale), sPV-2 (at 30 m2 scale) and sPV-3 (n = 3 at 30 m2 scale, and n = 1 at 150 m2 scale), respectively. For comparison, estimations regarding the reference process (for sPV-2 and sPV-3) and for literature data (for sPV-1, sPV-2 and sPV-3), both using 3 g/L Cytodex 1, were based on assuming the available surface area at 4400 cm2/g microcarriers [21]. (B) Infectious virus particles concentration in log10 TCID50/mL in the fixed-bed bioreactor harvest for sPV-1 (at 30 m2 scale), sPV-2 (at 30 m2 scale) and sPV-3 (n = 3 at 30 m2 scale, and n = 1 at 150 m2 scale), respectively. Error bars indicate standard deviation.