Next generation inactivated polio vaccine manufacturing to support post polio-eradication biosafety goals

Abstract

Worldwide efforts to eradicate polio caused a tipping point in polio vaccination strategies. A switch from the oral polio vaccine, which can cause circulating and virulent vaccine derived polioviruses, to inactivated polio vaccines (IPV) is scheduled. Moreover, a manufacturing process, using attenuated virus strains instead of wild-type polioviruses, is demanded to enhance worldwide production of IPV, especially in low- and middle income countries. Therefore, development of an IPV from attenuated (Sabin) poliovirus strains (sIPV) was pursued. Starting from the current IPV production process based on wild type Salk strains, adaptations, such as lower virus cultivation temperature, were implemented. sIPV was produced at industrial scale followed by formulation of both plain and aluminium adjuvanted sIPV. The final products met the quality criteria, were immunogenic in rats, showed no toxicity in rabbits and could be released for testing in the clinic. Concluding, sIPV was developed to manufacturing scale. The technology can be transferred worldwide to support post polio-eradication biosafety goals.

Figure 1. Process overview for preparation of trivalent IPV.

Monovalent bulks are prepared for each PV (type 1, 2 and 3) separately. During monovalent bulk preparation Vero cells are expanded using two pre-culture steps and a cell culture followed by virus culture. Virus is purified using normal flow filtration for clarification, tangential flow filtration for concentration and two chromatography units, size exclusion and ion exchange chromatography. Purified virus is subsequently inactivated using formaldehyde. Subsequently these are mixed to obtain trivalent bulk prior to formulation and filling.

Figure 2. Cell and virus culture.

Panel A shows the average Vero cell growth curve (n=12; error bars represent SD) in 350-L bioreactors. Photographs are light microscopy images (size bar 200 µm). Panel B shows the average (of the three subtypes) Vero cell death during virus culture determined microscopically (n=12; error bars represent SD). Photographs show corresponding images. Panel C shows average virus titers for Sabin PV type 1, 2 and 3 (n=4; error bars represent SD). Panel D shows average D-antigen concentrations after virus culture for Sabin PV type 1, 2 and 3 (n=4; error bars represent SD).

Figure 3. Purification of Sabin PV.

Panel A depicts a SEC chromatogram of Sabin PV type 1. The 1^st peak contained mostly large cell components; the 2^nd peak contained the majority of PV, following peaks consist of smaller components. Panel B shows a SDS-PAGE (4-20% gel); lanes represent (from left to right) the marker, the concentrated product, followed by the 1^st and 2^nd fraction of SEC and finally the IEX purified PV. Panel C shows chromatograms of Sabin PV type 1 (left) and Sabin PV type 2 (right) IEX purification. Panel D shows host cell protein (open) and DNA (solid) impurities. Panel E depicts the inactivation of PV, the gray area indicates the lower detection limit. In chromatograms A and C, the dotted and solid lines represent absorbance at respectively 254nm and 280nm. Gray dotted lines indicate peak fractioning.

Figure 4. Rat immunogenicity (VNT against wild-type viruses).

Panel A, B and C: VNT (log₂ titer) to immunization with plain sIPV (blue) and adjuvanted sIPV (red) for PV type 1, 2 and 3 respectively; Panel D, E and F: VNT of plain sIPV 20/32/64 (light blue), 10/16/32 (red), 5/8/16 (green) and plain IPV 40/8/32 (dark blue) for PV type 1, 2 and 3 respectively. Error bars in panel A-F depict standard deviation of the median (n=10 rats).

Figure 5. Stability of sIPV.

Panel A: PV type 1 VNT of plain sIPV 20/32/64 (blue), 10/16/32 (red), 5/8/16 (green) in time, error bars depict standard deviation of the median (n=10 rats); Panel B: slopes of linear regression lines determined for stability based on rat immunogenicity as illustrated in panel A, error bars depict 95% confidence interval; Panel C: Stability of PV type 1 D-antigen of sIPV 20/32/64; Panel D: slopes of linear regression lines determined for stability based on D-antigen as illustrated in panel C, error bars depict 95% confidence interval.