DEVELOPMENT OF INACTIVATED POLIO VACCINE FROM ATTENUATED SABIN STRAINS FOR CLINICAL STUDIES AND TECHNOLOGY-TRANSFER PURPOSES

Recently, responding to WHO’s call for new polio vaccines, the development of Sabin-IPV (injectable, formalin-Inactivated Polio Vaccine, based on attenuated ‘Sabin’ polio virus strains) was initated at NVI. This activity plays an important role in the WHO polio eradication strategy. The use of Sabin instead of wild-type Salk polio strains will provide additional safety during vaccine production. Initially, the Sabin-IPV production process will be based on the scale-down model of the current, and well-established, Salk-IPV process. In parallel, process development, optimization and formulation research is being carried out to further modernize the process and reduce cost per dose. The lab-scale accelerated process development, product characterization, clinical lot production, and preparations for technology transfer will be discussed. Multivariate data analysis (MVDA) was applied on data from current IPV production (more than 60 Vero cell culture based runs) to extract relevant information, like operating ranges. Subsequently, based on the MVDA analysis, a 3-L scale-down model of the current twin 750-L bioreactors has been setup. Currently, in this lab-scale process, cell and virus culture approximate the large-scale and process improvement studies are in progress. This includes the application of increased cell densities, animal component free media, and DOE optimization in multiple parallel bioreactors. Also, results will be shown from large-scale (to prepare for future technology transfer) generation and testing of Master- and Working virus seedlots, and clinical lot (for phase I studies) production under cGMP conditions. The obtained product was used for immunogenicity studies in rats. It was shown that Sabin-IPV induces a good immune response, and a comparison will be made to regular Salk-IPV. Finally, technology transfer to vaccine manufacturers in low and middle–income countries will take place. For that, an international Sabin-IPV manufacturing course, including practical training at pilot-scale, is being setup

Rat immunogenicity (VNT against wild-type viruses).

<p>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).</p

Stability of sIPV.

<p>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.</p

Process overview for preparation of trivalent IPV.

<p>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.</p

Purification of Sabin PV.

<p>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.</p

Cell and virus culture.

<p>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).</p