Manufacturing of the HPVB19 VLP vaccine using a two baculovirus system poses some challenges with respect to maintaining the ratios of the two viral proteins and in downstream purification. The process, however, has been sufficiently robust to allow the manufacture of three clinical lots by two different manufacturers, all of which were used in humans. After transfer of the process from the original manufacturer, initial pilot production studies were conducted to demonstrate reproducibility of the process and comparability of the resulting VLPs.
The process was then scaled to a 20 L Wave culture. The Sf9 working cell bank was expanded through a series of shake flask cultures and transferred to a 50 liter WaveBag® (GE Healthcare) at 1.5 x 106 cells/mL and allowed to expand until 20 liters of culture at a cell density of 1.5 to 2 x 106 were obtained. The culture was co-infected with bacVP1 and bacVP2 at an MOI of 1 and 0.5 respectively Figure 2.
The bioreactor was incubated at 26° to 28°C for four days and harvested when the cell viability dropped below 50%. The temperature, pH and rocking speed, and angle were monitored. Cell density and viability were monitored daily. The culture was harvested by centrifugation at 800 x g for 30 minutes after which the fluid was decanted and the cell paste stored at -60° to -80°C.
VLPs were purified by resuspending cell paste in a tris buffer with 1.6 µM leupeptin and microfluidized to prepare a cell lysate. Protocols transferred from the previous industry partner utilized filtration to remove insoluble particles with a single DEAE anion exchange chromatography step.
This filtration step was changed to improve manufacturability and product yields. Lysate was diluted with 20 mM Tris-HCL to reduce salt concentration and subjected to fluidized bed ion-exchange chromatography using Streamline DEAE media (GE Healthcare) in lieu of filtration. Eluate containing VLP’s was diluted with 20 mM Tris-HCl to reduce salt concentration and subjected to Fractogel DEAE ion-exchange column chromatography.
After washing with Tris-HCl buffer, the product was eluted with a phosphate buffer and the pH was adjusted for final polishing using Fractogel TMAE ion-exchange chromatography. The vaccine product was eluted and formulated with sucrose and Tween excipients. The final concentrated bulk product was filtered through a 0.22 µ filter.
Dilution of the vaccine to target concentrations of either 10 µg/mL or 100 µg/mL resulted in loss of the drug due to protein adherence to the vial. Formulation studies utilizing Schott Plus-1 vials combined with sucrose and Tween 80 minimized loss. Formulation and purification studies required a sensitive and specific method to track the vaccine.
A sandwich ELISA utilizing both polyclonal and monoclonal antibodies specific for HPVB19 was developed that demonstrated a sensitivity of £50 ng/mL and variability of under 20% within the range of quantitation (100 ng/mL to 750 ng/mL).