Continuous cell culture-based influenza vaccine production could significantly reduce footprint and manufacturing costs compared to current batch processing. However, yields of influenza virus in continuous mode can be affected by oscillations in virus titers caused by periodic accumulation of defective interfering particles. The generation of such particles has also been observed previously in cascades of continuous stirred tank reactors (CSTRs) and is known as the “von Magnus effect”. To improve virus yields and to avoid these oscillations, we have developed a novel continuous tubular bioreactor system for influenza A virus production. It was built using a 500 mL CSTR for cell growth linked to a 105 m long tubular plug-flow bioreactor (PFBR). Virus propagation took place only in the PFBR with a nominal residence time of 20 h and a production capacity of 0.2 mL/min. The bioreactor was first tested with suspension MDCK cells at different multiplicities of infection (MOI), and then with suspension avian AGE1.CR.pIX cells at a fixed nominal MOI of 0.02. Maximum hemagglutinin (HA) titers of 2.4 and 1.6 log10(HA units/100 μL) for suspension MDCK cells and AGE1.CR.pIX cells, respectively, were obtained. Flow cytometric analysis demonstrated that 100% infected cells with batch-like HA titers can be obtained at a MOI of at least 0.1. Stable HA and TCID50 titers over 18 days of production were confirmed using the AGE1.CR.pIX cell line, and PCR analysis demonstrated stable production of full-length genome. The contamination level of segments with deletions (potentially defective interfering particles), already present in the virus seed, was low and did not increase. Control experiments using batch and semi-continuous cultures confirmed these findings. A comparison showed that influenza virus production can be achieved with the tubular bioreactor system in about half the time with a space-time-yield up to two times higher than for typical batch cultures. In summary, a novel continuous tubular bioreactor system for cell culture-based influenza virus production was developed. One main advantage, an essentially single-passage amplification of viruses, should enable efficient production of vaccines as well as vectors for gene and cancer therapy.

The oncolytic virus H-1PV is a promising candidate for various cancer treatments. Therefore, production process needs to be optimized and scaled up for future market release. Currently, the virus is produced with minimum essential medium in 10-layer CellSTACK® chambers with limited scalability, requiring a minimum seeding density of 7.9E3 cells/cm2. Production also requires a 5% fetal bovine serum (FBS) supplementation and has a virus yield up to 3.1E7 plaque-forming units (PFU)/cm2. Using the animal-free cell culture medium VP-SFM™ and a new feeding strategy, we demonstrate a yield boost by a mean of 0.3 log while reducing seeding density to 5.0E3 cells/cm2 and cutting FBS supplementation by up to 40% during the production process. Additionally, FBS is completely removed at the time of harvest. Eleven commercial micro- and macrocarriers were screened regarding cell growth, bead-to-bead transfer capability, and virus yield. We present a proof-of-concept study for producing H-1PV on a large scale with the microcarrier Cytodex® 1 in suspension and a macrocarrier for a fixed-bed iCELLis® bioreactor. A carrier-based H-1PV production process combined with an optimized cell culture medium and feeding strategy can facilitate future upscaling to industrial-scale production. Key points • Virus yield increase and FBS-free harvest after switching to cell culture medium VP-SFM™. • We screened carriers for cell growth, bead-to-bead transfer capability, and H-1PV yield. • High virus yield is achieved with Cytodex® 1 and macrocarrier for iCellis® in Erlenmeyer flasks.

The oncolytic rodent protoparvovirus H-1PV has been successfully used in phase I/II clinical trials to treat recurrent glioblastoma multiforme and pancreatic cancer. The present work focuses on the stability and environmental safety of the H-1PV drug product from production up to its use in patients. We identified hold-steps in manufacturing for up to 3 months and showed 7-years stability for the optimal product formulation. Stress testing via UV, temperature, and pH also determined that the drug product is stable. De- and rehydration for lyophilization simulation are possible without infectious virus loss. Furthermore, we prove in-use stability for 4 days at room temperature and show no virus adsorption to injection devices, guaranteeing the correct administration dose. Iodixanol in the formulation, resulting in high viscosity, protects H-1PV against UV and some disinfectants. Nonetheless, H-1PV is depleted with rapid heat deactivation, autoclavation, and nanofiltration. Assessment of chemical disinfectants that are currently recommended by the Robert Koch-Institute demonstrated that ethanol-based hand disinfectants are not effective; however, aldehyde-based disinfectants for surfaces and instruments demonstrate sufficient H-1PV deactivation in aqueous formulations by 4 to 6 log10. With these results, we could establish a specific hygiene plan for all involved facilities from manufacturing to patient application. Overall, using 48% Iodixanol in Visipaque/Ringer as a drug formulation stabilizes H-1PV infectivity over years and protects against virus loss from short-term UV, low pH, and temperature exposure. Key points • Optimal formulation of drug product protects the H-1PV protoparvovirus against UV, temperatures up to 50 °C, and low pH (> 1.25), stabilizing the virus during manufacturing, storage, transport, and application. • H-1PV is stable during in-use and does not adsorb to injection devices during patient administration. • Hygiene plan for H-1PV with physicochemical methods has been established.