Datensatz

Zusammenfassung

The recent search of new cell lines for cell-based influenza virus vaccine production has identified possible host cell lines from duck, monkey, canine, chicken, mouse and human origin. However, the most logical choice for a human vaccine seems to be the use of human cell lines. Therefore, the availability of a new epithelial-like human suspension cell line, which is stable for far over 100 passages, motivated us to verify its capacity as a host cell line for influenza virus production. Similar to other designer cells currently available, the CAP cell line was obtained by transformation with adenoviral functions (E1A- E1B and pIX-functions) and subsequent adaptation to suspension growth. The amniocytes were collected from routine amniocentesis and are thus available without ethical concerns. To be comparable to the other proposed suspension host cell lines, yields of more than 6000 virions/cell should be achievable. Cell growth should allow for cell concentrations of at least 2-4 x 106 cells/mL after around 4 days, and virus production should be possible without medium exchange for process simplicity. Here, we present a first evaluation of the CAP cell line in small scale shaker flasks and stirred tank bioreactor. Cultivation in serum free medium showed that different influenza subtypes could be adapted to produce titers of at least 2.0 log10 HA units/100 µL without further optimizations. Adjusting trypsin concentration as well as infection conditions (moi, infection medium) resulted in cell specific virus yields up to 6400 virions/cell. Additionally, surface membrane expression of sialyloligosaccharides as well as HA N-glycosylation patterns were characterized. With respect to bioprocess development, the implication of the ATF2 system (Refine Technology) for a scalable perfusion system for high cell density cultivation of up to 5 x 107 cells/mL in chemically defined medium will be discussed. First virus particles (80-100 nm) will be released approximately 12 hours post infection. Thus, the choice of the hollow fiber cut-off and the design of the perfusion strategy during virus propagation will be crucial. Therefore, different process options taking also into account requirements in downstream processing will be evaluated. Overall, experimental data clearly shows the potential of CAP cells for influenza virus vaccine production.