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Zusammenfassung:
Epidemic spreading of avian influenza has once again stressed the importance of vaccination as the principal means of prophylaxis. For the production of vaccines propagation of virus in mammalian cell culture is getting more and more important. Here, we present a downstream processing scheme for the preparation of an inactivated whole-virion influenza vaccine from human influenza A virus propagated in serum-free mammalian cell culture. Human influenza virus A/PR/8/34 (H1N1) was replicated in adherent Madin-Darby canine kidney cells. Cell growth and virus propagation were conducted in roller bottles using serum-free cell culture medium. After pooling of the supernatants cell debris was removed by a combination of depth and membrane filters with an intermediate inactivation step using β-propiolactone. The virus was then concentrated 20-fold by cross-flow ultrafiltration resulting in a first reduction of impurities. Levels of host cell protein and genomic DNA were further depleted by two subsequent chromatography steps: size-exclusion chromatography (SEC) and anion-exchange chromatography (AEC). Yields of depth and membrane filtration were consistently high with average values of 85% and 93%, respectively (all yields based on HA activity). The yield of the concentration step was very sensitive to filtration flux. High flux led to membrane fouling and partial loss of the product. Limiting the flux to 28 l m‑2h‑1, however, resulted in an average yield of 97%. SEC was used to separate smaller colloids from virions and to adjust the salt concentration for AEC. Productivity was maximized to 0.15 column volumes of concentrate per hour. The average product yield was 85%. Total protein and DNA were reduced to 35% and 34% of the initial amount, respectively. AEC (run in negative mode; i.e. virus in flowthrough) was used to separate DNA from virions. The optimal salt concentration of 0.65 M NaCl was determined in batch adsorption experiments. The product yield of AEC depended on the amount of virus loaded. Satisfactory yields of 82% were obtained for viral loads > 160 kHAU per ml of resin while the reduction in DNA was 67-fold. Split-peak elution of virions in adsorptive AEC and bimodal particle volume distributions obtained by dynamic light scattering analysis suggested aggregation of virions. Co-elution with DNA and constant amounts of DNA per estimated dose indicated association of DNA to virus. An overall product yield of 53% was achieved. The amount of total protein and host cell DNA was reduced 19-fold and 500-fold, respectively. First estimations of the dose volume based on hemagglutination activity (assuming 15 µg of HA antigen per dose) indicated that a DNA burden in the range of 500 ng per dose can be achieved without DNAse-treatment. Further reduction by AEC may be achievable after breakage of presumable virus-DNA association. Current activities focus on the search for alternative capture strategies (including precipation and batch adsorption) and the enhancement of separation from DNA.
