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  A generic purification scheme for the production of human influenza vaccines derived from serum-free mammalian cell culture

Kalbfuss, B., Wolff, M. W., Genzel, Y., Zimmermann, A., Best, C., Morenweiser, R., et al. (2006). A generic purification scheme for the production of human influenza vaccines derived from serum-free mammalian cell culture. Talk presented at Vaccine Technology. Puerto Vallarta, Mexico. 2006-06-25 - 2006-06-30.

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Datensatz-Permalink: http://hdl.handle.net/11858/00-001M-0000-0013-9A27-2 Versions-Permalink: http://hdl.handle.net/11858/00-001M-0000-0025-0C0D-E
Genre: Vortrag

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 Urheber:
Kalbfuss, B.1, Autor              
Wolff, M. W.1, 2, Autor              
Genzel, Y.1, Autor              
Zimmermann, A.1, Autor              
Best, C.3, Autor
Morenweiser, R.3, Autor
Reichl, U.1, 4, Autor              
Affiliations:
1Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Max Planck Society, ou_1738140              
2Otto-von-Guericke-Universität Magdeburg, External Organizations, ou_1738156              
3Lehrstuhl für Bioverfahrenstechnik, Otto-von-Guericke Universität, Magdeburg/Germany Fast Trak Services Europe, GE Healthcare Europe GmbH, Munich/Germany, ou_persistent22              
4Otto-von-Guericke-Universität Magdeburg, ou_1738156              

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 Zusammenfassung: Epidemic spreading of avian influenza has once again stressed the importance of vaccination as the principal means of prophylaxis. The production of influenza vaccines in mammalian cell culture has been suggested before. Here, we present a downstream processing scheme for the production of an inactivated whole-virion human 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 (ECACC #841211903) grown 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 and anion-exchange chromatography. Yields of the depth and membrane filtration were consistently high with average values of 85% and 93%, respectively. In contrast, the yield of the concentration step was very sensitive to the filtration flux. A high flux led to membrane fouling and partial loss of the product. Limiting the flux to 28 l m-2 h-1, however, resulted in an average yield of 97%. The average yield of the size-exclusion chromatography was 85%. In order to maximize productivity, the injection volume and the fractionation were optimized in a modeling study. Anion-exchange chromatography was run in negative mode (virus in flowthrough). The optimal ionic strength to separate virus from DNA was determined in microtiter plate experiments. The product yield depended on the amount of virus loaded. Satisfactory yields of 82% were obtained for viral loads > 160 kHAU per ml of resin. An overall yield of 53% based on hemagglutination activity could be achieved. The amount of total protein (including the virus) and host cell DNA could be reduced to 3.5% and 0.19%, respectively. The protein purity of the final product was at least 94%. First estimations of the dose volume based on total protein measurements and hemagglutination activity (assuming 15 µg of HA antigen per dose) indicated that a DNA

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Sprache(n): eng - Englisch
 Datum: 2006
 Publikationsstatus: Keine Angabe
 Seiten: -
 Ort, Verlag, Ausgabe: -
 Inhaltsverzeichnis: -
 Art der Begutachtung: -
 Identifikatoren: eDoc: 285547
 Art des Abschluß: -

Veranstaltung

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Titel: Vaccine Technology
Veranstaltungsort: Puerto Vallarta, Mexico
Start-/Enddatum: 2006-06-25 - 2006-06-30

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