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Downstream Process of Influenza Viruses

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http://pubman.mpdl.mpg.de/cone/persons/resource/persons86517

Wolff,  M. W.
Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Max Planck Society;
Otto-von-Guericke-Universität Magdeburg, External Organizations;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons86343

Kalbfuss,  B.
Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons86422

Opitz,  L.
Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons86531

Zimmermann,  A.
Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons86386

Lehmann,  S.
Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons86448

Reichl,  U.
Otto-von-Guericke-Universität Magdeburg;
Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Max Planck Society;

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Citation

Wolff, M. W., Kalbfuss, B., Opitz, L., Zimmermann, A., Lehmann, S., & Reichl, U. (2006). Downstream Process of Influenza Viruses. Talk presented at European BioPharm Scale-Up Congress 2006. Geneva, Switzerland. 2006-09-12 - 2006-09-14.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0013-99CC-5
Abstract
Due to its annual death rate and the potential to cause pandemics, Influenza remains a major public health concern. Efforts to control the annual spread of influenza have centered on prophylactic vaccinations in combination with antiviral medications. The majority of licensed vaccines for human influenza are still produced in embryonated eggs. However, this production process conveys major drawbacks such as lack of scalability, difficulties of some strains to replicate on eggs to high enough yields, and possible allergic reactions induced by egg proteins. These limitations emphasize the need for an alternative process. In recent years several continuous cell lines such as the Madin-Darby canine kidney (MDCK), the African green monkey kidney Vero or the human fetal retinoblast PER.C6 have been successfully established for the production of influenza vaccines. These processes require the adaptation of existing downstream processing strategies to account for the modified upstream technology. The presented study focuses on the development of two downstream processing schemes for the purification of human Influenza A/PR/8/34 (H1N1) virus. The virus was replicated in adherent MDCK cells in roller bottles or in small scale (5 and 10L) bioreactors (Genzel et al., 2006). Cell debris and microcarriers were removed by a combination of depth and membrane filters with an intermediate ï¢-propiolactone inactivation step. Yields of the depth and membrane filtration were consistently high with average values of 85% and 93%, respectively. Subsequently, the virus was concentrated approximately 20-fold via ultrafiltration resulting in a first reduction of host cell proteins and genomic DNA (Kalbfuss et al., 2006a; Wickramasinghe et al., 2005). In a next step the concentrated virus samples were purified via two process schemes. Size exclusion and anion exchange chromatography Purification scheme 1 was based on a combination of size exclusion and anion exchange chromatography. Several chromatography media have been screened to improve the overall yield of the process and the product purity. Including filtration and concentration the resulting scheme gained an overall yield of 53% based on the hemagglutination activity assay. The amount of total protein (including virus) and host cell DNA could be reduced to 3.5% and 0.19%, respectively (Kalbfuss, B. et al.; 2006b). Lectin affinity chromatography Scheme 2 centers on a lectin affinity capture step. Lectins are a class of carbohydrate specific proteins of non-immune origin that have a selective affinity for a carbohydrate or a group of carbohydrates. The influenza A virus contains two spike glycoproteins on its surface: hemagglutinin (HA) and neuraminidase (NA). HA is the most abundant and immunogenic surface glycoprotein. It is a trimeric glycoprotein containing 3 to 9 N-linked glycosylation sites per subunit. These glycans are mainly of the complex type of N-glycans, which are usually terminating in sialic acid or galactose, if fully elaborated. However, the degree of glycan sialylation depends on the viral neuraminidase activity. Hence, the number of terminal sialic acid or ï¢-linked galactose residues on complex glycans of influenza envelope glycoproteins is highly process dependent. Some complex glycan structures of HA contain terminal ï¡-linked galactose. These glycan branches are not affected by the viral neuraminidase activity. Lectin screening has revealed, that the most specific lectin binding can be achieved via the two galactose specific lectins Erythrina cristagalli lectin (ECL, gal (ï¢1, 4)GlcNAc) and Euonymus europaeus lectin (EEL), gal(ï¡1,3)gal) (Opitz et al. 2006) . A comparison of different EEL-support matrices have shown that membrane adsorbers and a specific polymer can achieve a total viral recovery of up to 80% based on hemagglutination activity with a high degree of host cell protein and nucleic acid removal. References: Genzel, Y. et al. (2006) Vaccine 24, 3261-3272 Kalbfuss, B. et al. (2006a) Biotechnology and Bioengineering, in press Kalbfuss, B. et al. (2006b) Biotechnology and Bioengineering, in press Opitz, L. et al. (2006) Vaccine, submitted Wickramasinghe, S. et al. (2005) Biotechnology and Bioengineering 92 (2), 199-208