de.mpg.escidoc.pubman.appbase.FacesBean
English
 
Help Guide Disclaimer Contact us Login
  Advanced SearchBrowse

Item

ITEM ACTIONSEXPORT

Released

Poster

Lectin-affinity purification of cell culture derived human Influenza A viruses

MPS-Authors
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/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/persons86460

Salaklang,  J.
Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Max Planck Society;
Univ. of Applied Sciences, Muenster, Germany;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons86442

Rapp,  E.
Physical and Chemical Foundations of Process Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Max Planck Society;

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/persons86448

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

Locator
There are no locators available
Fulltext (public)
There are no public fulltexts available
Supplementary Material (public)
There is no public supplementary material available
Citation

Opitz, L., Wolff, M. W., Salaklang, J., Rapp, E., Kalbfuss, B., & Reichl, U. (2006). Lectin-affinity purification of cell culture derived human Influenza A viruses. Poster presented at Vaccine Technology 2006, Puerto Vallarta, Mexico.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0013-9A25-6
Abstract
Influenza remains due to its annual death rate and potential to cause pandemics a major public health concern. Strategies to control influenza outbreaks are mainly focused on prophylactic vaccinations in conjuction with antiviral medications. Human Influenza vaccines are conventionally produced in embryonated hen's eggs. However, major constraints with this method, e.g. allergic reactions induced by egg proteins and lack of scalability have lead to the development of cell culture based production processes. These upstream modifications demand the development of appropriate downstream procedures. Purification of biological products is generally divided into three parts: capture or concentration, separation or fractionation and polishing. The capture step is the most important unit operation concerning the overall process economics. The product selectivity and the concentration factor of the capture step define the efforts required for all further purification steps. The presented study aims on the development of a capture step for the purification of human Influenza viruses using lectin-affinity chromatography. Lectins are proteins, which specifically bind to a carbohydrate or a group of carbohydrates. Introduction of an affinity chromatography step at the beginning of the downstream process enables a high product concentration factor. Furthermore, it separates the target biomolecule from the major bulk of contaminants with a single step. In the following studies a Madin Darby canine kidney (MDCK) cell produced human Influenza A/PR/8/34 virus has been chosen as a model strain. The Influenza A virus surface possesses two viral spike glycoproteins: the hemagglutinin (HA) and the neuraminidase (NA). The HA is the most abundant and immunogenic surface glycoprotein. Based on HA glycoanalysis some adequate lectins have been chosen for lectin binding screening via lectins blots. Therefore, the proteins of the concentrated fermentation broth were separated by SDS-PAGE analysis and transferred onto PVDF-membranes. Treatment of the membrane with biotinylated lectins and detection by chemiluminescence indicated the lectin affinity to viral and host cell glycoproteins. The most specific binding to the viral glycoprotein HA was achieved via the galactose specific lectins Erythrina cristagalli lectin (ECL, gal(beta1,4)glcNAc) and Euonymous Europaeus Lectin (EEL, gal(alpha1,3)gal). These lectins have been selected for further investigations of chromatographic separations. The virus and the viral membrane glycoproteins from concentrated cell culture broth adsorbed to the lectin-polymer-matrix specifically. The majority of host cell proteins did not bind to the lectins. These proteins were washed out from the columns. Viral proteins were desorbted from the column by competitive elution with appropriate carbohydrate. HA recoveries of up to 80% based on activity measurements have been achieved with this procedure with a high degree of removal of host cell proteins and nucleic acids. On that account, lectin affinity chromatography represents a promissing tool for an effective capture step for the downstream processing of MDCK-cell derived Influenza vaccines