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Elucidation of the Influenza A Virus Glycosylation-Patterns with Respect to Vaccine-Production Down to the Zeptomolar Range

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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/persons86475

Schwarzer,  J.
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

Rapp, E., Schwarzer, J., & Reichl, U. (2007). Elucidation of the Influenza A Virus Glycosylation-Patterns with Respect to Vaccine-Production Down to the Zeptomolar Range. Poster presented at 3rd Glycan Forum Berlin 2007, Berlin-Dahlem, Germany.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0013-9843-1
Abstract
The ability of monitoring and thereby quality controlling the glycosylation-pattern of viral membrane-proteins during the vaccine-production process (USP & DSP), is a prerequisite to ensure sufficient immune-response after vaccination. The glycosylation-pattern of viral glycoproteins can be affected by virus strain, host cell glycosylation machinery, cultivation conditions and via incipient degradation of the glycosylation during virus inactivation and downstream-processing. The presented approach allows the characterization of N-glycosylation-patterns, shown exemplarily on hemagglutinin of influenza-A/PR/8/34(H1N1)-virus, produced in mammalian cells. The procedure includes: virus purification and concentration directly from cell-culture supernatant, followed by preparation, separation and identification of viral (glyco-)proteins and their N-glycan pools, utilizing: g-force-step-gradient-centrifugation, SDS-PAGE, enzymatical cleavage, fluorescent labeling, CGE-LIF (employing a DNA-sequenzer) and MALDI-TOF-MS. The N-glycans are analyzed in two stages: glycan pool fingerprints and structural identification. Generation of fingerprints with CGE-LIF, enables LODs down to the zeptomolar range. Additional structural information are obtained, spiking the samples with a series of N-glycan standards, measured with CGE-LIF and MALDI-TOF-MS. This proceeding enables monitoring of N-glycosylation-patterns of relevant glycoproteins during major steps of upstream- and downstream-processing in influenza virus vaccine production.