Quantitative Mass Spectrometry in Cell Culture-Based Vaccine Production

Motivation

For influenza vaccination, the cell-culture based production of virus particles has become an alternative approach to the propagation in embryonated chicken eggs. Major advantages are the potential for fast start-up of production, the straight-forward scale-up, and no risk of allergic reactions from egg components. Success of this approach can only be ensured with optimized strategies of up- and downstream processes supported by adequate process models. During all stages of production a reliable absolute quantification of the viral proteins is required. Furthermore, absolute influenza virus protein data could help monitoring the dynamics of the viral reproduction cycle on a functional level, which has a high potential to improve existing mathematical models on the single cell level. Finally, absolute virus quantification based on proteome data could help to overcome limitations in analytics, i.e. replace imprecise assays such as the hemagglutination assay.

Protein AQUA strategy for quantification of proteins using mass spectrometry.

© MPI Magdeburg (BPE)

Protein AQUA strategy for quantification of proteins using mass spectrometry.

© MPI Magdeburg (BPE)

Aim of the project

The focus of this project is to establish an assay for absolute quantification of influenza virus proteins using mass spectrometry. Label-free methods as well as stable isotope (¹³C, ¹⁵N) labeling methods using signature peptides with a known mass difference to the viral peptides will be compared and validated [1]. Signature peptides can be derived either via chemical synthesis or using expression vectors in E. coli [2,3].

References

[1] Vaudel, M.; Sickmann, A.; Martens, L. Peptide and protein quantification: A map of the minefield. Proteomics 10 (10), pp. 650-670 (2010)

[2] Gerber, S. A.; Rush, J.; Stemman, O.; Kirschner, M. W. & Gygi, S. P. Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. Proceedings of the National Academy of Sciences, 100 (12), 6940-6945 (2003)

[3] Beynon, R. J.; Doherty, M. K.; Pratt, J. M. & Gaskell, S. J. Multiplexed absolute quantification in proteomics using artificial QCAT proteins of concatenated signature peptides. Nature methods, 2(8), 587-589 (2005)