Max Planck Institute for Dynamics of Complex Technical Systems
Comprehensive Structural and Site-Specific Glycan Analysis of Viral Glycoproteins
Glycosylation is considered as a critical quality attribute for the production of biopharmaceuticals, i.e. recombinant proteins such as monoclonal antibodies. In recent years, glycosylation of antigens is also considered for the manufacturing of viral vaccines. As yet, however, glycosylation and immunogenicity of only few viral antigens was characterized in the context of host cell selection, cultivation parameters, processes mode (batch, fed-batch, perfusion), and purification. Using xCGE-LIF based glycoanalysis , the glycosylation patterns of viral surface proteins from unit operations in up- and downstream processing can be characterized (Figure 1). One example is the glycosylation of the membrane protein hemagglutinin (HA), which is the most abundant and immunogenic influenza virus glycoprotein. Here, the influence of host cell lines and virus strains [2,3], the impact of adaptation processes [4,5], changes due to modifications of cultivation conditions [6,7,8], and questions regarding its immunogenicity  were characterized.
Glycoproteins can be considered as a collection of different glycoforms varying in structure of attached sugar residues (micro-heterogeneity) as well as in glycosylation site occupancy (macro-heterogeneity). Our current work focuses on the characterization of viral antigens on the glycopeptide/glycoprotein level. Using state-of-the-art mass spectrometry-based techniques, our group is working on in-depth structural and site-specific glycoanalysis of viral antigens, e.g. from influenza A virus (Figure 2). The methods developed offer the flexibility to be applied for the analysis of other viral glycoproteins relevant in science and industry, too.
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Hennig, R.; Rapp, E.; Kottler, R.; Cajic, S.; Reichl, U.: N -Glycosylation Fingerprinting of Viral Glycoproteins by xCGE-LIF. In: Carbohydrate-Based Vaccines: Methods and Protocols, pp. 123 - 143. Springer, New York (2015)
Genzel, Y.; Dietzsch, C.; Rapp, E.; Schwarzer, J.; Reichl, U.: MDCK and Vero cells for influenza virus vaccine production: a one-to-one comparison up to lab-scale bioreactor cultivation. Applied Microbiology and Biotechnology 88 (2), pp. 461 - 475 (2010)
Roedig, J.; Rapp, E.; Höper, D.; Genzel, Y.; Reichl, U.: Impact of Host Cell Line Adaptation on Quasispecies Composition and Glycosylation of Influenza A Virus Hemagglutinin. PLoS One 6 (12), p. e27989 (2011)
Rödig, J. V.; Rapp, E.; Bohne, J.; Kampe, M.; Kaffka, H.; Bock, A.; Genzel, Y.; Reichl, U.: Impact of Cultivation Conditions on N-glycosylation of Influenza Virus A Hemagglutinin Produced in MDCK Cell Culture. Biotechnology and Bioengineering 110 (6), pp. 1691 - 1703 (2013)