Comprehensive Structural and Site-Specific Glycan Analysis of Viral Glycoproteins

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 [1], 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 [9] were characterized.

Figure 1: Shifted overlay of HA N-glycosylation patterns during adaptation of influenza A virus PR/8/34 (H1N1) from MDCK to Vero cells and back (A) or to AGE1.CR.pIX host cells (B). Relative fluorescence units (RFU) are plotted over normalized migration times (tmig) in base pairs (bp). (A) The normalized capillary electropherograms altogether represent 11 successive virus passages: (1.) virus seed, (2.) to (6.) adaptation to Vero cells, (7.) to (11.) back-adaptation to MDCK cells. MDCK and Vero cell-derived HA N-glycosylation patterns dominated by 15 peaks and 16 peaks, respectively. (B) MDCK-derived virus seed (red) was adapted over four passages (passage 1-4) to replicate in AGE1.CR.pIX cells (black). Numbered peaks have a minimum RFU of 20 % of the highest peak (threshold, - - -): red, only in MDCK cells; black, only in AGE1.CR.pIX cells; blue, in both cell lines, both with a minimum of 20 %; green, in both cell lines, but only one cell line exceeding the threshold of 20 %.

Figure 2: Top: N-glycomics and N-glycoproteomics analysis workflow developed for the structural and site-specific analysis of influenza A virus glycoproteins. Bottom: Hemagglutinin molecule with attached N-glycans (Taken from Butler, M., & Reichl, U. (2017). Animal Cell Expression Systems.)

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.

References

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)
Schwarzer, J.; Rapp, E.; Reichl, U.: N-Glycan Analysis by CGE-LIF – Profiling Influenza A Virus Hemagglutinin N-Glycosylation during Vaccine Production. Electrophoresis 29 (20), pp. 4203 - 4214 (2008)
Schwarzer, J.; Rapp, E.; Hennig, R.; Genzel, Y.; Jordan, I.; Sandig, V.; Reichl, U.: Glycan analysis in cell culture-based influenza vaccine production: Influence of host cell line and virus strain on the glycosylation pattern of viral hemagglutinin. Vaccine 27 (32), pp. 4325 - 4336 (2009)
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)
Rödig, J.; Rapp, E.; Djeljadini, S.; Lohr, V.; Genzel, Y.; Jordan, I.; Sandig, V.; Reichl, U.: Impact of Influenza Virus Adaptation Status on HA N-Glycosylation Patterns in Cell Culture-Based Vaccine Production. Journal of Carbohydrate Chemistry 30 (4-6), pp. 281 - 290 (2011)
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)
Bock, A.; Schulze-Horsel, J.; Schwarzer, J.; Rapp, E.; Genzel, Y.; Reichl, U.: High-density microcarrier cell cultures for influenza virus production. Biotechnology Progress 27 (1), pp. 241 - 250 (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)
Hütter, J.; Rödig, J. V.; Höper, D.; Seeberger, P. H.; Reichl, U.; Rapp, E.; Lepenies, B.: Toward Animal Cell Culture-based Influenza Vaccine Design: Viral Hemagglutinin N-Glycosylation Markedly Impacts Immunogenicity. The Journal of Immunology 190 (1), pp. 220 - 230 (2013)
Pralow, A., Nguyen-Khuong, T., Pioch, M., Hennig, R., Hoffmann, M., Rapp, E. & Reichl, U.
Comprehensive structural and site-specific N-glycan analysis of Influenza H1N1 propagated in adherent and suspension MDCK cells.
Manuscript in preparation. (2018)

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