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.


Pralow, A.; Nikolay, A.; Leon, A.; Genzel, Y.; Rapp, E.; Reichl, U.: Site-specific N-glycosylation analysis of animal cell culture-derived Zika virus proteins. Scientific Reports 11 (1), 5147 (2021)
Pralow, A.; Hoffmann, M.; Nguyen-Khuong, T.; Pioch, M.; Hennig, R.; Genzel, Y.; Rapp, E.; Reichl, U.: Comprehensive N‐glycosylation analysis of the influenza A virus proteins HA and NA from adherent and suspension MDCK cells. The FEBS Journal 288 (16), pp. 4869 - 4891 (2021)
Gränicher, G.; Coronel, J.; Pralow, A.; Marichal-Gallardo, P.; Wolff, M. W.; Rapp, E.; Karlas, A.; Sandig, V.; Genzel, Y.; Reichl, U.: Efficient influenza A virus production in high cell density using the novel porcine suspension cell line PBG.PK2.1. Vaccine 37 (47), pp. 7019 - 7028 (2019)

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