Viral Glycocalix in Dependence on Host Cell Line, Influenza Virus Strain and Production Process Parameters

Viral Glycocalix in Dependence on Host Cell Line, Influenza Virus Strain and Production Process Parameters

Motivation

Glycoproteins, such as viral membrane protein hemagglutinin (HA) - the main antigen of influenza vaccines - can be considered as a collection of different glycoforms varying in structure of attached sugar residues (microheterogeneity) as well as in glycosylation site occupancy (macroheterogeneity). Since it is known that the glycosylation may significantly impact protein characteristics such as protein stability, antigenicity, immunogenicity, activity regulatory agencies usually demand glycosylation profiles for biopharmaceutical quality assurance. Since so far, there is only little known about the impact of glycosylation on vaccine efficiency and safety such regulations not yet exist for vaccines. However, first studies indicate a significant impact of glycosylation on vaccine immunogenicity, too.

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 %.

Aim of the project

Using the developed high performance glycoanalysis system, the glycosylation patterns of viral surface proteins from unit operations in up- and downstream processing can be characterized. The main questions addressed regarding the glycosylation of HA, which is the most abundant and immunogenic influenza glycoprotein, are e.g.: the influence of host cell lines and virus strains [1,2], the impact of adaptation processes [3,4], changes due to modifications in cultivation conditions [5,6,7] and its impact on immunogenicity [8].

References

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)
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)

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