Impact of cultivation conditions on N‐glycosylation of influenza virus a hemagglutinin produced in MDCK cell culture

Abstract

Manufacturers worldwide produce influenza vaccines in different host systems. So far, either fertilized chicken eggs or mammalian cell lines are used. In all these vaccines, hemagglutinin (HA) and neuraminidase are the major components. Both are highly abundant glycoproteins in the viral envelope, and particularly HA is able to induce a strong and protective immune response. The quality characteristics of glycoproteins, such as specific activity, antigenicity, immunogenicity, binding avidity, and receptor-binding specificity can strongly depend on changes or differences in their glycosylation pattern (potential N-glycosylation occupancy as well as glycan composition). In this study, capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF) based glycoanalysis (N-glycan fingerprinting) was used to determine the impact of cultivation conditions on the HA N-glycosylation pattern of Madin-Darby canine kidney (MDCK) cell-derived influenza virus A PR/8/34 (H1N1). We found that adaptation of adherent cells to serum-free growth has only a minor impact on the HA N-glycosylation pattern. Only relative abundances of N-glycan structures are affected. In contrast, host cell adaptation to serum-free suspension growth resulted in significant changes in the HA N-glycosylation pattern regarding the presence of specific N-glycans as well as their abundance. Further controls such as different suppliers for influenza virus A PR/8/34 (H1N1) seed strains, different cultivation scales and vessels in standard or high cell density mode, different virus production media varying in either composition or trypsin activity, different temperatures during virus replication and finally, the impact of β-propiolactone inactivation resulted-at best-only in minor changes in the relative N-glycan structure abundances of the HA N-glycosylation pattern. Surprisingly, these results demonstrate a rather stable HA N-glycosylation pattern despite various (significant) changes in upstream processing. Only the adaptation of the production host cell line to serum-free suspension growth significantly influenced HA N-glycosylation regarding both, the type of attached glycan structures as well as their abundances.

Development of a high‐throughput glycoanalysis method for the characterization of oligosaccharides in human milk utilizing multiplexed capillary gel electrophoresis with laser‐induced fluorescence detection

“…HMOS concentration changes during lactation were analyzed by calculating relative PHP (as published previously for N-glycosylation patterns [92,[98][99][100][101][102][103]). Briefly, the same set of 18 peaks was picked in all HMOS fingerprints of the samples from the LTP series (peak picking criteria: peaks > 200 relative fluorescence units (RFU) or already known peaks).…”

Influence of the production system on the surface properties of influenza A virus particles

“…Besides the buffer conditions, e.g., pH value, ionic strength, osmolarity, and the presence of excipients [2,3], the structural characteristics of VP surface proteins, e.g., mutations or glycosylation strongly influence the aggregation propensity [7]. It has already been shown that the N-glycosylation of the influenza A virus surface protein hemagglutinin (HA) depends strongly on the host cell line [8][9][10][11], which might change the physicochemical properties of the whole VP. In a previous study [12] we investigated ion-specific effects on the particle size distributions and aggregation of influenza A (A/Puerto Rico/8/34 H1N1, A/PR) VP produced in adherent and suspension Madin Darby canine kidney (MDCK) cells.…”

Ezrin and HNRNP expression correlate with increased virus release rate and early onset of virus‐induced apoptosis of MDCK suspension cells

“…Previous studies regarding the adaptation of MDCK cells to suspension growth revealed a clear impact on cellular physiology and influenza virus replication, i.e. changes in glycosylation and onset of apoptosis were observed. In particular, suspension cells showed an earlier induction of apoptosis and a decreased cell‐specific virus yield .…”

“…Changes in the physiological status of cells after adaptation can have a significant impact, not only on growth properties and process robustness, but also on virus replication. For the MDCK suspension cell line established recently (MDCK SUS2 ) , it was shown that adaptation also influences the glycosylation of 0hemagglutinin (HA) , the main immunogenic membrane protein of influenza A virus. Additionally, differences for MDCK ADH and MDCK SUS2 cells were observed regarding the onset of virus‐induced cell death determined by late apoptosis markers (DNA fragmentation) .…”