Glycosylation Pattern Analysis of Viral Antigens

Mammalian cell culture processes are commonly used for production of recombinant glycoproteins, antibodies and viral vaccines. Since several years there is an increasing interest in cell culture-based influenza vaccine production to overcome limitations of egg-based production systems, to improve vaccine supply and to increase flexibility in vaccine manufacturing. With the switch of the production system several key questions concerning the possible impact of host cell lines on antigen quality, passage dependent selection of certain viral phenotypes or changes in hemagglutinin (HA) conformation have to be addressed to guarantee safety and efficiency of vaccines. In contrast to the production of recombinant glycoproteins, comparatively little is known regarding glycosylation of HA, derived from mammalian cell cultures. To ensure consisting quality of the corresponding products, glycosylation profiles have to be tightly controlled, as glycosylation may affect important properties of the corresponding proteins, including bioactivity and antigenicity. A method for high-throughput analysis of N-glycosylation patterns of cell culture derived influenza A virus glycoproteins with respect to vaccine manufacturing will be presented. It comprises virus purification directly from cell culture supernatant, protein isolation, deglycosylation, and clean-up steps as well as ‘‘fingerprint’’ analysis of N-glycan pools by capillary gel electrophoresis with laser induced fluorescence detection (CGE-LIF), utilizing a capillary DNA-sequencer. This method allows characterization of variations in protein glycosylation patterns, directly by N-glycan ‘fingerprint’’ comparison. Results concerning the influence of the host cell line on complexity and composition of the HA N-glycosylation pattern are presented for different influenza virus strains, which were replicated in different cell lines. Strong host cell but also virus type and subtype dependence of HA N-glycosylation was found. It revealed, that host cell dependence of HA N-glycosylation is mainly related to minor variations of the (monomeric) constitution of single N-glycans. To some extent, shifts in the N-glycan pool composition regarding the proportion of different N-glycan types were observed. In contrast to this, a principal switch of the N-glycan type attached to HA was observed, when comparing different virus types (A and B) and subtypes (H1N1 and H3N2)