J. Schwarzer

N-Glycan Analysis by CGE-LIF – Profiling Influenza A Virus Hemagglutinin N-Glycosylation during Vaccine Production

Glycoproteins, such as monoclonal antibodies as well as recombinant and viral proteins produced in mammalian cell culture play an important role in manufacturing of many biopharmaceuticals. To ensure consisting quality of the corresponding products, glycosylation profiles have to be tightly controlled, as glycosylation affects important properties of the corresponding proteins, including bioactivity and antigenicity. This study describes the establishment of a method for analyzing N‐glycosylation patterns of mammalian cell culture‐derived influenza A virus glycoproteins used in vaccine manufacturing. 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 CGE‐LIF, using a capillary DNA‐sequencer. Reproducibility studies of CGE‐LIF, virus purification, and sample preparation have been performed. For demonstrating its applicability, the method was exemplarily used for monitoring batch‐to‐batch reproducibility in vaccine production, with respect to the glycosylation pattern of the membrane protein hemagglutinin of influenza A/PR/8/34 (H1N1) virus. This method allows characterization of variations in protein glycosylation patterns, directly by N‐glycan “fingerprint” alignment.

Glycan analysis in cell culture-based influenza vaccine production: Influence of host cell line and virus strain on the glycosylation pattern of viral hemagglutinin

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. Within this study, a capillary DNA-sequencer (based on CGE-LIF technology), was utilized for N-glycan analysis of three different influenza virus strains, which were replicated in six different cell lines. Detailed results concerning the influence of the host cell line on complexity and composition of the HA N-glycosylation pattern, are presented. Strong host cell but also virus type and subtype dependence of HA N-glycosylation was found. Clear differences were already observed, by N-glycan fingerprint comparison. Further structural investigations of the N-glycan pools revealed that host cell dependence of HA N-glycosylation was 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).

High-density microcarrier cell cultures for influenza virus production

Influenza virus A/PR/8/34 virus propagation in adherent Madin–Darby canine kidney cells in high‐density microcarrier cultures is described. To improve virus yields, perfusion and repeated fed‐batch modes were applied using cell‐specific feed rates. Cell densities up to 1.1 × 107 cells/mL were achieved. Cell‐specific virus yields in high‐density cultures were at similar levels compared with standard, low‐density cultivations. In the average 2,400 and 3,300 virions per cell were obtained for two variants of the virus strain A/PR/8/34, PR8‐National Institute for Biological Standards and Control (NIBSC) and PR8‐Robert Koch Institute, respectively. Maximum virus titer (HA activity = 1,778 HAU/100 μL) for virus variant PR8‐NIBSC was obtained for a cultivation infected before maximum cell concentration was reached.

Optimized CGE-LIF-based glycan analysis for high-throughput applications

This study describes a robust but sensitive method for fast analysis of protein N-glycosylation patterns, which is based on capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF). Compared to our previous work (Schwarzer et al. (2008); Schwarzer et al. (2009); Rapp et al. (2008)) sample preparation was optimized and the method adjusted to high-throughput applications with reduced hands-on-time per sample. Excellent long-term reproducibility of this method is shown, regarding migration times for specific N-glycan structures of immunoglobulin G (IgG). Furthermore, data are presented concerning N-glycosylation variants of influenza virus hemagglutinin produced in different cell lines considered for vaccine manufacturing.