Udo Reichl

Continuous cell lines as a production system for influenza vaccines.

The declaration of pandemic alert Phase 6 for human influenza A (H1N1) by the WHO and the measures taken by individual countries in June 2009 has shown the world how fragile today’s resources in pandemic and prepandemic, but also seasonal, vaccines are. Conventionally, human influenza vaccines are produced in embryonated chicken eggs. However, significant efforts of authorities and vaccine manufacturers over the last decade have led to the establishment of cell culture-derived vaccines. Currently, vaccines produced in three different host cell lines (Madin–Darby Canine Kidney, Vero and PER.C6) are in clinical trials, and the first licenses for seasonal as well as pandemic H5N1 vaccines have been granted. These encouraging developments for cell culture-based influenza virus production are summarized and an overview on potential cell substrates is given.

Substitution of Glutamine by Pyruvate To Reduce Ammonia Formation and Growth Inhibition of Mammalian Cells

In mammalian cell culture technology glutamine is required for biomass synthesis and as a major energy source together with glucose. Different pathways for glutamine metabolism are possible, resulting in different energy output and ammonia release. The accumulation of ammonia in the medium can limit cell growth and product formation. Therefore, numerous ideas to reduce ammonia concentration in cultivation broths have been developed. Here we present new aspects on the energy metabolism of mammalian cells. The replacement of glutamine (2 mM) by pyruvate (10 mM) supported cell growth without adaptation for at least 19 passages without reduction in growth rate of different adherent commercial cell lines (MDCK, BHK21, CHO‐K1) in serum‐containing and serum‐free media. The changes in metabolism of MDCK cells due to pyruvate uptake instead of glutamine were investigated in detail (on the amino acid level) for an influenza vaccine production process in large‐scale microcarrier culture. In addition, metabolite profiles from variations of this new medium formulation (1–10 mM pyruvate) were compared for MDCK cell growth in roller bottles. Even at very low levels of pyruvate (1 mM) MDCK cells grew to confluency without glutamine and accumulation of ammonia. Also glucose uptake was reduced, which resulted in lower lactate production. However, pyruvate and glutamine were both metabolized when present together. Amino acid profiles from the cell growth phase for pyruvate medium showed a reduced uptake of serine, cysteine, and methionine, an increased uptake of leucine and isoleucine and a higher release of glycine compared to glutamine medium. After virus infection completely different profiles were found for essential and nonessential amino acids.

Metabolic effects of influenza virus infection in cultured animal cells: Intra- and extracellular metabolite profiling

BackgroundMany details in cell culture-derived influenza vaccine production are still poorly understood and approaches for process optimization mainly remain empirical. More insights on mammalian cell metabolism after a viral infection could give hints on limitations and cell-specific virus production capacities. A detailed metabolic characterization of an influenza infected adherent cell line (MDCK) was carried out based on extracellular and intracellular measurements of metabolite concentrations.ResultsFor most metabolites the comparison of infected (human influenza A/PR/8/34) and mock-infected cells showed a very similar behavior during the first 10-12 h post infection (pi). Significant changes were observed after about 12 h pi: (1) uptake of extracellular glucose and lactate release into the cell culture supernatant were clearly increased in infected cells compared to mock-infected cells. At the same time (12 h pi) intracellular metabolite concentrations of the upper part of glycolysis were significantly increased. On the contrary, nucleoside triphosphate concentrations of infected cells dropped clearly after 12 h pi. This behaviour was observed for two different human influenza A/PR/8/34 strains at slightly different time points.ConclusionsComparing these results with literature values for the time course of infection with same influenza strains, underline the hypothesis that influenza infection only represents a minor additional burden for host cell metabolism. The metabolic changes observed after12 h pi are most probably caused by the onset of apoptosis in infected cells. The comparison of experimental data from two variants of the A/PR/8/34 virus strain (RKI versus NIBSC) with different productivities and infection dynamics showed comparable metabolic patterns but a clearly different timely behavior. Thus, infection dynamics are obviously reflected in host cell metabolism.

Metagenome and metaproteome analyses of microbial communities in mesophilic biogas-producing anaerobic batch fermentations indicate concerted plant carbohydrate degradation

Microbial communities in biogas batch fermentations, using straw and hay as co-substrates, were analyzed at the gene and protein level by metagenomic and metaproteomic approaches. The analysis of metagenomic data revealed that the Clostridiales and Bacteroidales orders were prevalent in the community. However, the number of sequences assigned to the Clostridiales order decreased during fermentation, whereas the number of sequences assigned to the Bacteroidales order increased. In addition, changes at the functional level were monitored and the metaproteomic analyses detected transporter proteins and flagellins, which were expressed mainly by members of the Bacteroidetes and Firmicutes phyla. A high number of sugar transporters, expressed by members of the Bacteroidetes, proved their potential to take up various glycans efficiently. Metagenome data also showed that methanogenic organisms represented less than 4% of the community, while 20-30% of the identified proteins were of archeal origin. These data suggested that methanogens were disproportionally active. In conclusion, the community studied was capable of digesting the recalcitrant co-substrate. Members of the Firmicutes phylum seemed to be the main degraders of cellulose, even though expression of only a few glycoside hydrolases was detected. The Bacteroidetes phylum expressed a high number of sugar transporters and seemed to specialize in the digestion of other polysaccharides. Finally, it was found that key enzymes of methanogenesis were expressed in high quantities, indicating the high metabolic activity of methanogens, although they only represented a minor group within the microbial community.

Comparative Performance of Four Methods for High-throughput Glycosylation Analysis of Immunoglobulin G in Genetic and Epidemiological Research

The biological and clinical relevance of glycosylation is becoming increasingly recognized, leading to a growing interest in large-scale clinical and population-based studies. In the past few years, several methods for high-throughput analysis of glycans have been developed, but thorough validation and standardization of these methods is required before significant resources are invested in large-scale studies. In this study, we compared liquid chromatography, capillary gel electrophoresis, and two MS methods for quantitative profiling of N-glycosylation of IgG in the same data set of 1201 individuals. To evaluate the accuracy of the four methods we then performed analysis of association with genetic polymorphisms and age. Chromatographic methods with either fluorescent or MS-detection yielded slightly stronger associations than MS-only and multiplexed capillary gel electrophoresis, but at the expense of lower levels of throughput. Advantages and disadvantages of each method were identified, which should inform the selection of the most appropriate method in future studies.

New avian suspension cell lines provide production of influenza virus and MVA in serum-free media: Studies on growth, metabolism and virus propagation

Few suspension cells can be used for vaccine manufacturing today as they either do not meet requirements from health regulatory authorities or do not produce high virus titres. Two new avian designer cell lines (AGE1.CR and AGE1.CR.pIX) that have been adapted to grow in suspension in serum-free medium were evaluated for their potential as host cells for influenza and modified vaccinia Ankara (MVA, wild type) vaccine production. Their metabolism was studied during growth in static (T-flasks) and dynamic cultivation systems (roller bottles, stirred tank reactor, wave bioreactor). High cell concentrations up to 5.8x10(6)cells/mL were obtained with doubling times of 23h for AGE1.CR and 35h for AGE1.CR.pIX, respectively. Both viruses were produced to high titres (3.5 logHA/100 microL for influenza virus, 3.2x10(8)pfu/mL for MVA). Hence, the CR cell lines are an appropriate substrate for pharmaceutical influenza and MVA production.

Purification of cell culture-derived human influenza A virus by size-exclusion and anion-exchange chromatography.

A process comprising of size‐exclusion chromatography (SEC) and anion‐exchange chromatography (AEC) was investigated for downstream processing of cell culture‐derived influenza A virus. Human influenza virus A/PR/8/34 (H1N1) was propagated in serum‐free medium using MDCK cells as a host. Concentrates of the virus were prepared from clarified and inactivated cell culture supernatants by cross‐flow ultrafiltration as described before. SEC on Sepharose 4 FF resulted in average product yields of 85% based on hemagglutination (HA) activity. Productivity was maximized to 0.15 column volumes (cv) of concentrate per hour yielding a reduction in total protein and host cell DNA (hcDNA) to 35 and 34%, respectively. AEC on Sepharose Q XL was used to separate hcDNA from virus at a salt concentration of 0.65 M sodium chloride. Product yields >80% were achieved for loads >160 kHAU/mL of resin. The reduction in hcDNA was 67‐fold. Split peak elution and bimodal particle volume distributions suggested aggregation of virions. Co‐elution with hcDNA and constant amounts of hcDNA per dose indiciated association of virions to hcDNA. An overall product yield of 52% was achieved. Total protein was reduced more than 19‐fold; hcDNA more than 500‐fold by the process. Estimation of the dose volume from HA activity predicted a protein content at the limit for human vaccines. Reduction of hcDNA was found insufficient (about 500 ng per dose) requiring further optimization of AEC or additional purification steps. All operations were selected to be scalable and independent of the virus strain rendering the process suitable for vaccine production. Biotechnol. Bioeng. 2007;96:932–944.

Quantitative analysis of cellular proteome alterations in human influenza A virus-infected mammalian cell lines.

Over the last years virus–host cell interactions were investigated in numerous studies. Viral strategies for evasion of innate immune response, inhibition of cellular protein synthesis and permission of viral RNA and protein production were disclosed. With quantitative proteome technology, comprehensive studies concerning the impact of viruses on the cellular machinery of their host cells at protein level are possible. Therefore, 2‐D DIGE and nanoHPLC‐nanoESI‐MS/MS analysis were used to qualitatively and quantitatively determine the dynamic cellular proteome responses of two mammalian cell lines to human influenza A virus infection. A cell line used for vaccine production (MDCK) was compared with a human lung carcinoma cell line (A549) as a reference model. Analyzing 2‐D gels of the proteomes of uninfected and influenza‐infected host cells, 16 quantitatively altered protein spots (at least ±1.7‐fold change in relative abundance, p<0.001) were identified for both cell lines. Most significant changes were found for keratins, major components of the cytoskeleton system, and for Mx proteins, interferon‐induced key components of the host cell defense. Time series analysis of infection processes allowed the identification of further proteins that are described to be involved in protein synthesis, signal transduction and apoptosis events. Most likely, these proteins are required for supporting functions during influenza viral life cycle or host cell stress response. Quantitative proteome‐wide profiling of virus infection can provide insights into complexity and dynamics of virus–host cell interactions and may accelerate antiviral research and support optimization of vaccine manufacturing processes.

Infection dynamics and virus-induced apoptosis in cell culture-based influenza vaccine production – flow cytometry and mathematical modeling

Cell culture-based influenza vaccine manufacturing is of growing importance. Depending on virus strains, differences in infection dynamics, virus-induced apoptosis, cell lysis and virus yields are observed. Comparatively little is known concerning details of virus-host cell interaction on a cellular level and virus spreading in a population of cells in bioreactors. In this study, the infection of MDCK cells with different influenza A virus strains in lab-scale microcarrier culture was investigated by flow cytometry. Together with the infection status of cells, virus-induced apoptosis was monitored. A mathematical model has been formulated to describe changes in the concentration of uninfected and infected adherent cells, dynamics of virus particle release (infectious virions, hemagglutinin content), and the time course of the percentage composition of the cell population.

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.