Verena Lohr

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.

A new MDCK suspension line cultivated in a fully defined medium in stirred-tank and wave bioreactor

An adherently growing MDCK cell line was adapted in a two-step process in a fully defined medium and in suspension. The resulting MDCK.SUS2 cells were subsequently evaluated for their potential as host cells for influenza vaccine production in two lab-scale bioreactors (wave and stirred-tank). Cell concentrations up to 2.3 x 10(6)cells/mL were obtained after 96 h, which is slightly higher than cell concentrations obtained with adherent MDCK cells cultivated on microcarriers (2g/L). Infections with influenza A/PR/8/34 and B/Malaysia resulted in high virus titers (2.90 and 2.75 log HA units/100 microL, respectively). The monitoring of extracellular metabolites, including amino acids, revealed a change in some of the metabolite consumption or release profiles, which indicates changes in metabolism during the adaptation process. Overall, the MDCK.SUS2 cell line represents a new cell substrate for a robust influenza vaccine production in a fully defined process.

Metabolic and Kinetic analyses of influenza production in perfusion HEK293 cell culture

BackgroundCell culture-based production of influenza vaccine remains an attractive alternative to egg-based production. Short response time and high production yields are the key success factors for the broader adoption of cell culture technology for industrial manufacturing of pandemic and seasonal influenza vaccines. Recently, HEK293SF cells have been successfully used to produce influenza viruses, achieving hemagglutinin (HA) and infectious viral particle (IVP) titers in the highest ranges reported to date. In the same study, it was suggested that beyond 4 × 106 cells/mL, viral production was limited by a lack of nutrients or an accumulation of toxic products.ResultsTo further improve viral titers at high cell densities, perfusion culture mode was evaluated. Productivities of both perfusion and batch culture modes were compared at an infection cell density of 6 × 106 cells/mL. The metabolism, including glycolysis, glutaminolysis and amino acids utilization as well as physiological indicators such as viability and apoptosis were extensively documented for the two modes of culture before and after viral infection to identify potential metabolic limitations. A 3 L bioreactor with a perfusion rate of 0.5 vol/day allowed us to reach maximal titers of 3.3 × 1011 IVP/mL and 4.0 logHA units/mL, corresponding to a total production of 1.0 × 1015 IVP and 7.8 logHA units after 3 days post-infection. Overall, perfusion mode titers were higher by almost one order of magnitude over the batch culture mode of production. This improvement was associated with an activation of the cell metabolism as seen by a 1.5-fold and 4-fold higher consumption rates of glucose and glutamine respectively. A shift in the viral production kinetics was also observed leading to an accumulation of more viable cells with a higher specific production and causing an increase in the total volumetric production of infectious influenza particles.ConclusionsThese results confirm that the HEK293SF cell is an excellent substrate for high yield production of influenza virus. Furthermore, there is great potential in further improving the production yields through better control of the cell culture environment and viral production kinetics. Once accomplished, this cell line can be promoted as an industrial platform for cost-effective manufacturing of the influenza seasonal vaccine as well as for periods of peak demand during pandemics.

Live attenuated influenza viruses produced in a suspension process with avian AGE1.CR.pIX cells

BackgroundCurrent influenza vaccines are trivalent or quadrivalent inactivated split or subunit vaccines administered intramuscularly, or live attenuated influenza vaccines (LAIV) adapted to replicate at temperatures below body temperature and administered intranasally. Both vaccines are considered safe and efficient, but due to differences in specific properties may complement each other to ensure reliable vaccine coverage. By now, licensed LAIV are produced in embryonated chicken eggs. In the near future influenza vaccines for human use will also be available from adherent MDCK or Vero cell cultures, but a scalable suspension process may facilitate production and supply with vaccines.ResultsWe evaluated the production of cold-adapted human influenza virus strains in the duck suspension cell line AGE1.CR.pIX using a chemically-defined medium. One cold-adapted A (H1N1) and one cold-adapted B virus strain was tested, as well as the reference strain A/PR/8/34 (H1N1). It is shown that a medium exchange is not required for infection and that maximum virus titers are obtained for 1 × 10-6 trypsin units per cell. 1 L bioreactor cultivations showed that 4 × 106 cells/mL can be infected without a cell density effect achieving titers of 1 × 108 virions/mL after 24 h.ConclusionsOverall, this study demonstrates that AGE1.CR.pIX cells support replication of LAIV strains in a chemically-defined medium using a simple process without medium exchanges. Moreover, the process is fast with peak titers obtained 24 h post infection and easily scalable to industrial volumes as neither microcarriers nor medium replacements are required.

The avian cell line AGE1.CR.pIX characterized by metabolic flux analysis

BackgroundIn human vaccine manufacturing some pathogens such as Modified Vaccinia Virus Ankara, measles, mumps virus as well as influenza viruses are still produced on primary material derived from embryonated chicken eggs. Processes depending on primary cell culture, however, are difficult to adapt to modern vaccine production. Therefore, we derived previously a continuous suspension cell line, AGE1.CR.pIX, from muscovy duck and established chemically-defined media for virus propagation.ResultsTo better understand vaccine production processes, we developed a stoichiometric model of the central metabolism of AGE1.CR.pIX cells and applied flux variability and metabolic flux analysis. Results were compared to literature dealing with mammalian and insect cell culture metabolism focusing on the question whether cultured avian cells differ in metabolism. Qualitatively, the observed flux distribution of this avian cell line was similar to distributions found for mammalian cell lines (e.g. CHO, MDCK cells). In particular, glucose was catabolized inefficiently and glycolysis and TCA cycle seem to be only weakly connected.ConclusionsA distinguishing feature of the avian cell line is that glutaminolysis plays only a minor role in energy generation and production of precursors, resulting in low extracellular ammonia concentrations. This metabolic flux study is the first for a continuous avian cell line. It provides a basis for further metabolic analyses to exploit the biotechnological potential of avian and vertebrate cell lines and to develop specific optimized cell culture processes, e.g. vaccine production processes.

Impact of Influenza Virus Adaptation Status on HA N-Glycosylation Patterns in Cell Culture-Based Vaccine Production

The highly abundant and strongly immunogenic influenza envelope glycoprotein hemagglutinin (HA) represents the main component of influenza vaccines. Human influenza vaccines are typically produced in embryonated chicken eggs. In addition, cell culture-derived vaccine production systems are currently being established. Since characteristics of glycoproteins such as the HA can be significantly influenced by N-glycosylation, the impact of host cells considered for vaccine manufacturing needs to be addressed. In this study MDCK cell-derived influenza A/PR/8/34 (H1N1) virus was adapted over four passages in AGE1.CR.pIX-cells. HA N-glycosylation patterns (normalized capillary electropherograms) were determined and analyzed using capillary gel electrophoresis with laser-induced fluorescence detection (each peak represents at least one distinct N-glycan structure). During the adaptation to AGE1.CR.pIX-cells, virus titers 24 hours postinfection improved. HA N-glycosylation patterns of MDCK and AGE1.CR.pIX-derived virus particles differed significantly after the first adaptation step. This clearly suggests that the structure of the viral antigens is strongly influenced by the host cell. Furthermore, AGE1.CR.pIX-derived antigens showed a tendency toward small glycans. Differences between glycan patterns of the four successive passages in AGE1.CR.pIX cell were minor; only low variability in relative peak height was noted in the HA N-glycosylation pattern.

Elements in the Development of a Production Process for Modified Vaccinia Virus Ankara

The production of several viral vaccines depends on chicken embryo fibroblasts or embryonated chicken eggs. To replace this logistically demanding substrate, we created continuous anatine suspension cell lines (CR and CR.pIX), developed chemically-defined media, and established production processes for different vaccine viruses. One of the processes investigated in greater detail was developed for modified vaccinia virus Ankara (MVA). MVA is highly attenuated for human recipients and an efficient vector for reactogenic expression of foreign genes. Because direct cell-to-cell spread is one important mechanism for vaccinia virus replication, cultivation of MVA in bioreactors is facilitated if cell aggregates are induced after infection. This dependency may be the mechanism behind our observation that a novel viral genotype (MVA-CR) accumulates with serial passage in suspension cultures. Sequencing of a major part of the genomic DNA of the new strain revealed point mutations in three genes. We hypothesize that these changes confer an advantage because they may allow a greater fraction of MVA-CR viruses to escape the host cells for infection of distant targets. Production and purification of MVA-based vaccines may be simplified by this combination of designed avian cell line, chemically defined media and the novel virus strain.

Evaluating differences of metabolic performances: Statistical methods and their application to animal cell cultivations

In cell culture process development, monitoring and analyzing metabolic key parameters is routinely applied to demonstrate specific advantages of one experimental setup over another. It is of great importance that the observed differences and expected improvements are practically relevant and statistically significant. However, a systematic assessment whether observed differences in metabolic rates are statistically significant or not is often missing. This can lead to time‐consuming and costly changes of an established biotechnological process due to false positive results. In the present work we demonstrate how well‐established statistical tools can be employed to analyze systematically different sources of variations in metabolic rate determinations and to assess, in an unbiased way, their implications on the significance of the observed differences. As a case study, we evaluate differing growth characteristics and metabolic rates of the avian designer cell line AGE1.CR.pIX cultivated in a stirred tank reactor and in a wave bioreactor. Although large differences in metabolic rates and cell growth were expected (due to different aeration, agitation, pH‐control, etc.) and partially observed (up to 79%), our results show that the inter‐experimental variance between experiments performed under identical conditions but with different pre‐cultures is a major contributor to the overall variance of metabolic rates. The lower bounds of the overall relative standard deviations for specific metabolic rates were between 4% and 73%. The application of available statistical methods revealed that the observed differences were statistically not significant and consequently insufficient to confirm relevant differences between both cultivation systems. Our study provides a general guideline for statistical analyses in comparative cultivation studies and emphasizes the necessity to account for the inter‐experimental variance (mainly caused by biological variation) to avoid false‐positive results. Biotechnol. Bioeng. 2013;110: 2633–2642.

Avian Designer Cells AGE1.CR as Candidates for MVA and Influenza Vaccine Production

In the last years, cells specifically designed for vaccine production have been developed in order to improve industrial vaccine production. However, only few candidates for the propagation of a broad range of virus strains, including influenza viruses and viral vectors derived from modified vaccinia virus Ankara (MVA), are at hand. The avian designer cells AGE1.CR and AGE1.CR.pIX from ProBioGen AG belong to these candidates and were evaluated in this study for their potential to produce influenza virus and MVA. The growth behaviour of these cells was studied in T-flasks and shaker flasks. Thereby, shaker flasks were shown to be favourable for cell growth, providing cell concentrations up to 9.3 × 106 and 5.6 × 106 cells/mL for AGE1.CR and AGE1.CR.pIX, respectively. Staining experiments showed that sialic acids in both, α2,3 and α2,6 conformation, are present on the cell surface so that propagation of avian and human influenza strains is most likely supported. Replication of several influenza virus strains (type A and B) showed that both cell lines lead to maximum HA titers in the same range than MDCK and Vero cells for most of the tested virus strains. Moreover, virus propagation of a typical influenza strain (A/PuertoRico/8/34) was significantly faster compared to MDCK and Vero cells. As a second product, MVA can be produced in AGE1.CR cells. Besides the standard parameters routinely monitored during the virus propagation phase in cell culture, flow cytometry was used in a first approach to investigate cell populations with different DNA content (measured with propidiumiodide). These experiments in lab-scale stirred tank bioreactors showed that apoptosis is induced during infection. Taken together, results indicate that AGE1.CR and AGE1.CR.pIX cells are excellent candidates for large-scale production of influenza virus and MVA.

Purification of modified vaccinia virus Ankara from suspension cell culture

Background A spectrum of viral vaccines, including the annual 620 million doses of trivalent influenza vaccines, are being produced in embryonated eggs of pharmaceutical quality, or with primary cells derived from such eggs [1,2]. Regulatory guidelines and experience for these processes are established and proven against time for 75 years [1]. However, production with galline primary material is not optimal and producers sometimes struggle to provide needed vaccine doses. Among the challenges are limitations in supply [3] and that rigid intervalls between husbandry, harvest of eggs and inoculation with vaccine seed must be accomodated [4]. Manipulation of embryonated eggs and disposal of solid biohazardous waste that accumulates if vaccines are being produced in egg cavities come at considerable costs [5,6]. Finally, risk of contamination with environmental and endogenous agents is high [7-9]. Such issues can be circumvented if a continuous cell line is used to propagate viral vaccines [1]. Master Cell Banks can be prepared in sufficient amounts and tested for presence of adventitious agents ahead of production, chemically defined media obviate dependence on animal derived components, and predictable seed trains towards a wide range of bioreactor volumes allow flexible and fast response times for vaccine production [10]. However, there is a regulatory concern that DNA derived from the immortal production substrate may be transferred to vaccine recipients [1]. Risk calculations that relate values for the length of typical oncogenes, number of such genes in the genome and fragmentation of DNA during purification have arrived at a permissive threshold of 10 ng of nucleic acid per vaccine dose [1]. Materials and methods We investigated purification of modified vaccinia virus Ankara (MVA) produced on the continuous avian cell line CR.pIX. MVA is a versatile and highly immunogenic viral vector, but also known to pose unique challenges in production processes [11]. For example, the majority of the infectious units of MVA remain cellassociated so that downstream purification must initiate with a complete lysate of the infected cultures (rather than cell-free supernatant). The viral particles are furthermore too large for conventional filtration, centrifugation and chromatographic separation. Finally, because MVA cannot amplify in human recipients, a desired safety feature, each vaccine dose requires 108 infectious units for full efficacy. This dose is 400-fold above that recommended for replication-competent poxviruses and necessitates efficient and robust manufacturing processes [10]. Derivation and properties of the anatine CR.pIX cell line have been described previously [12], as well as cellassociated propagation of poxviruses in suspension cultures in chemically-defined media by induction of CR. pIX aggregates [10]. We used GFP-recombinant versions of both genetically stable strains of MVA, wildtype and strain MVA-CR that was isolated previously with help of the suspension process [13]. Purification of MVA was performed with CIM monolithic chromatography columns from Bia Separations [14]. The CIM monoliths consist of a single piece of highly porous material with a network of branched channels. These channels can be provided with large 6 μm-diameters and can be functionalized with various ion exchange groups.