Alexander Rath

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.

Evaluation of criteria for bioreactor comparison and operation standardization for mammalian cell culture

Development of bioprocesses with mammalian cell culture deals with different bioreactor types and scales. The bioreactors might be intended for generation of cell inoculum and production, research, process development, validation, or transfer purposes. During these activities, not only the difficulty of up and downscaling might lead to failure of consistency in cell growth, but also the use of different bioreactor geometries and operation conditions. In such cases, criteria for bioreactor design and process transfer should be carefully evaluated in order to select appropriate cultivation parameters.

Changes in intracellular metabolite pools during growth of adherent MDCK cells in two different media

In bioprocess engineering, the growth of continuous cell lines is mainly studied with respect to the changes in cell concentration, the resulting demand for substrates, and the accumulation of extracellular metabolites. The underlying metabolic process rests upon intracellular metabolite pools and their interaction with enzymes in the form of substrates, products, or allosteric effectors. Here, we quantitatively analyze time courses of 29 intracellular metabolites of adherent Madin–Darby canine kidney cells during cultivation in a serum-containing medium and a serum-free medium. The cells, which originated from the same pre-culture, showed similar overall growth behavior and only slight differences in their demand for the substrates glucose (GLC), glutamine (GLN), and glutamate (GLU). Analysis of intracellular metabolites, which mainly cover the glycolytic pathway, the citric acid cycle, and the nucleotide pools, revealed surprisingly similar dynamics for both cultivation conditions. Instead of a strong influence of the medium, we rather observed a growth phase-specific behavior in glycolysis and in the lower citric acid cycle. Furthermore, analysis of the lower part of glycolysis suggests the well-known regulation of pyruvate kinase by fructose 1,6-bisphosphate. The upper citric acid cycle (citrate, cis-aconitate, and isocitrate) is apparently uncoupled from the lower part (α-ketoglutarate, succinate, fumarate, and malate), which is in line with the characteristics of a truncated cycle. Decreased adenosine triphosphate and guanosine triphosphate pools, as well as a relatively low energy charge soon after inoculation of cells, indicate a high demand for cellular energy and the consumption of nucleotides for biosynthesis. We finally conclude that, with sufficient availability of substrates, the dynamics of GLC and GLN/GLU metabolism is influenced mainly by the cellular growth regime and regulatory function of key enzymes.

Identification of Growth Phases and Influencing Factors in Cultivations with AGE1.HN Cells Using Set-Based Methods

Production of bio-pharmaceuticals in cell culture, such as mammalian cells, is challenging. Mathematical models can provide support to the analysis, optimization, and the operation of production processes. In particular, unstructured models are suited for these purposes, since they can be tailored to particular process conditions. To this end, growth phases and the most relevant factors influencing cell growth and product formation have to be identified. Due to noisy and erroneous experimental data, unknown kinetic parameters, and the large number of combinations of influencing factors, currently there are only limited structured approaches to tackle these issues. We outline a structured set-based approach to identify different growth phases and the factors influencing cell growth and metabolism. To this end, measurement uncertainties are taken explicitly into account to bound the time-dependent specific growth rate based on the observed increase of the cell concentration. Based on the bounds on the specific growth rate, we can identify qualitatively different growth phases and (in-)validate hypotheses on the factors influencing cell growth and metabolism. We apply the approach to a mammalian suspension cell line (AGE1.HN). We show that growth in batch culture can be divided into two main growth phases. The initial phase is characterized by exponential growth dynamics, which can be described consistently by a relatively simple unstructured and segregated model. The subsequent phase is characterized by a decrease in the specific growth rate, which, as shown, results from substrate limitation and the pH of the medium. An extended model is provided which describes the observed dynamics of cell growth and main metabolites, and the corresponding kinetic parameters as well as their confidence intervals are estimated. The study is complemented by an uncertainty and outlier analysis. Overall, we demonstrate utility of set-based methods for analyzing cell growth and metabolism under conditions of uncertainty.

The influence of cell growth and enzyme activity changes on intracellular metabolite dynamics in AGE1.HN.AAT cells

Optimization of bioprocesses with mammalian cells mainly concentrates on cell engineering, cell screening and medium optimization to achieve enhanced cell growth and productivity. For improving cell lines by cell engineering techniques, in-depth understandings of the regulation of metabolism and product formation as well as the resulting demand for the different medium components are needed. In this work, the relationship of cell specific growth and uptake rates and of changes in maximum in vitro enzyme activities with intracellular metabolite pools of glycolysis, pentose phosphate pathway, citric acid cycle and energy metabolism were determined for batch cultivations with AGE1.HN.AAT cells. Results obtained by modeling cell growth and consumption of main substrates showed that the dynamics of intracellular metabolite pools is primarily linked to the dynamics of specific glucose and glutamine uptake rates. By analyzing maximum in vitro enzyme activities we found low activities of pyruvate dehydrogenase and pyruvate carboxylase which suggest a reduced metabolite transfer into the citric acid cycle resulting in lactate release (Warburg effect). Moreover, an increase in the volumetric lactate production rate during the transition from exponential to stationary growth together with a transient accumulation of fructose 1,6-bisphosphate, fructose 1-phosphate and ribose 5-phosphate point toward an upregulation of PK via FBP. Glutaminase activity was about 44-fold lower than activity of glutamine synthetase. This seemed to be sufficient for the supply of intermediates for biosynthesis but might lead to unnecessary dissipation of ATP. Taken together, our results elucidate regulation of metabolic networks of immortalized mammalian cells by changes of metabolite pools over the time course of batch cultivations. Eventually, it enables the use of cell engineering strategies to improve the availability of building blocks for biomass synthesis by increasing glucose as well as glutamine fluxes. An additional knockdown of the glutamine synthetase might help to prevent unnecessary dissipation of ATP, to yield a cell line with optimized growth characteristics and increased overall productivity.

Batch-to-batch variability of two human designer cell lines - AGE1.HN and AGE1.HN.AAT - carried out by different laboratories under defined culture conditions using a mathematical model

Systems biology approaches involve collaboration of a larger number of research groups. Experiments are being performed in different laboratories dealing with different aspects of the topic of interest. Therefore, comparability of data collected for further analysis and modeling needs critical assessment. Here, growth and product formation of two human designer cell lines (AGE1.HN and its α1‐antitrypsin producing clone AGE1.HN.AAT) were investigated by four research laboratories. Cell lines were cultivated in shake flasks and stirred tank bioreactors operated at standardized conditions using a chemically defined medium, and a simple mathematical model was used to estimate characteristic process parameters. Results obtained for 35 batches showed that neither the initial viable cell concentration nor the initial concentration of glucose and glutamine showed significant differences between laboratories. For these measurements with low variations, specific growth rate and yields varied between 8.5 and 26% (relative standard error), indicating comparability of cultivations between laboratories for exponential growth. Higher variations of fitted parameters related to measurements with high initial variation. Comparing the nonproducing with the producing cell line, no significant differences were found regarding growth dynamics and metabolism. Overall, it seems justified to draw conclusions based on the entire experimental dataset of this systems biology project.

Criteria for bioreactor comparison and operation standardisation during process development for mammalian cell culture

BackgroundDevelopment of bioprocesses for animal cells has to dealwith different bioreactor types and scales. Bioreactorsmight be intended for generation of cell inoculum andproduction, research, process development, validation ortransfer purposes. During these activities, not only thedifficulty of up- and downscaling might lead to failureof consistency in cell growth, but also the use of differ-ent bioreactor geometries and operation conditions. Insuch cases, the criteria for bioreactor design and processtransfer should be carefully evaluated in order to avoidan erroneous transfer of cultivation parameters.In this work, power input, mixing time, impeller tipspeed, and Reynolds number have been compared sys-tematically for the cultivation of the human cell lineAGE1.HN

Early changes in the metabolic profile of activated CD8+ T cells

BackgroundAntigenic stimulation of the T cell receptor (TCR) initiates a change from a resting state into an activated one, which ultimately results in proliferation and the acquisition of effector functions. To accomplish this task, T cells require dramatic changes in metabolism. Therefore, we investigated changes of metabolic intermediates indicating for crucial metabolic pathways reflecting the status of T cells. Moreover we analyzed possible regulatory molecules required for the initiation of the metabolic changes.ResultsWe found that proliferation inducing conditions result in an increase in key glycolytic metabolites, whereas the citric acid cycle remains unaffected. The upregulation of glycolysis led to a strong lactate production, which depends upon AKT/PKB, but not mTOR. The observed upregulation of lactate dehydrogenase results in increased lactate production, which we found to be dependent on IL-2 and to be required for proliferation. Additionally we observed upregulation of Glucose-transporter 1 (GLUT1) and glucose uptake upon stimulation, which were surprisingly not influenced by AKT inhibition.ConclusionsOur findings suggest that AKT plays a central role in upregulating glycolysis via induction of lactate dehydrogenase expression, but has no impact on glucose uptake of T cells. Furthermore, under apoptosis inducing conditions, T cells are not able to upregulate glycolysis and induce lactate production. In addition maintaining high glycolytic rates strongly depends on IL-2 production.

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.

Characterisation of cultivation of the human cell line AGE1.HN.AAT

Background Human cell lines are an interesting alternative to CHO cells for the production of recombinant proteins and monoclonal antibodies, because of their ability to produce genuine human posttranslational modifications. The human cell line AGE1.HN.AAT (ProBioGen, Berlin, Germany), that originated from human neural precursor tissue, has been adapted to serum-free conditions and cultivated in many different systems. Here we present our results using this cell line in a scale-up of batch cultivation from 50 mL vented polypropylene tube on a shaking platform, polycarbonate shakeflask (cultivation volume from 50 mL up to 300 mL), a 2 L-glass vessel stirred tank reactor and a 20 L-stainless steel stirred tank reactor (both Sartorius Stedim, Goettingen, Germany).