Increased titer and reduced lactate accumulation in recombinant retrovirus production through the down-regulation of HIF1 and PDK.

Many mammalian cell lines used in the manufacturing of biopharmaceuticals exhibit high glycolytic flux predominantly channeled to the production of lactate. The accumulation of lactate in culture reduces cell viability and may also decrease product quality. In this work, we engineered a HEK 293 derived cell line producing a recombinant gene therapy retroviral vector, by down-regulating hypoxia inducible factor 1 (HIF1) and pyruvate dehydrogenase kinase (PDK). Specific productivity of infectious viral titers could be increased more than 20-fold for single gene knock-down (HIF1 or PDK) and more than 30-fold under combined down-regulation. Lactate production was reduced up to 4-fold. However, the reduction in lactate production, alone, was not sufficient to enhance the titer: high-titer clones also showed significant enrollment of metabolic routes not related to lactate production. Transcriptome analysis indicated activation of biological amines metabolism, detoxification routes, including glutathione metabolism, pentose phosphate pathway, glycogen biosynthesis and amino acid catabolism. The latter were validated by enzyme activity assays and metabolite profiling, respectively. High-titer clones also presented substantially increased transcript levels of the viral genes expression cassettes. The results herein presented demonstrate the impact of HIF1 and PDK down-regulation on the production performance of a mammalian cell line, reporting one of the highest fold-increase in specific productivity of infectious virus titers achieved by metabolic engineering. They additionally highlight the contribution of secondary pathways, beyond those related to lactate production, that can be also explored to pursue improved metabolic status favoring a high-producing phenotype.

Production of canine adenovirus type 2 in serum-free suspension cultures of MDCK cells.

The potential of adherent Madin Darby Canine Kidney (MDCK) cells for the production of influenza viruses and canine adenovirus type 2 (CAV-2) for vaccines or gene therapy approaches has been shown. Recently, a new MDCK cell line (MDCK.SUS2) that was able to grow in suspension in a fully defined system was established. In this work, we investigated whether the new MDCK.SUS2 suspension cell line is suitable for the amplification of CAV-2 under serum-free culture conditions. Cell growth performance and CAV-2 production were evaluated in three serum-free media: AEM, SMIF8, and EXCELL MDCK. CAV-2 production in shake flasks was maximal when AEM medium was used, resulting in an amplification ratio of infectious particles (IP) of 142 IP out/IP in and volumetric and cell-specific productivities of 2.1 × 10(8) IP/mL and 482 IP/cell, respectively. CAV-2 production was further improved when cells were cultivated in a 0.5-L stirred tank bioreactor. To monitor infection and virus production, cells were analyzed by flow cytometry. A correlation between the side scatter measurement and CAV-2 productivity was found, which represents a key feature to determine the best harvesting time during process development of gene therapy vectors that do not express reporter genes. This work demonstrates that MDCK.SUS2 is a suitable cell substrate for CAV-2 production, constituting a step forward in developing a production process transferable to industrial scales. This could allow for the production of high CAV-2 titers either for vaccination or for gene therapy purposes.

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.

Comparison of influenza virus yields and apoptosis-induction in an adherent and a suspension MDCK cell line.

Cell culture-based manufacturing of influenza vaccines is ideally based on easily scalable platforms using suspension cells that grow in chemically defined media. Consequently, different adherent cell lines selected for high virus yields were adapted to grow in suspension culture. This includes the MDCK suspension cell line MDCK.SUS2, which was shown to be a suitable substrate for influenza virus propagation in previous studies. In this study, we investigated options for further improvement of influenza A/PR/8 (H1N1) virus titres and cell-specific virus yields. Best results were achieved by performing a 1:2 dilution with fresh medium at time of infection. In shake flask cultivations, even for multiplicities of infection as low as 10⁻5, all cells were infected at 36 h post infection as determined by flow cytometry. In addition, these cells showed a better viability than cells infected without previous washing steps, which was reflected by a reduced level of apoptotic cells, and virus yields exceeding 3 log10 HAU/100 µL. Comparison of bioreactor infections of MDCK.SUS2 cells to the parental adherent MDCK cells showed similar HA titres of 2.94 and 3.15 log10 HAU/100 µL and TCID50 of 1 × 109 and 2.37 × 109 infectious virions/mL. Surprisingly, virus-induced apoptosis differed between the two cell lines, with the MDCK.SUS2 cells showing a much stronger apoptosis induction than the adherent MDCK cells. Obviously, despite their resistance to anoikis, the suspension cells were more susceptible to virus-induced apoptosis. Whether this is related to the adaptation process itself and/or to changes in cell survival pathways influenced by adhesion molecules or influenza virus proteins needs to be clarified in additional studies.

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.

The relation between growth phases, cell volume changes and metabolism of adherent cells during cultivation.

In biotechnology, mathematical models often consider changes in cell numbers as well as in metabolite conversion to describe different cell growth phases. It has been frequently observed that the cell number is only a delayed indicator of cell growth compared to the biomass, which challenges the principle structure of corresponding models. Here, we evaluate adherent cell growth phases in terms of cell number and biomass increase on the basis of detailed experimental data of three independent cultivations for Madin Darby canine kidney cells. We develop a model linking cell numbers and mean cell diameters to estimate cell volume changes during growth without the need for diameter distribution measurements. It simultaneously describes the delay between cell number and cell volume increase, cell-specific volume changes and the transition from growth to maintenance metabolism while taking different pre-culture conditions, which affect the cell diameter, into account. In addition, inspection of metabolite uptake and release rates reveals that glucose is mainly used for generation of cellular energy and glutamine is not required for cellular maintenance. Finally, we conclude that changes in cell number, cell diameter and metabolite uptake during cultivation contribute to the understanding of the time course of intracellular metabolites during the cultivation process.

Productivity, apoptosis, and infection dynamics of influenza A/PR/8 strains and A/PR/8-based reassortants.

In cell culture-based influenza vaccine production significant efforts are directed towards virus seed optimization for maximum yields. Typically, high growth reassortants (HGR) containing backbones of six gene segments of e.g. influenza A/PR/8, are generated from wild type strains. Often, however, HA and TCID50 titres obtained do not meet expectations and further optimization measures are required. Flow cytometry is an invaluable tool to improve our understanding of mechanism related to progress of infection, virus-induced apoptosis, and cell-specific productivity. In this study, we performed infections with two influenza A/PR/8 variants (from NIBSC and RKI) and two A/PR/8-based HGRs (Wisconsin-like and Uruguay-like) to investigate virus replication, apoptosis and virus titres at different multiplicities of infection (MOI 0.0001, 0.1, 3). Flow cytometric analyses showed similar dynamics in the time course of infected and apoptotic cell populations for all four tested strains at MOI 0.0001. Interestingly, higher MOI resulted in an earlier increase of the populations of infected and apoptotic cells and showed strain-specific differences. Infections with A/PR/8 NIBSC resulted in an earlier increase in both cell populations compared to A/PR/8 RKI. The Uruguay-like reassortant showed the earliest increase in the concentration of infected cells and a late induction of apoptosis at all tested MOIs. In contrast, the Wisconsin-like reassortant showed strong apoptosis induction at high MOIs resulting in reduced titres compared to lower MOI. Maximum HA titres were unaffected by changes in the MOI for the two A/PR/8 and the Uruguay-like reassortant. Maximum TCID50 titres, however, decreased with increasing MOI for all strains. Overall, infections at very low MOI (0.0001) resulted not only in similar dynamics concerning progress of infection and induction of apoptosis but also in maximum virus yields. Highest HA titres were obtained for virus seed strains combining a fast progress in infection with a late onset of apoptosis. Therefore, both factors should be considered for the establishment of robust influenza vaccine production processes.

Metabolic adaptation of MDCK cells to different growth conditions: effects on catalytic activities of central metabolic enzymes.

Lactate and ammonia are the most important waste products of central carbon metabolism in mammalian cell cultures. In particular during batch and fed-batch cultivations these toxic by-products are excreted into the medium in large amounts, and not only affect cell viability and productivity but often also prevent growth to high cell densities. The most promising approach to overcome such a metabolic imbalance is the replacement of one or several components in the culture medium. It has been previously shown that pyruvate can be substituted for glutamine in cultures of adherent Madin-Darby canine kidney (MDCK) cells. As a consequence, the cells not only released no ammonia but glucose consumption and lactate production were also reduced significantly. In this work, the impact of media changes on glucose and glutamine metabolism was further elucidated by using a high-throughput platform for enzyme activity measurements of mammalian cells. Adherent MDCK cells were grown to stationary and exponential phase in six-well plates in serum-containing GMEM supplemented with glutamine or pyruvate. A total number of 28 key metabolic enzyme activities of cell extracts were analyzed. The overall activity of the pentose phosphate pathway was up-regulated during exponential cell growth in pyruvate-containing medium suggesting that more glucose-6-phosphate was channeled into the oxidative branch. Furthermore, the anaplerotic enzymes pyruvate carboxylase and pyruvate dehydrogenase showed higher cell specific activities with pyruvate. An increase in cell specific activity was also found for NAD(+)-dependent isocitrate dehydrogenase, glutamate dehydrogenase, and glutamine synthetase in MDCK cells grown with pyruvate. It can be assumed that the increase in enzyme activities was required to compensate for the energy demand and to replenish the glutamine pool. On the other hand, the activities of glutaminolytic enzymes (e.g., alanine and aspartate transaminase) were decreased in cells grown with pyruvate, which seems to be related to a decreased glutamine metabolism.

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 × 10(7) 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.

Expression, purification, and characterization of a His6-tagged glycerokinase from Pichia farinosa for enzymatic cycling assays in mammalian cells.

The GUT1 gene of the halotolerant yeast Pichia farinosa, encoding glycerokinase (EC 2.7.1.30), was expressed in Pichia pastoris. A purification factor of approximately 61-fold was achieved by a combination of nickel affinity and anion exchange chromatography. The specific activity of the final preparation was 201.6 units per mg protein with a yield of about 21%. A nearly homogeneous enzyme preparation was confirmed by SDS-polyacrylamide gels and mass spectrometry analysis. Glycerol stabilized the purified enzyme for long-term storage at -80°C. The pH and temperature optima were in the range of 6.5-7.0 and 45-50°C, respectively. ATP was the most effective phosphoryl group donor tested. Additionally, the enzyme phosphorylated glycerol also with ITP, UTP, GTP and CTP. The K(m) values of the enzyme for ATP and ITP were 0.428 and 0.845 mM, respectively. The kinetic properties of the enzyme with respect to UTP, GTP, and CTP suggested that glycerokinase exhibited negative cooperativity as double reciprocal plots showed a biphasic response to increasing nucleoside triphosphate concentrations. The application as a coupling enzyme in the determination of pyruvate kinase activity in cell extracts of Madin-Darby canine kidney cells showed good reproducibility when compared with a commercially available preparation of bacterial glycerokinase.

Differential activation of host cell signalling pathways through infection with two variants of influenza A/Puerto Rico/8/34 (H1N1) in MDCK cells.

In cell culture-based influenza vaccine production, few efforts have been undertaken to characterise virus-host cell interactions in detail. Two influenza virus strains that grew to different virus titres, and differed in virus dynamics, apoptosis induction and proteome changes were observed. In order to elucidate biological mechanisms related to these differences, the induction of signalling cascades in adherent MDCK cells infected with two variants of influenza A/PuertoRico/8/34 (H1N1) was analysed. The pathways chosen for analysis are key components of the innate immune response and crucial for influenza A virus replication (NF-κB, IRF-3, PI3K-Akt, Jak-Stat, Raf/MEK/ERK, PKR/eIF2α). Interestingly, all investigated pathways were induced stronger by PR8-NIBSC than by PR8-RKI, the virus variant which results in higher virus titres. In particular, PR8-NIBSC infection lead to a higher induction of IFN-beta as well as IFN-stimulated gene expression, which was confirmed by Western blot as well as real-time PCR. Overall, results obtained clearly facilitate interpretation of observations regarding proteome changes and virus-induced apoptosis in cell culture-based vaccine manufacturing processes and support efforts towards design of improved host cell lines.

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.

Measurement of key metabolic enzyme activities in mammalian cells using rapid and sensitive microplate-based assays.

Sensitive microplate-based assays to determine low levels of key enzyme activities in mammalian cells are presented. The enzyme platform consists of four cycling assays to measure the activity of 28 enzymes involved in central carbon and glutamine metabolism. The sensitivity limit of all cycling assays was between 0.025 and 0.4 nmol product. For the detection of glutaminase activity, a new glutamate cycle system involving the enzymes glutamate dehydrogenase and aspartate transaminase was established. The relative standard deviation of the method was found to be 1.7% with a limit of detection of 8.2 pmol and a limit of quantitation of 24.8 pmol. Hence, cell extracts could be highly diluted to reduce interferences caused by other components in the extract, which in addition minimized underestimates or overestimates of actual enzyme activities. Since substrate concentrations could be maintained at a nearly constant level throughout the assay product accumulation during the reaction was low, which minimized product inhibition. As an example, the enzyme platform was used to investigate maximum enzyme activities of stationary-phase MDCK cells grown in serum-containing GMEM medium as typically used in influenza vaccine production.

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.

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.

Establishment of a mink enteritis vaccine production process in stirred-tank reactor and Wave Bioreactor microcarrier culture in 1-10 L scale.

A scale-up and process optimization scheme for the growth of adherent embryonic feline lung fibroblasts (E-FL) on microcarriers and the propagation of a mink enteritis virus (MEV) strain for the production of an inactivated vaccine is shown. Stirred-tank cultivations are compared with results obtained from Wave Bioreactors. Transfer from a roller bottle-based production process into large-scale microcarrier culture with starting concentrations of 2g/L Cytodex 1 microcarriers and 2.0 x 10(5)cells/mL was successful. A maximum cell yield of 1.2 x 10(6)cells/mL was obtained in stirred-tank microcarrier batch culture while cell numbers in the Wave Bioreactor could not be determined accurately due to the fast sedimentation of microcarriers under non-rocking conditions required for sampling. Detailed off-line analysis was carried out to understand the behaviour of the virus-host cell system in both cultivation systems. Metabolic profiles for glucose, lactate, glutamine, and ammonium showed slight differences for both systems. E-FL cell growth was on the same level in stirred-tank and Wave Bioreactor with a higher volumetric cell yield compared to roller bottles. Propagation of MEV, which can only replicate efficiently in mitotic cells, was characterized in the Wave Bioreactor using a multiple harvest strategy. Maximum virus titres of 10(6.6) to 10(6.8) TCID(50)/mL were obtained, which corresponds to an increase in virus yield by a factor of about 10 compared to cultivations in roller bottles. As a consequence, a single Wave Bioreactor cultivation of appropriate scale can replace hundreds of roller bottles. Thus, the Wave Bioreactor proved to be a suitable system for large-scale production of an inactivated MEV vaccine.

Wave microcarrier cultivation of MDCK cells for influenza virus production in serum containing and serum-free media.

A process for equine influenza virus vaccine production using a microcarrier system (Cytodex 1) in a 2 L Wave bioreactor is described. Growth of Madin Darby canine kidney (MDCK) cells in serum containing GMEM medium (SC) is compared to growth in serum-free Ex-Cell MDCK medium (SF) without washing steps and medium exchange before infection. Cultivations with microcarrier concentrations of 2 and 4 g/L for both media are shown. Metabolic data from carbon and amino acid metabolism are discussed. Additionally, in roller bottle experiments the influence of multiplicity of infection (moi) and trypsin concentration on the HA value was investigated. Analysis of HA and TCID(50) at 37 degrees C showed a stable HA of maximum 2.6 log HA/100 microL for 2 weeks. Peak TCID(50) titers of 10(7.7) viruses/mL were achieved 20h post infection, but infectivity was below detection limit after 150 h. Cell attachment onto microcarriers under serum-free conditions was improved by Ca(2+) addition and by cell harvesting without trypsin using only an EDTA/PBS solution. For the wave cultivation maximum virus titers of 2.3-2.6 log HA units/100 microL were reached from infection with a moi of 0.05. However, in SF medium pH dropped to less than pH 6.8 which resulted in lower HA titers of 1.7 log HA units/100 microL. For the higher microcarrier concentration (4 g/L) medium exchange steps (500 mL) were needed for both media. Omission of the washing step and medium exchange before infection in SF medium clearly simplified the influenza production process; however, for higher virus yields a better pH control of the wave bioreactor would be required. Higher cell densities (2.8 x 10(6) cells/mL for 2 g/L microcarrier) and better attachment compared to stirred tank bioreactors showed, that the wave bioreactor is a good alternative to stirred tank processes for expanding production capacities in case of a pandemic.

Serum-free influenza virus production avoiding washing steps and medium exchange in large-scale microcarrier culture.

A complete serum-free process without washing steps and medium exchange before infection for influenza A virus vaccine production (equine and human) is described for cultivation in roller bottles and in a 5-L stirred tank microcarrier system. Adherent Madin-Darby canine kidney cells (MDCK) were adapted from growth in serum containing GMEM medium to growth in serum-free Ex-Cell MDCK medium. Roller bottle experiments showed that the medium exchange step, typically required for serum containing vaccine production processes, could be omitted without losses in virus titre and without limitations in glucose or glutamine supply in the cultivation medium. The serum-free medium could even be used glutamine-free as it contained pyruvate, resulting in very low levels of ammonia. Cell attachment onto microcarriers was critical. Therefore, microcarriers had to be preconditioned in medium. Also, trypsin concentration used for inoculum preparation had to be reduced. After these modifications 1.3 x 10(6)cells/mL were obtained after 97 h (2g/L Cytodex 1) of cell growth. Maximum virus titres of 2.3-2.9 log HA units/100 microL were obtained from infections with a multiplicity of infection (moi) of 0.05-0.10 for human and equine influenza A virus. Metabolite and amino acid profiles as well as on-line data for the serum-free process are compared with the serum containing process. Omission of the medium exchange before infection clearly simplified the process and reduced sterility risks due to washing steps.

Metabolism of MDCK cells during cell growth and influenza virus production in large-scale microcarrier culture.

The production of equine influenza in Madin-Darby canine kidney (MDCK) cells in large-scale microcarrier culture is described with detailed on- and off-line analytical data during cell growth and virus replication. Metabolite concentration profiles for glucose, glutamine, lactate and ammonium are shown. Lactate and ammonium concentrations were always below inhibiting levels. Concentration profiles for essential and non-essential amino acids of the cell culture medium are discussed. During cell growth proline was released into the medium with a significant rate while two amino acids, serine and methionine were almost depleted. After infection, virus titer increased after a delay of 10-16 h whereas first changes in amino acid metabolism could be observed within 4h post-infection. Here, glutamate and aspartate increase correlated to virus release kinetics, indicating cell disruption and apoptosis. Starting with a moi of 0.025 resulted in a maximum virus yield of 2.4 log HA/100 microl at 44 h post-infection.

Microbiological transformations. 47. A step toward a green chemistry preparation of enantiopure (S)-2-, -3-, and -4-pyridyloxirane via an epoxide hydrolase catalyzed kinetic resolution.

The biocatalyzed hydrolytic kinetic resolution of 2-, 3-, and 4-pyridyloxirane by the Aspergillus niger epoxide hydrolase (EH) has been explored. This was used to perform a gram scale preparation of these epoxides of (S) absolute configuration using a process performed at a concentration as high as 10 g/L (82 mM). All three epoxides have been obtained in a nearly enantiopure form (ee > 98%). Interestingly, it was shown that this biotransformation could be achieved using plain water instead of buffer solution, an important improvement as far as downstream processing of an eventual industrial process is concerned. Neither of these substrates could be obtained in reasonable enantiomeric purity and yield using the nowadays most efficient metal-based catalysts.