Characterization of a quail suspension cell line for production of a fusogenic oncolytic virus

The development of efficient processes for the production of oncolytic viruses (OV) plays a crucial role regarding the clinical success of virotherapy. Although many different OV platforms are currently under investigation, manufacturing of such viruses still mainly relies on static adherent cell cultures, which bear many challenges, particularly for fusogenic OVs. Availability of GMP-compliant continuous cell lines is limited, further complicating the development of commercially viable products. BHK21, AGE1. CR and HEK293 cells were previously identified as possible cell substrates for the recombinant vesicular stomatitis virus (rVSV)-based fusogenic OV, rVSV-NDV. Now, another promising cell substrate was identified, the CCX.E10 cell line, developed by Nuvonis Technologies. This suspension cell line is considered non-GMO as no foreign genes or viral sequences were used for its development. The CCX.E10 cells were thus thoroughly investigated as a potential candidate for OV production. Cell growth in the chemically defined medium in suspension resulted in concentrations up to 8.9 × 10⁶ cells/mL with a doubling time of 26.6 h in batch mode. Cultivation and production of rVSV-NDV, was demonstrated successfully for various cultivation systems (ambr15, shake flask, stirred tank reactor, and orbitally shaken bioreactor) at vessel scales ranging from 15 mL to 10 L. High infectious virus titers of up to 4.2 × 10⁸ TCID₅₀/mL were reached in orbitally shaken bioreactors and stirred tank reactors in batch mode, respectively. Our results suggest that CCX.E10 cells are a very promising option for industrial production of OVs, particularly for fusogenic VSV-based constructs.     

Process intensification strategies toward cell culture-based high-yield production of a fusogenic oncolytic virus

We present a proof-of-concept study for production of a recombinant vesicular stomatitis virus (rVSV)-based fusogenic oncolytic virus (OV), rVSV-Newcastle disease virus (NDV), at high cell densities (HCD). Based on comprehensive experiments in 1 L stirred tank reactors (STRs) in batch mode, first optimization studies at HCD were carried out in semi-perfusion in small-scale cultivations using shake flasks. Further, a perfusion process was established using an acoustic settler for cell retention. Growth, production yields, and process-related impurities were evaluated for three candidate cell lines (AGE1.CR, BHK-21, HEK293SF)infected at densities ranging from 15 to 30 × 10⁶ cells/mL. The acoustic settler allowed continuous harvesting of rVSV-NDV with high cell retention efficiencies (above 97%) and infectious virus titers (up to 2.4 × 10⁹ TCID₅₀/mL), more than 4–100 times higher than for optimized batch processes. No decrease in cell-specific virus yield (CSVY) was observed at HCD, regardless of the cell substrate. Taking into account the accumulated number of virions both from the harvest and bioreactor, a 15–30 fold increased volumetric virus productivity for AGE1.CR and HEK293SF was obtained compared to batch processes performed at the same scale. In contrast to all previous findings, formation of syncytia was observed at HCD for the suspension cells BHK 21 and HEK293SF. Oncolytic potency was not affected compared to production in batch mode. Overall, our study describes promising options for the establishment of perfusion processes for efficient large-scale manufacturing of fusogenic rVSV-NDV at HCD for all three candidate cell lines.     

Measurement and simulation of the morphological development of filamentous microorganisms

Growth of Streptomyces tendae was investigated in submerged culture. Images of several mycelia were analyzed by means of an image-processing system. The studies revealed that tip growth angles and branching outgrowth angles could be regarded as normally distributed. Based on these results, a random model for directional growth of hyphal tips as well as directional growth of branches is proposed. This model shows curved elongation of hyphal tips, so that the morphological development of a mycelium up to the formation of a pellet is predicted, similar to that observed in nature.     

A dynamic model linking cell growth to intracellular metabolism and extracellular by-product accumulation

Mathematical modeling of animal cell growth and metabolism is essential for the understanding and improvement of the production of biopharmaceuticals. Models can explain the dynamic behavior of cell growth and product formation, support the identification of the most relevant parameters for process design, and significantly reduce the number of experiments to be performed for process optimization. Few dynamic models have been established that describe both extracellular and intracellular dynamics of growth and metabolism of animal cells. In this study, a model was developed, which comprises a set of 33 ordinary differential equations to describe batch cultivations of suspension AGE1.HN.AAT cells considered for the production of α1-antitrypsin. This model combines a segregated cell growth model with a structured model of intracellular metabolism. Overall, it considers the viable cell concentration, mean cell diameter, viable cell volume, concentration of extracellular substrates, and intracellular concentrations of key metabolites from the central carbon metabolism. Furthermore, the release of metabolic by-products such as lactate and ammonium was estimated directly from the intracellular reactions. Based on the same set of parameters, this model simulates well the dynamics of four independent batch cultivations. Analysis of the simulated intracellular rates revealed at least two distinct cellular physiological states. The first physiological state was characterized by a high glycolytic rate and high lactate production. Whereas the second state was characterized by efficient adenosine triphosphate production, a low glycolytic rate, and reactions of the TCA cycle running in the reverse direction from α-ketoglutarate to citrate. Finally, we show possible applications of the model for cell line engineering and media optimization with two case studies.     

Ezrin and HNRNP expression correlate with increased virus release rate and early onset of virus‐induced apoptosis of MDCK suspension cells

With the aim to adapt high‐yield adherent cell lines to suspension growth, Madin Darby canine kidney (MDCK) suspension cells were developed recently that achieved comparable influenza virus yields despite an early induction of apoptosis compared to the parental adherent cell line. For both cell lines, a comprehensive study under comparable infection conditions is performed comprising information on: time course of viral infection, antiviral state of cells, virus‐induced apoptosis, and virus‐induced cellular protein expression for early and late infection with influenza A/PuertoRico/8/34 H1N1. The proteomic analysis is performed with 2D differential gel electrophoreses followed by mass spectrometry. Based on flow cytometric data and on the differential expression of various stress and apoptosis‐related proteins, the earlier onset of virus‐induced apoptosis is confirmed for suspension cells. Surprisingly, the data indicated an increased virus release rate for suspension cells. These observations correlate with an increased expression of the apical marker protein ezrin, known to play a role in influenza‐induced cytoskeletal rearrangement, and the differential expression of heterogeneous nuclear ribonucleoproteins, known to bind actively influenza viral proteins and play a central role in regulating gene expression. Based on these findings, additional studies towards the design of MDCK suspension cells with further increase in influenza virus yields will be performed.     

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.     

The influence of various storage conditions on cell viability in amniotic membrane

Up to now freeze-dried, gamma-sterilised or glycerol-preserved amniotic membranes (AMs) have widely been used in the field of ophthalmology and wound care (e.g. leg ulcers, burns). After some preservation processes in use, like freeze-drying or glycerol-preserving, the cells in the AM are no longer viable. Within this study we evaluated the influence of different short-term and long-term storage conditions on cell viability in AM. Therefore AMs from cesarean section placentae were washed and biopsied to evaluate the microbiological status and to determine the viability of the tissue. Additionally, viability under various storage conditions was examined by assessment of mitochondrial activity. Preservation included temperatures above and below 0°C as well as various media compositions. As expected, cell viability in amnion decreases during storage, in fact the effect was more pronounced when stored frozen, but the higher viability of amnion obtained by storage above 0°C with medium is associated with the limitation to a short period of storage of about 28 days. The evaluated preservation methods are the basis for future non-clinical in-vivo studies in which the possible benefit of amnion as a viable biomaterial in wound healing will be investigated. A part of this work was presented at the World Congress on Tissue Banking in Rio in May 2005 and was honoured by the Poster Award Commission.     

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.