From single-cell cloning to high-yield influenza virus production - implementing advanced technologies in vaccine process development.

Innovations in viral vaccine manufacturing are crucial for pandemic preparedness and to meet ever-rising global demands. For influenza, however, production still mainly relies on technologies established decades ago. Although modern production shifts from egg-based towards cell culture technologies, the full potential has not yet been fully exploited. Here, we evaluate whether implementation of state-of-the-art technologies for cell culture-based recombinant protein production are capable to challenge outdated approaches in viral vaccine process development. For this, a fully automated single-cell cloning strategy was established to generate monoclonal suspension Madin-Darby canine kidney (MDCK) cells. Among selected cell clones, we could observe distinct metabolic and growth characteristics, with C59 reaching a maximum viable cell concentration of 17.3 × 106 cells/mL and low doubling times in batch mode. Screening for virus production using a panel of human vaccine-relevant influenza A and B viruses in an ambr15 system revealed high titers with yields competing or even outperforming available MDCK cell lines. With C113, we achieved cell-specific virus yields of up to 25,000 virions/cell, making this cell clone highly attractive for vaccine production. Finally, we confirmed process performance at a 50-fold higher working volume. In summary, we present a scalable and powerful approach for accelerated development of high-yield influenza virus production in chemically defined medium starting from a single cell.

Mathematical model calibrated to in vitro data predicts mechanisms of antiviral action of the influenza defective interfering particle "OP7".

Defective interfering particles (DIPs) are regarded as potent broad-spectrum antivirals. We developed a mathematical model that describes intracellular co-infection dynamics of influenza standard virus (STV) and "OP7", a new type of influenza DIP discovered recently. Based on experimental data from in vitro studies to calibrate the model and confirm its predictions, we deduce OP7's mechanisms of interference, which were yet unknown. Simulations suggest that the "superpromoter" on OP7 genomic viral RNA enhances its replication and results in a depletion of viral proteins. This reduces STV genomic RNA replication, which appears to constitute an antiviral effect. Further, a defective viral protein (M1-OP7) likely causes the deficiency of OP7's replication. It appears unable to bind to genomic viral RNAs to facilitate their nuclear export, a critical step in the viral life cycle. An improved understanding of OP7's antiviral mechanism is crucial toward application in humans as a prospective antiviral treatment strategy.

Production of recombinant vesicular stomatitis virus-based vectors by tangential flow depth filtration.

Cell culture-based production of vector-based vaccines and virotherapeutics is of increasing interest. The vectors used not only retain their ability to infect cells but also induce robust immune responses. Using two recombinant vesicular stomatitis virus (rVSV)-based constructs, we performed a proof-of-concept study regarding an integrated closed single-use perfusion system that allows continuous virus harvesting and clarification. Using suspension BHK-21 cells and a fusogenic oncolytic hybrid of vesicular stomatitis virus and Newcastle disease virus (rVSV-NDV), a modified alternating tangential flow device (mATF) or tangential flow depth filtration (TFDF) systems were used for cell retention. As the hollow fibers of the former are characterized by a large internal lumen (0.75 mm; pore size 0.65 µm), membrane blocking by the multi-nucleated syncytia formed during infection could be prevented. However, virus particles were completely retained. In contrast, the TFDF filter unit (lumen 3.15 mm, pore size 2-5 µm) allowed not only to achieve high viable cell concentrations (VCC, 16.4-20.6×106 cells/mL) but also continuous vector harvesting and clarification. Compared to an optimized batch process, 11-fold higher infectious virus titers were obtained in the clarified permeate (maximum 7.5×109 TCID50/mL). Using HEK293-SF cells and a rVSV vector expressing a green fluorescent protein, perfusion cultivations resulted in a maximum VCC of 11.3×106 cells/mL and infectious virus titers up to 7.1×1010 TCID50/mL in the permeate. Not only continuous harvesting but also clarification was possible. Although the cell-specific virus yield decreased relative to a batch process established as a control, an increased space-time yield was obtained. KEY POINTS: • Viral vector production using a TFDF perfusion system resulted in a 460% increase in space-time yield • Use of a TFDF system allowed continuous virus harvesting and clarification • TFDF perfusion system has great potential towards the establishment of an intensified vector production.

Production of antiviral "OP7 chimera" defective interfering particles free of infectious virus.

Defective interfering particles (DIPs) of influenza A virus (IAV) are suggested for use as broad-spectrum antivirals. We discovered a new type of IAV DIP named "OP7" that carries point mutations in its genome segment (Seg) 7 instead of a deletion as in conventional DIPs (cDIPs). Recently, using genetic engineering tools, we generated "OP7 chimera DIPs" that carry point mutations in Seg 7 plus a deletion in Seg 1. Together with cDIPs, OP7 chimera DIPs were produced in shake flasks in the absence of infectious standard virus (STV), rendering UV inactivation unnecessary. However, only part of the virions harvested were OP7 chimera DIPs (78.7%) and total virus titers were relatively low. Here, we describe the establishment of an OP7 chimera DIP production process applicable for large-scale production. To increase total virus titers, we reduced temperature from 37 to 32 °C during virus replication. Production of almost pure OP7 chimera DIP preparations (99.7%) was achieved with a high titer of 3.24 log10(HAU/100 µL). This corresponded to an 11-fold increase relative to the initial process. Next, this process was transferred to a stirred tank bioreactor resulting in comparable yields. Moreover, DIP harvests purified and concentrated by steric exclusion chromatography displayed an increased interfering efficacy in vitro. Finally, a perfusion process with perfusion rate control was established, resulting in a 79-fold increase in total virus yields compared to the original batch process in shake flasks. Again, a very high purity of OP7 chimera DIPs was obtained. This process could thus be an excellent starting point for good manufacturing practice production of DIPs for use as antivirals. KEY POINTS: • Scalable cell culture-based process for highly effective antiviral OP7 chimera DIPs • Production of almost pure OP7 chimera DIPs in the absence of infectious virus • Perfusion mode production and purification train results in very high titers.

Breakdown of hardly degradable carbohydrates (lignocellulose) in a two-stage anaerobic digestion plant is favored in the main fermenter.

The yield and productivity of biogas plants depend on the degradation performance of their microbiomes. The spatial separation of the anaerobic digestion (AD) process into a separate hydrolysis and a main fermenter should improve cultivation conditions of the microorganisms involved in the degradation of complex substrates like lignocellulosic biomass (LCB) and, thus, the performance of anaerobic digesters. However, relatively little is known about such two-stage processes. Here, we investigated the process performance of a two-stage agricultural AD over one year, focusing on chemical and technical process parameters and metagenome-centric metaproteomics. Technical and chemical parameters indicated stable operation of the main fermenter but varying conditions for the open hydrolysis fermenter. Matching this, the microbiome in the hydrolysis fermenter has a higher dynamic than in the main fermenter. Metaproteomics-based microbiome analysis revealed a partial separation between early and common steps in carbohydrate degradation and primary fermentation in the hydrolysis fermenter but complex carbohydrate degradation, secondary fermentation, and methanogenesis in the main fermenter. Detailed metagenomics and metaproteomics characterization of the single metagenome-assembled genomes showed that the species focus on specific substrate niches and do not utilize their full genetic potential to degrade, for example, LCB. Overall, it seems that a separation of AD in a hydrolysis and a main fermenter does not improve the cleavage of complex substrates but significantly improves the overall process performance. In contrast, the remaining methanogenic activity in the hydrolysis fermenter may cause methane losses.

Generation of "OP7 chimera" defective interfering influenza A particle preparations free of infectious virus that show antiviral efficacy in mice.

Influenza A virus (IAV) defective interfering particles (DIPs) are considered as new promising antiviral agents. Conventional DIPs (cDIPs) contain a deletion in the genome and can only replicate upon co-infection with infectious standard virus (STV), during which they suppress STV replication. We previously discovered a new type of IAV DIP "OP7" that entails genomic point mutations and displays higher antiviral efficacy than cDIPs. To avoid safety concerns for the medical use of OP7 preparations, we developed a production system that does not depend on infectious IAV. We reconstituted a mixture of DIPs consisting of cDIPs and OP7 chimera DIPs, in which both harbor a deletion in their genome. To complement the defect, the deleted viral protein is expressed by the suspension cell line used for production in shake flasks. Here, DIP preparations harvested are not contaminated with infectious virions, and the fraction of OP7 chimera DIPs depended on the multiplicity of infection. Intranasal administration of OP7 chimera DIP material was well tolerated in mice. A rescue from an otherwise lethal IAV infection and no signs of disease upon OP7 chimera DIP co-infection demonstrated the remarkable antiviral efficacy. The clinical development of this new class of broad-spectrum antiviral may contribute to pandemic preparedness.

Broad-Spectrum Antiviral Activity of Influenza A Defective Interfering Particles against Respiratory Syncytial, Yellow Fever, and Zika Virus Replication In Vitro.

New broadly acting and readily available antiviral agents are needed to combat existing and emerging viruses. Defective interfering particles (DIPs) of influenza A virus (IAV) are regarded as promising options for the prevention and treatment of IAV infections. Interestingly, IAV DIPs also inhibit unrelated viral infections by stimulating antiviral innate immunity. Here, we tested the ability of IAV DIPs to suppress respiratory syncytial, yellow fever and Zika virus infections in vitro. In human lung (A549) cells, IAV DIP co-infection inhibited the replication and spread of all three viruses. In contrast, we observed no antiviral activity in Vero cells, which are deficient in the production of interferon (IFN), demonstrating its importance for the antiviral effect. Further, in A549 cells, we observed an enhanced type-I and type-III IFN response upon co-infection that appears to explain the antiviral potential of IAV DIPs. Finally, a lack of antiviral activity in the presence of the Janus kinase 1/2 (JAK1/2) inhibitor ruxolitinib was detected. This revealed a dependency of the antiviral activity on the JAK/signal transducers and activators of transcription (STAT) signaling pathway. Overall, this study supports the notion that IAV DIPs may be used as broad-spectrum antivirals to treat infections with a variety of IFN-sensitive viruses, particularly respiratory viruses.

Cell-free N-glycosylation of peptides using synthetic lipid-linked hybrid and complex N-glycans.

Cell-free, chemoenzymatic platforms are emerging technologies towards generating glycoconjugates with defined and homogeneous glycoforms. Recombinant oligosaccharyltransferases can be applied to glycosylate "empty," i.e., aglycosyalted, peptides and proteins. While bacterial oligosaccharlytransferases have been extensively investigated, only recently a recombinant eukaryotic single-subunit oligosaccharyltransferase has been successfully used to in vitro N-glycosylate peptides. However, its applicability towards synthesizing full-length glycoproteins and utilizing glycans beyond mannose-type glycans for the transfer have not be determined. Here, we show for the first time the synthesis of hybrid- and complex-type glycans using synthetic lipid carriers as substrates for in vitro N-glycosylation reactions. For this purpose, transmembrane-deleted human ß-1,2 N-acetylglucosamintransferase I and II (MGAT1ΔTM and MGAT2ΔTM) and ß-1,4-galactosyltransferase (GalTΔTM) have been expressed in Escherichia coli and used to extend an existing multi-enzyme cascade. Both hybrid and agalactosylated complex structures were transferred to the N-glycosylation consensus sequence of peptides (10 amino acids: G-S-D-A-N-Y-T-Y-T-Q) by the recombinant oligosaccharyltransferase STT3A from Trypanosoma brucei.

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.

A Cell-Free Multi-enzyme Cascade Reaction for the Synthesis of CDP-Glycerol.

CDP-glycerol is a nucleotide-diphosphate-activated version of glycerol. In nature, it is required for the biosynthesis of teichoic acid in Gram-positive bacteria, which is an appealing target epitope for the development of new vaccines. Here, a cell-free multi-enzyme cascade was developed to synthetize nucleotide-activated glycerol from the inexpensive and readily available substrates cytidine and glycerol. The cascade comprises five recombinant enzymes expressed in Escherichia coli that were purified by immobilized metal affinity chromatography. As part of the cascade, ATP is regenerated in situ from polyphosphate to reduce synthesis costs. The enzymatic cascade was characterized at the laboratory scale, and the products were analyzed by high-performance anion-exchange chromatography (HPAEC)-UV and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). After the successful synthesis had been confirmed, a design-of-experiments approach was used to screen for optimal operation conditions (temperature, pH value and MgCl2 concentration). Overall, a substrate conversion of 89 % was achieved with respect to the substrate cytidine.

Production of retroviral vectors in continuous high cell density culture.

Retroviral vectors derived from murine leukemia virus (MLV) are used in somatic gene therapy applications e.g. for genetic modification of hematopoietic stem cells. Recently, we reported on the establishment of a suspension viral packaging cell line (VPC) for the production of MLV vectors. Human embryonic kidney 293-F (HEK293-F) cells were genetically modified for this purpose using transposon vector technology. Here, we demonstrate the establishment of a continuous high cell density (HCD) process using this cell line. First, we compared different media regarding the maximum achievable viable cell concentration (VCC) in small scale. Next, we transferred this process to a stirred tank bioreactor before we applied intensification strategies. Specifically, we established a perfusion process using an alternating tangential flow filtration system. Here, VCCs up to 27.4E + 06 cells/mL and MLV vector titers up to 8.6E + 06 transducing units/mL were achieved. Finally, we established a continuous HCD process using a tubular membrane for cell retention and continuous viral vector harvesting. Here, the space-time yield was 18-fold higher compared to the respective batch cultivations. Overall, our results clearly demonstrate the feasibility of HCD cultivations for high yield production of viral vectors, especially when combined with continuous viral vector harvesting. KEY POINTS: • A continuous high cell density process for MLV vector production was established • The tubular cell retention membrane allowed for continuous vector harvesting • The established process had a 18-fold higher space time yield compared to a batch.

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.

Reliable N-Glycan Analysis-Removal of Frequently Occurring Oligosaccharide Impurities by Enzymatic Degradation.

Glycosylation, especially N-glycosylation, is one of the most common protein modifications, with immense importance at the molecular, cellular, and organismal level. Thus, accurate and reliable N-glycan analysis is essential in many areas of pharmaceutical and food industry, medicine, and science. However, due to the complexity of the cellular glycosylation process, in-depth glycoanalysis is still a highly challenging endeavor. Contamination of samples with oligosaccharide impurities (OSIs), typically linear glucose homo-oligomers, can cause further complications. Due to their physicochemical similarity to N-glycans, OSIs produce potentially overlapping signals, which can remain unnoticed. If recognized, suspected OSI signals are usually excluded in data evaluation. However, in both cases, interpretation of results can be impaired. Alternatively, sample preparation can be repeated to include an OSI removal step from samples. However, this significantly increases sample amount, time, and effort necessary. To overcome these issues, we investigated the option to enzymatically degrade and thereby remove interfering OSIs as a final sample preparation step. Therefore, we screened ten commercially available enzymes concerning their potential to efficiently degrade maltodextrins and dextrans as most frequently found OSIs. Of these enzymes, only dextranase from Chaetomium erraticum and glucoamylase P from Hormoconis resinae enabled a degradation of OSIs within only 30 min that is free of side reactions with N-glycans. Finally, we applied the straightforward enzymatic degradation of OSIs to N-glycan samples derived from different standard glycoproteins and various stem cell lysates.

Adaptation of a microbial community to demand-oriented biological methanation.

BACKGROUND: Biological conversion of the surplus of renewable electricity and carbon dioxide (CO2) from biogas plants to biomethane (CH4) could support energy storage and strengthen the power grid. Biological methanation (BM) is linked closely to the activity of biogas-producing Bacteria and methanogenic Archaea. During reactor operations, the microbiome is often subject to various changes, e.g., substrate limitation or pH-shifts, whereby the microorganisms are challenged to adapt to the new conditions. In this study, various process parameters including pH value, CH4 production rate, conversion yields and final gas composition were monitored for a hydrogenotrophic-adapted microbial community cultivated in a laboratory-scale BM reactor. To investigate the robustness of the BM process regarding power oscillations, the biogas microbiome was exposed to five hydrogen (H2)-feeding regimes lasting several days. RESULTS: Applying various "on-off" H2-feeding regimes, the CH4 production rate recovered quickly, demonstrating a significant resilience of the microbial community. Analyses of the taxonomic composition of the microbiome revealed a high abundance of the bacterial phyla Firmicutes, Bacteroidota and Thermotogota followed by hydrogenotrophic Archaea of the phylum Methanobacteriota. Homo-acetogenic and heterotrophic fermenting Bacteria formed a complex food web with methanogens. The abundance of the methanogenic Archaea roughly doubled during discontinuous H2-feeding, which was related mainly to an increase in acetoclastic Methanothrix species. Results also suggested that Bacteria feeding on methanogens could reduce overall CH4 production. On the other hand, using inactive biomass as a substrate could support the growth of methanogenic Archaea. During the BM process, the additional production of H2 by fermenting Bacteria seemed to support the maintenance of hydrogenotrophic methanogens at non-H2-feeding phases. Besides the elusive role of Methanothrix during the H2-feeding phases, acetate consumption and pH maintenance at the non-feeding phase can be assigned to this species. CONCLUSIONS: Taken together, the high adaptive potential of microbial communities contributes to the robustness of BM processes during discontinuous H2-feeding and supports the commercial use of BM processes for energy storage. Discontinuous feeding strategies could be used to enrich methanogenic Archaea during the establishment of a microbial community for BM. Both findings could contribute to design and improve BM processes from lab to pilot scale.

Human 3D Airway Tissue Models for Real-Time Microscopy: Visualizing Respiratory Virus Spreading.

Our knowledge about respiratory virus spreading is mostly based on monolayer cultures that hardly reflect the complex organization of the airway epithelium. Thus, there is a strong demand for biologically relevant models. One possibility to study virus spreading at the cellular level is real-time imaging. In an attempt to visualize virus spreading under somewhat more physiological conditions, Calu-3 cells and human primary fibroblasts were co-cultured submerged or as air-liquid interface (ALI). An influenza A virus (IAV) replicating well in cell culture, and carrying a red fluorescent protein (RFP) reporter gene was used for real-time imaging. Our three-dimensional (3D) models exhibited important characteristics of native airway epithelium including a basement membrane, tight junctions and, in ALI models, strong mucus production. In submerged models, first fluorescence signals appeared between 9 and 12 h post infection (hpi) with a low multiplicity of infection of 0.01. Virus spreading further proceeded in the immediate vicinity of infected cells. In ALI models, RFP was found at 22 hpi and later. Consequently, the progression of infection was delayed, in contrast to the submerged model. With these features, we believe that our 3D airway models can deliver new insights in the spreading of IAV and other respiratory viruses.

Discovering Biomarkers for Non-Alcoholic Steatohepatitis Patients with and without Hepatocellular Carcinoma Using Fecal Metaproteomics.

High-calorie diets lead to hepatic steatosis and to the development of non-alcoholic fatty liver disease (NAFLD), which can evolve over many years into the inflammatory form of non-alcoholic steatohepatitis (NASH), posing a risk for the development of hepatocellular carcinoma (HCC). Due to diet and liver alteration, the axis between liver and gut is disturbed, resulting in gut microbiome alterations. Consequently, detecting these gut microbiome alterations represents a promising strategy for early NASH and HCC detection. We analyzed medical parameters and the fecal metaproteome of 19 healthy controls, 32 NASH patients, and 29 HCC patients, targeting the discovery of diagnostic biomarkers. Here, NASH and HCC resulted in increased inflammation status and shifts within the composition of the gut microbiome. An increased abundance of kielin/chordin, E3 ubiquitin ligase, and nucleophosmin 1 represented valuable fecal biomarkers, indicating disease-related changes in the liver. Although a single biomarker failed to separate NASH and HCC, machine learning-based classification algorithms provided an 86% accuracy in distinguishing between controls, NASH, and HCC. Fecal metaproteomics enables early detection of NASH and HCC by providing single biomarkers and machine learning-based metaprotein panels.

Cell-line screening and process development for a fusogenic oncolytic virus in small-scale suspension cultures.

Oncolytic viruses (OVs) represent a novel class of immunotherapeutics under development for the treatment of cancers. OVs that express a cognate or transgenic fusion protein is particularly promising as their enhanced intratumoral spread via syncytia formation can be a potent mechanism for tumor lysis and induction of antitumor immune responses. Rapid and efficient fusion of infected cells results in cell death before high titers are reached. Although this is an attractive safety feature, it also presents unique challenges for large-scale clinical-grade manufacture of OVs. Here we evaluate the use of four different suspension cell lines for the production of a novel fusogenic hybrid of vesicular stomatitis virus and Newcastle disease virus (rVSV-NDV). The candidate cell lines were screened for growth, metabolism, and virus productivity. Permissivity was evaluated based on extracellular infectious virus titers and cell-specific virus yields (CSVYs). For additional process optimizations, virus adaptation and multiplicity of infection (MOI) screenings were performed and confirmed in a 1 L bioreactor. BHK-21 and HEK293SF cells infected at concentrations of 2 × 106 cells/mL were identified as promising candidates for rVSV-NDV production, leading to infectious titers of 3.0 × 108 TCID50/mL and 7.5 × 107 TCID50/mL, and CSVYs of 153 and 9, respectively. Compared to the AGE1.CR.pIX reference produced in adherent cultures, oncolytic potency was not affected by production in suspension cultures and possibly even increased in cultures of HEK293SF and AGE1.CR.pIX. Our study describes promising suspension cell-based processes for efficient large-scale manufacturing of rVSV-NDV. KEY POINTS: • Cell contact-dependent oncolytic virus (OV) replicates in suspension cells. • Oncolytic potency is not encompassed during suspension cultivation. • Media composition, cell line, and MOI are critical process parameters for OV production. • The designed process is scalable and shows great promise for manufacturing clinical-grade material.

Impact of Influenza A Virus Infection on Growth and Metabolism of Suspension MDCK Cells Using a Dynamic Model.

Cell cultured-based influenza virus production is a viable option for vaccine manufacturing. In order to achieve a high concentration of viable cells, is requirement to have not only optimal process conditions, but also an active metabolism capable of intracellular synthesis of viral components. Experimental metabolic data collected in such processes are complex and difficult to interpret, for which mathematical models are an appropriate way to simulate and analyze the complex and dynamic interaction between the virus and its host cell. A dynamic model with 35 states was developed in this study to describe growth, metabolism, and influenza A virus production in shake flask cultivations of suspension Madin-Darby Canine Kidney (MDCK) cells. It considers cell growth (concentration of viable cells, mean cell diameters, volume of viable cells), concentrations of key metabolites both at the intracellular and extracellular level and virus titers. Using one set of parameters, the model accurately simulates the dynamics of mock-infected cells and correctly predicts the overall dynamics of virus-infected cells for up to 60 h post infection (hpi). The model clearly suggests that most changes observed after infection are related to cessation of cell growth and the subsequent transition to apoptosis and cell death. However, predictions do not cover late phases of infection, particularly for the extracellular concentrations of glutamate and ammonium after about 12 hpi. Results obtained from additional in silico studies performed indicated that amino acid degradation by extracellular enzymes resulting from cell lysis during late infection stages may contribute to this observed discrepancy.

Absolute quantification of viral proteins during single-round replication of MDCK suspension cells.

Madin-Darby canine kidney (MDCK) cells are widely used in basic research and for the propagation of influenza A viruses (IAV) for vaccine production. To identify targets for antiviral therapies and to optimize vaccine manufacturing, a detailed understanding of the viral life cycle is important. This includes the characterization of virus entry, the synthesis of the various viral RNAs and proteins, the transfer of viral compounds in the cell and virus budding. In case quantitative information is available, the analysis can be complemented by mathematical modelling approaches. While comprehensive studies focusing on IAV entry as well as viral mRNA, vRNA and cRNA accumulation in the nucleus of cells have been performed, quantitative data regarding IAV protein synthesis and accumulation was mostly lacking. In this study, we present a mass spectrometry (MS)-based method to evaluate whether an absolute quantification of viral proteins is possible for single-round replication in suspension MDCK cells. Using influenza A/PR/8/34 (H1N1, RKI) as a model strain at a multiplicity of infection of ten, defined amounts of isotopically labelled peptides of synthetic origin of four IAV proteins (hemagglutinin, neuraminidase, nucleoprotein, matrix protein 1) were added as an internal standard before tryptic digestion of samples for absolute quantification (AQUA). The first intracellular protein detected was NP at 1 h post infection (hpi). A maximum extracellular concentration of 7.7E+12 copies/mL was achieved. This was followed by hemagglutinin (3 hpi, maximum 4.1E+12 copies/mL at 13 hpi), matrix protein 1 (5 hpi, maximum 2.2E+12 copies/mL at 13 hpi) and neuraminidase (5 hpi, 6.0E+11 copies/mL at 13 hpi). In sum, for the first time absolute IAV protein copy numbers were quantified by a MS-based method for infected MDCK cells providing important insights into viral protein dynamics during single-round virus replication. SIGNIFICANCE: Influenza A virus is a significant human pathogen worldwide. To improve therapies against influenza and overcome bottlenecks in vaccine production in cell culture, it is critical to gain a detailed understanding of the viral life cycle. In addition to qPCR-based models, this study will examine the dynamics of influenza virus proteins during infection of producer cells to gain initial insights into changes in absolute copy numbers.

High-Titer Hepatitis C Virus Production in a Scalable Single-Use High Cell Density Bioreactor.

Hepatitis C virus (HCV) infections pose a major public health burden due to high chronicity rates and associated morbidity and mortality. A vaccine protecting against chronic infection is not available but would be important for global control of HCV infections. In this study, cell culture-based HCV production was established in a packed-bed bioreactor (CelCradle™) aiming to further the development of an inactivated whole virus vaccine and to facilitate virological and immunological studies requiring large quantities of virus particles. HCV was produced in human hepatoma-derived Huh7.5 cells maintained in serum-free medium on days of virus harvesting. Highest virus yields were obtained when the culture was maintained with two medium exchanges per day. However, increasing the total number of cells in the culture vessel negatively impacted infectivity titers. Peak infectivity titers of up to 7.2 log10 focus forming units (FFU)/mL, accumulated virus yields of up to 5.9 × 1010 FFU, and a cell specific virus yield of up to 41 FFU/cell were obtained from one CelCradle™. CelCradle™-derived and T flask-derived virus had similar characteristics regarding neutralization sensitivity and buoyant density. This packed-bed tide-motion system is available with larger vessels and may thus be a promising platform for large-scale HCV production.