Publications by authors named "Yvonne Genzel"

76 Publications

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

Metabolites 2022 Mar 12;12(3). Epub 2022 Mar 12.

Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstrasse 1, 39106 Magdeburg, Germany.

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.
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http://dx.doi.org/10.3390/metabo12030239DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8950586PMC
March 2022

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

J Proteomics 2022 05 1;259:104544. Epub 2022 Mar 1.

Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany; Bioprocess Engineering, Otto von Guericke University Magdeburg, Magdeburg, Germany.

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.
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http://dx.doi.org/10.1016/j.jprot.2022.104544DOI Listing
May 2022

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

Vaccines (Basel) 2022 Feb 7;10(2). Epub 2022 Feb 7.

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital-Hvidovre, 2650 Hvidovre, Denmark.

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 log focus forming units (FFU)/mL, accumulated virus yields of up to 5.9 × 10 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.
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http://dx.doi.org/10.3390/vaccines10020249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8880717PMC
February 2022

Semi-continuous Propagation of Influenza A Virus and Its Defective Interfering Particles: Analyzing the Dynamic Competition To Select Candidates for Antiviral Therapy.

J Virol 2021 11 22;95(24):e0117421. Epub 2021 Sep 22.

Max Planck Institute for Dynamics of Complex Technical Systemsgrid.419517.f, Bioprocess Engineering, Magdeburg, Germany.

Defective interfering particles (DIPs) of influenza A virus (IAV) are naturally occurring mutants that have an internal deletion in one of their eight viral RNA (vRNA) segments, rendering them propagation-incompetent. Upon coinfection with infectious standard virus (STV), DIPs interfere with STV replication through competitive inhibition. Thus, DIPs are proposed as potent antivirals for treatment of the influenza disease. To select corresponding candidates, we studied generation of DIPs and propagation competition between different defective interfering (DI) vRNAs in an STV coinfection scenario in cell culture. A small-scale two-stage cultivation system that allows long-term semi-continuous propagation of IAV and its DIPs was used. Strong periodic oscillations in virus titers were observed due to the dynamic interaction of DIPs and STVs. Using next-generation sequencing, we detected a predominant formation and accumulation of DI vRNAs on the polymerase-encoding segments. Short DI vRNAs accumulated to higher fractions than longer ones, indicating a replication advantage, yet an optimum fragment length was observed. Some DI vRNAs showed breaking points in a specific part of their bundling signal (belonging to the packaging signal), suggesting its dispensability for DI vRNA propagation. Over a total cultivation time of 21 days, several individual DI vRNAs accumulated to high fractions, while others decreased. Using reverse genetics for IAV, purely clonal DIPs derived from highly replicating DI vRNAs were generated. We confirm that these DIPs exhibit a superior interfering efficacy compared to DIPs derived from lowly accumulated DI vRNAs and suggest promising candidates for efficacious antiviral treatment. Defective interfering particles (DIPs) emerge naturally during viral infection and typically show an internal deletion in the viral genome. Thus, DIPs are propagation-incompetent. Previous research suggests DIPs as potent antiviral compounds for many different virus families due to their ability to interfere with virus replication by competitive inhibition. For instance, the administration of influenza A virus (IAV) DIPs resulted in a rescue of mice from an otherwise lethal IAV dose. Moreover, no apparent toxic effects were observed when only DIPs were administered to mice and ferrets. IAV DIPs show antiviral activity against many different IAV strains, including pandemic and highly pathogenic avian strains, and even against nonhomologous viruses, such as SARS-CoV-2, by stimulation of innate immunity. Here, we used a cultivation/infection system, which exerted selection pressure toward accumulation of highly competitive IAV DIPs. These DIPs showed a superior interfering efficacy , and we suggest them for effective antiviral therapy.
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http://dx.doi.org/10.1128/JVI.01174-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8610589PMC
November 2021

Cell culture-based production of defective interfering influenza A virus particles in perfusion mode using an alternating tangential flow filtration system.

Appl Microbiol Biotechnol 2021 Oct 14;105(19):7251-7264. Epub 2021 Sep 14.

Chair of Bioprocess Engineering, Otto-Von-Guericke-University Magdeburg, Magdeburg, Germany.

Respiratory diseases including influenza A virus (IAV) infections represent a major threat to human health. While the development of a vaccine requires a lot of time, a fast countermeasure could be the use of defective interfering particles (DIPs) for antiviral therapy. IAV DIPs are usually characterized by a large internal deletion in one viral RNA segment. Consequentially, DIPs can only propagate in presence of infectious standard viruses (STVs), compensating the missing gene function. Here, they interfere with and suppress the STV replication and might act "universally" against many IAV subtypes. We recently reported a production system for purely clonal DIPs utilizing genetically modified cells. In the present study, we established an automated perfusion process for production of a DIP, called DI244, using an alternating tangential flow filtration (ATF) system for cell retention. Viable cell concentrations and DIP titers more than 10 times higher than for a previously reported batch cultivation were observed. Furthermore, we investigated a novel tubular cell retention device for its potential for continuous virus harvesting into the permeate. Very comparable performances to typically used hollow fiber membranes were found during the cell growth phase. During the virus replication phase, the tubular membrane, in contrast to the hollow fiber membrane, allowed 100% of the produced virus particles to pass through. To our knowledge, this is the first time a continuous virus harvest was shown for a membrane-based perfusion process. Overall, the process established offers interesting possibilities for advanced process integration strategies for next-generation virus particle and virus vector manufacturing.Key points• An automated perfusion process for production of IAV DIPs was established.• DIP titers of 7.40E + 9 plaque forming units per mL were reached.• A novel tubular cell retention device enabled continuous virus harvesting.
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http://dx.doi.org/10.1007/s00253-021-11561-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8437742PMC
October 2021

A high cell density perfusion process for Modified Vaccinia virus Ankara production: Process integration with inline DNA digestion and cost analysis.

Biotechnol Bioeng 2021 12 23;118(12):4720-4734. Epub 2021 Sep 23.

Bioprocess Engineering Group, Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany.

By integrating continuous cell cultures with continuous purification methods, process yields and product quality attributes have been improved over the last 10 years for recombinant protein production. However, for the production of viral vectors such as Modified Vaccinia virus Ankara (MVA), no such studies have been reported although there is an increasing need to meet the requirements for a rising number of clinical trials against infectious or neoplastic diseases. Here, we present for the first time a scalable suspension cell (AGE1.CR.pIX cells) culture-based perfusion process in bioreactors integrating continuous virus harvesting through an acoustic settler with semi-continuous chromatographic purification. This allowed obtaining purified MVA particles with a space-time yield more than 600% higher for the integrated perfusion process (1.05 × 10 TCID /L /day) compared to the integrated batch process. Without further optimization, purification by membrane-based steric exclusion chromatography resulted in an overall product recovery of 50.5%. To decrease the level of host cell DNA before chromatography, a novel inline continuous DNA digestion step was integrated into the process train. A detailed cost analysis comparing integrated production in batch versus production in perfusion mode showed that the cost per dose for MVA was reduced by nearly one-third using this intensified small-scale process.
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http://dx.doi.org/10.1002/bit.27937DOI Listing
December 2021

Towards integrated production of an influenza A vaccine candidate with MDCK suspension cells.

Biotechnol Bioeng 2021 10 20;118(10):3996-4013. Epub 2021 Jul 20.

Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany.

Seasonal influenza epidemics occur both in northern and southern hemispheres every year. Despite the differences in influenza virus surface antigens and virulence of seasonal subtypes, manufacturers are well-adapted to respond to this periodical vaccine demand. Due to decades of influenza virus research, the development of new influenza vaccines is relatively straight forward. In similarity with the ongoing coronavirus disease 2019 pandemic, vaccine manufacturing is a major bottleneck for a rapid supply of the billions of doses required worldwide. In particular, egg-based vaccine production would be difficult to schedule and shortages of other egg-based vaccines with high demands also have to be anticipated. Cell culture-based production systems enable the manufacturing of large amounts of vaccines within a short time frame and expand significantly our options to respond to pandemics and emerging viral diseases. In this study, we present an integrated process for the production of inactivated influenza A virus vaccines based on a Madin-Darby Canine Kidney (MDCK) suspension cell line cultivated in a chemically defined medium. Very high titers of 3.6 log (HAU/100 µl) were achieved using fast-growing MDCK cells at concentrations up to 9.5 × 10 cells/ml infected with influenza A/PR/8/34 H1N1 virus in 1 L stirred tank bioreactors. A combination of membrane-based steric-exclusion chromatography followed by pseudo-affinity chromatography with a sulfated cellulose membrane adsorber enabled full recovery for the virus capture step and up to 80% recovery for the virus polishing step. Purified virus particles showed a homogenous size distribution with a mean diameter of 80 nm. Based on a monovalent dose of 15 µg hemagglutinin (single-radial immunodiffusion assay), the level of total protein and host cell DNA was 58 µg and 10 ng, respectively. Furthermore, all process steps can be fully scaled up to industrial quantities for commercial manufacturing of either seasonal or pandemic influenza virus vaccines. Fast production of up to 300 vaccine doses per liter within 4-5 days makes this process competitive not only to other cell-based processes but to egg-based processes as well.
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http://dx.doi.org/10.1002/bit.27876DOI Listing
October 2021

SARS-CoV-2 Production in a Scalable High Cell Density Bioreactor.

Vaccines (Basel) 2021 Jun 29;9(7). Epub 2021 Jun 29.

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital-Hvidovre, 2650 Hvidovre, Denmark.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has demonstrated the value of pursuing different vaccine strategies. Vaccines based on whole viruses, a widely used vaccine technology, depend on efficient virus production. This study aimed to establish SARS-CoV-2 production in the scalable packed-bed CelCradle 500-AP bioreactor. CelCradle 500-AP bottles with 0.5 L working volume and 5.5 g BioNOC™ II carriers were seeded with 1.5 × 10 Vero (WHO) cells, approved for vaccine production, in animal component-free medium and infected at a multiplicity of infection of 0.006 at a total cell number of 2.2-2.5 × 10 cells/bottle seven days post cell seeding. Among several tested conditions, two harvests per day and a virus production temperature of 33 °C resulted in the highest virus yield with a peak SARS-CoV-2 infectivity titer of 7.3 log 50% tissue culture infectious dose (TCID)/mL at 72 h post-infection. Six harvests had titers of ≥6.5 log TCID/mL, and a total of 10.5 log TCID were produced in ~5 L. While trypsin was reported to enhance virus spread in cell culture, addition of 0.5% recombinant trypsin after infection did not improve virus yields. Overall, we demonstrated successful animal component-free production of SARS-CoV-2 in well-characterized Vero (WHO) cells in a scalable packed-bed bioreactor.
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http://dx.doi.org/10.3390/vaccines9070706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8310283PMC
June 2021

Cell culture-based production and in vivo characterization of purely clonal defective interfering influenza virus particles.

BMC Biol 2021 05 3;19(1):91. Epub 2021 May 3.

Otto-von-Guericke-University Magdeburg, Chair of Bioprocess Engineering, Magdeburg, Germany.

Background: Infections with influenza A virus (IAV) cause high morbidity and mortality in humans. Additional to vaccination, antiviral drugs are a treatment option. Besides FDA-approved drugs such as oseltamivir or zanamivir, virus-derived defective interfering (DI) particles (DIPs) are considered promising new agents. IAV DIPs typically contain a large internal deletion in one of their eight genomic viral RNA (vRNA) segments. Consequently, DIPs miss the genetic information necessary for replication and can usually only propagate by co-infection with infectious standard virus (STV), compensating for their defect. In such a co-infection scenario, DIPs interfere with and suppress STV replication, which constitutes their antiviral potential.

Results: In the present study, we generated a genetically engineered MDCK suspension cell line for production of a purely clonal DIP preparation that has a large deletion in its segment 1 (DI244) and is not contaminated with infectious STV as egg-derived material. First, the impact of the multiplicity of DIP (MODIP) per cell on DI244 yield was investigated in batch cultivations in shake flasks. Here, the highest interfering efficacy was observed for material produced at a MODIP of 1E-2 using an in vitro interference assay. Results of RT-PCR suggested that DI244 material produced was hardly contaminated with other defective particles. Next, the process was successfully transferred to a stirred tank bioreactor (500 mL working volume) with a yield of 6.0E+8 PFU/mL determined in genetically modified adherent MDCK cells. The produced material was purified and concentrated about 40-fold by membrane-based steric exclusion chromatography (SXC). The DI244 yield was 92.3% with a host cell DNA clearance of 97.1% (99.95% with nuclease digestion prior to SXC) and a total protein reduction of 97.2%. Finally, the DIP material was tested in animal experiments in D2(B6).A2G-Mx1 mice. Mice infected with a lethal dose of IAV and treated with DIP material showed a reduced body weight loss and all animals survived.

Conclusion: In summary, experiments not only demonstrated that purely clonal influenza virus DIP preparations can be obtained with high titers from animal cell cultures but confirmed the potential of cell culture-derived DIPs as an antiviral agent.
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http://dx.doi.org/10.1186/s12915-021-01020-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8091782PMC
May 2021

Site-specific N-glycosylation analysis of animal cell culture-derived Zika virus proteins.

Sci Rep 2021 03 4;11(1):5147. Epub 2021 Mar 4.

Bioprocess Engineering Group, Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany.

Here, we present for the first time, a site-specific N-glycosylation analysis of proteins from a Brazilian Zika virus (ZIKV) strain. The virus was propagated with high yield in an embryo-derived stem cell line (EB66, Valneva SE), and concentrated by g-force step-gradient centrifugation. Subsequently, the sample was proteolytically digested with different enzymes, measured via a LC-MS/MS-based workflow, and analyzed in a semi-automated way using the in-house developed glyXtool software. The viral non-structural protein 1 (NS1) was glycosylated exclusively with high-mannose structures on both potential N-glycosylation sites. In case of the viral envelope (E) protein, no specific N-glycans could be identified with this method. Nevertheless, N-glycosylation could be proved by enzymatic de-N-glycosylation with PNGase F, resulting in a strong MS-signal of the former glycopeptide with deamidated asparagine at the potential N-glycosylation site N444. This confirmed that this site of the ZIKV E protein is highly N-glycosylated but with very high micro-heterogeneity. Our study clearly demonstrates the progress made towards site-specific N-glycosylation analysis of viral proteins, i.e. for Brazilian ZIKV. It allows to better characterize viral isolates, and to monitor glycosylation of major antigens. The method established can be applied for detailed studies regarding the impact of protein glycosylation on antigenicity and human pathogenicity of many viruses including influenza virus, HIV and corona virus.
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http://dx.doi.org/10.1038/s41598-021-84682-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7933209PMC
March 2021

Comprehensive N-glycosylation analysis of the influenza A virus proteins HA and NA from adherent and suspension MDCK cells.

FEBS J 2021 08 15;288(16):4869-4891. Epub 2021 Mar 15.

Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany.

Glycosylation is considered as a critical quality attribute for the production of recombinant biopharmaceuticals such as hormones, blood clotting factors, or monoclonal antibodies. In contrast, glycan patterns of immunogenic viral proteins, which differ significantly between the various expression systems, are hardly analyzed yet. The influenza A virus (IAV) proteins hemagglutinin (HA) and neuraminidase (NA) have multiple N-glycosylation sites, and alteration of N-glycan micro- and macroheterogeneity can have strong effects on virulence and immunogenicity. Here, we present a versatile and powerful glycoanalytical workflow that enables a comprehensive N-glycosylation analysis of IAV glycoproteins. We challenged our workflow with IAV (A/PR/8/34 H1N1) propagated in two closely related Madin-Darby canine kidney (MDCK) cell lines, namely an adherent MDCK cell line and its corresponding suspension cell line. As expected, N-glycan patterns of HA and NA from virus particles produced in both MDCK cell lines were similar. Detailed analysis of the HA N-glycan microheterogeneity showed an increasing variability and a higher complexity for N-glycosylation sites located closer to the head region of the molecule. In contrast, NA was found to be exclusively N-glycosylated at site N73. Almost all N-glycan structures were fucosylated. Furthermore, HA and NA N-glycan structures were exclusively hybrid- and complex-type structures, to some extent terminated with alpha-linked galactose(s) but also with blood group H type 2 and blood group A epitopes. In contrast to the similarity of the overall glycan pattern, differences in the relative abundance of individual structures were identified. This concerned, in particular, oligomannose-type, alpha-linked galactose, and multiantennary complex-type N-glycans.
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http://dx.doi.org/10.1111/febs.15787DOI Listing
August 2021

Tracking changes in adaptation to suspension growth for MDCK cells: cell growth correlates with levels of metabolites, enzymes and proteins.

Appl Microbiol Biotechnol 2021 Mar 13;105(5):1861-1874. Epub 2021 Feb 13.

Bioprocess Engineering, Otto von Guericke University Magdeburg, Magdeburg, Germany.

Adaptations of animal cells to growth in suspension culture concern in particular viral vaccine production, where very specific aspects of virus-host cell interaction need to be taken into account to achieve high cell specific yields and overall process productivity. So far, the complexity of alterations on the metabolism, enzyme, and proteome level required for adaptation is only poorly understood. In this study, for the first time, we combined several complex analytical approaches with the aim to track cellular changes on different levels and to unravel interconnections and correlations. Therefore, a Madin-Darby canine kidney (MDCK) suspension cell line, adapted earlier to growth in suspension, was cultivated in a 1-L bioreactor. Cell concentrations and cell volumes, extracellular metabolite concentrations, and intracellular enzyme activities were determined. The experimental data set was used as the input for a segregated growth model that was already applied to describe the growth dynamics of the parental adherent cell line. In addition, the cellular proteome was analyzed by liquid chromatography coupled to tandem mass spectrometry using a label-free protein quantification method to unravel altered cellular processes for the suspension and the adherent cell line. Four regulatory mechanisms were identified as a response of the adaptation of adherent MDCK cells to growth in suspension. These regulatory mechanisms were linked to the proteins caveolin, cadherin-1, and pirin. Combining cell, metabolite, enzyme, and protein measurements with mathematical modeling generated a more holistic view on cellular processes involved in the adaptation of an adherent cell line to suspension growth. KEY POINTS: • Less and more efficient glucose utilization for suspension cell growth • Concerted alteration of metabolic enzyme activity and protein expression • Protein candidates to interfere glycolytic activity in MDCK cells.
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http://dx.doi.org/10.1007/s00253-021-11150-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7907048PMC
March 2021

High cell density perfusion process for high yield of influenza A virus production using MDCK suspension cells.

Appl Microbiol Biotechnol 2021 Feb 30;105(4):1421-1434. Epub 2021 Jan 30.

State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China.

Similar to the recent COVID-19 pandemic, influenza A virus poses a constant threat to the global community. For the treatment of flu disease, both antivirals and vaccines are available with vaccines the most effective and safest approach. In order to overcome limitations in egg-based vaccine manufacturing, cell culture-based processes have been established. While this production method avoids egg-associated risks in face of pandemics, process intensification using animal suspension cells in high cell density perfusion cultures should allow to further increase manufacturing capacities worldwide. In this work, we demonstrate the development of a perfusion process using Madin-Darby canine kidney (MDCK) suspension cells for influenza A (H1N1) virus production from scale-down shake flask cultivations to laboratory scale stirred tank bioreactors. Shake flask cultivations using semi-perfusion mode enabled high-yield virus harvests (4.25 log(HAU/100 μL)) from MDCK cells grown up to 41 × 10 cells/mL. Scale-up to bioreactors with an alternating tangential flow (ATF) perfusion system required optimization of pH control and implementation of a temperature shift during the infection phase. Use of a capacitance probe for on-line perfusion control allowed to minimize medium consumption. This contributed to a better process control and a more economical performance while maintaining a maximum virus titer of 4.37 log(HAU/100 μL) and an infectious virus titer of 1.83 × 10 virions/mL. Overall, this study clearly demonstrates recent advances in cell culture-based perfusion processes for next-generation high-yield influenza vaccine manufacturing for pandemic preparedness. KEY POINTS: • First MDCK suspension cell-based perfusion process for IAV produciton was established. • "Cell density effect" was overcome and process was intensified by reduction of medium use and automated process control. • The process achieved cell density over 40 × 10 cells/mL and virus yield over 4.37 log(HAU/100 μL).
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http://dx.doi.org/10.1007/s00253-020-11050-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7847233PMC
February 2021

OP7, a novel influenza A virus defective interfering particle: production, purification, and animal experiments demonstrating antiviral potential.

Appl Microbiol Biotechnol 2021 Jan 4;105(1):129-146. Epub 2020 Dec 4.

Bioprocess Engineering, Otto von Guericke University Magdeburg, Magdeburg, Germany.

The novel influenza A virus (IAV) defective interfering particle "OP7" inhibits IAV replication in a co-infection and was previously suggested as a promising antiviral agent. Here, we report a batch-mode cell culture-based production process for OP7. In the present study, a seed virus containing standard virus (STV) and OP7 was used. The yield of OP7 strongly depended on the production multiplicity of infection. To inactivate infectious STV in the OP7 material, which may cause harm in a potential application, UV irradiation was used. The efficacy of OP7 in this material was preserved, as shown by an in vitro interference assay. Next, steric exclusion chromatography was used to purify and to concentrate (~ 13-fold) the UV-treated material. Finally, administration of produced OP7 material in mice did not show any toxic effects. Furthermore, all mice infected with a lethal dose of IAV survived the infection upon OP7 co-treatment. Thus, the feasibility of a production workflow for OP7 and its potential for antiviral treatment was demonstrated. KEY POINTS: • OP7 efficacy strongly depended on the multiplicity of infection used for production • Purification by steric exclusion chromatography increased OP7 efficacy • OP7-treated mice were protected against a lethal infection with IAV.
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http://dx.doi.org/10.1007/s00253-020-11029-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7778630PMC
January 2021

Cell Bank Origin of MDCK Parental Cells Shapes Adaptation to Serum-Free Suspension Culture and Canine Adenoviral Vector Production.

Int J Mol Sci 2020 Aug 25;21(17). Epub 2020 Aug 25.

iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal.

Phenotypic variation in cultured mammalian cell lines is known to be induced by passaging and culture conditions. Yet, the effect these variations have on the production of viral vectors has been overlooked. In this work we evaluated the impact of using Madin-Darby canine kidney (MDCK) parental cells from American Type Culture Collection (ATCC) or European Collection of Authenticated Cell Cultures (ECACC) cell bank repositories in both adherent and suspension cultures for the production of canine adenoviral vectors type 2 (CAV-2). To further explore the differences between cells, we conducted whole-genome transcriptome analysis. ECACC's MDCK showed to be a less heterogeneous population, more difficult to adapt to suspension and serum-free culture conditions, but more permissive to CAV-2 replication progression, enabling higher yields. Transcriptome data indicated that this increased permissiveness is due to a general down-regulation of biological networks of innate immunity in ECACC cells, including apoptosis and death receptor signaling, Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling, toll-like receptors signaling and the canonical pathway of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling. These results show the impact of MDCK source on the outcome of viral-based production processes further elucidating transcriptome signatures underlying enhanced adenoviral replication. Following functional validation, the genes and networks identified herein can be targeted in future engineering approaches aiming at improving the production of CAV-2 gene therapy vectors.
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http://dx.doi.org/10.3390/ijms21176111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7504089PMC
August 2020

Production of Modified Vaccinia Ankara Virus by Intensified Cell Cultures: A Comparison of Platform Technologies for Viral Vector Production.

Biotechnol J 2021 Jan 8;16(1):e2000024. Epub 2020 Sep 8.

Max Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering, Sandtorstr. 1, Magdeburg, 39106, Germany.

Modified Vaccinia Ankara (MVA) virus is a promising vector for vaccination against various challenging pathogens or the treatment of some types of cancers, requiring a high amount of virions per dose for vaccination and gene therapy. Upstream process intensification combining perfusion technologies, the avian suspension cell line AGE1.CR.pIX and the virus strain MVA-CR19 is an option to obtain very high MVA yields. Here the authors compare different options for cell retention in perfusion mode using conventional stirred-tank bioreactors. Furthermore, the authors study hollow-fiber bioreactors and an orbital-shaken bioreactor in perfusion mode, both available for single-use. Productivity for the virus strain MVA-CR19 is compared to results from batch and continuous production reported in literature. The results demonstrate that cell retention devices are only required to maximize cell concentration but not for continuous harvesting. Using a stirred-tank bioreactor, a perfusion strategy with working volume expansion after virus infection results in the highest yields. Overall, infectious MVA virus titers of 2.1-16.5 × 10  virions/mL are achieved in these intensified processes. Taken together, the study shows a novel perspective on high-yield MVA virus production in conventional bioreactor systems linked to various cell retention devices and addresses options for process intensification including fully single-use perfusion platforms.
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http://dx.doi.org/10.1002/biot.202000024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7435511PMC
January 2021

Application of an Inclined Settler for Cell Culture-Based Influenza A Virus Production in Perfusion Mode.

Front Bioeng Biotechnol 2020 2;8:672. Epub 2020 Jul 2.

Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany.

Influenza viruses have been successfully propagated using a variety of animal cell lines in batch, fed-batch, and perfusion culture. For suspension cells, most studies reported on membrane-based cell retention devices typically leading to an accumulation of viruses in the bioreactor in perfusion mode. Aiming at continuous virus harvesting for improved productivities, an inclined settler was evaluated for influenza A virus (IAV) production using the avian suspension cell line AGE1.CR.pIX. Inclined settlers present many advantages as they are scalable, robust, and comply with cGMP regulations, e.g., for recombinant protein manufacturing. Perfusion rates up to 3000 L/day have been reported. In our study, successful growth of AGE1.CR.pIX cells up to 50 × 10 cells/mL and a cell retention efficiency exceeding 96% were obtained with the settler cooled to room temperature. No virus retention was observed. A total of 5.4-6.5 × 10 virions were produced while a control experiment with an ATF system equaled to 1.9 × 10 virions. For infection at 25 × 10 cells/mL, cell-specific virus yields up to 3474 virions/cell were obtained, about 5-fold higher than for an ATF based cultivation performed as a control (723 virions/cell). Trypsin activity was shown to have a large impact on cell growth dynamics after infection following the cell retention device, especially at a cell concentration of 50 × 10 cells/mL. Further control experiments performed with an acoustic settler showed that virus production was improved with a heat exchanger of the inclined settler operated at 27°C. In summary, cell culture-based production of viruses in perfusion mode with an inclined settler and continuous harvesting can drastically increase IAV yields and possibly the yield of other viruses. To our knowledge, this is the first report to show the potential of this device for viral vaccine production.
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http://dx.doi.org/10.3389/fbioe.2020.00672DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7343718PMC
July 2020

Virus harvesting in perfusion culture: Choosing the right type of hollow fiber membrane.

Biotechnol Bioeng 2020 10 2;117(10):3040-3052. Epub 2020 Jul 2.

Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany.

The use of bioreactors coupled to membrane-based perfusion systems enables very high cell and product concentrations in vaccine and viral vector manufacturing. Many virus particles, however, are not stable and either lose their infectivity or physically degrade resulting in significant product losses if not harvested continuously. Even hollow fiber membranes with a nominal pore size of 0.2 µm can retain much smaller virions within a bioreactor. Here, we report on a systematic study to characterize structural and physicochemical membrane properties with respect to filter fouling and harvesting of yellow fever virus (YFV; ~50 nm). In tangential flow filtration perfusion experiments, we observed that YFV retention was only marginally determined by nominal but by effective pore sizes depending on filter fouling. Evaluation of scanning electron microscope images indicated that filter fouling can be reduced significantly by choosing membranes with (i) a flat inner surface (low boundary layer thickness), (ii) a smooth material structure (reduced deposition), (iii) a high porosity (high transmembrane flux), (iv) a distinct pore size distribution (well-defined pore selectivity), and (v) an increased fiber wall thickness (larger effective surface area). Lowest filter fouling was observed with polysulfone (PS) membranes. While the use of a small-pore PS membrane (0.08 µm) allowed to fully retain YFV within the bioreactor, continuous product harvesting was achieved with the large-pore PS membrane (0.34 µm). Due to the low protein rejection of the latter, this membrane type could also be of interest for other applications, that is, recombinant protein production in perfusion cultures.
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http://dx.doi.org/10.1002/bit.27470DOI Listing
October 2020

Performance of an acoustic settler versus a hollow fiber-based ATF technology for influenza virus production in perfusion.

Appl Microbiol Biotechnol 2020 Jun 15;104(11):4877-4888. Epub 2020 Apr 15.

Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstr. 1, 39106, Magdeburg, Germany.

Process intensification and integration is crucial regarding an ever increasing pressure on manufacturing costs and capacities in biologics manufacturing. For virus production in perfusion mode, membrane-based alternating tangential flow filtration (ATF) and acoustic settler are the commonly described cell retention technologies. While acoustic settlers allow for continuous influenza virus harvesting, the use of commercially available membranes for ATF systems typically results in the accumulation of virus particles in the bioreactor vessel. Accordingly, with one single harvest at the end of a cultivation, this increases the risk of lowering the product quality. To assess which cell retention device would be most suitable for influenza A virus production, we compared various key performance figures using AGE1.CR.pIX cells at concentrations between 25 and 50 × 10 cells/mL at similar infection conditions using either an ATF system or an acoustic settler. Production yields, process-related impurities, and aggregation of viruses and other large molecules were evaluated. Taking into account the total number of virions from both the bioreactor and the harvest vessel, a 1.5-3.0-fold higher volumetric virus yield was obtained for the acoustic settler. In addition, fewer large-sized aggregates (virus particles and other molecules) were observed in the harvest taken directly from the bioreactor. In contrast, similar levels of process-related impurities (host cell dsDNA, total protein) were obtained in the harvest for both retention systems. Overall, a clear advantage was observed for continuous virus harvesting after the acoustic settler operation mode was optimized. This development may also allow direct integration of subsequent downstream processing steps. KEY POINTS: • High suspension cell density, immortalized avian cell line, influenza vaccine.
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http://dx.doi.org/10.1007/s00253-020-10596-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7228903PMC
June 2020

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

Biotechnol Bioeng 2020 05 15;117(5):1533-1553. Epub 2020 Feb 15.

Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany.

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.
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http://dx.doi.org/10.1002/bit.27288DOI Listing
May 2020

Perfusion Control for High Cell Density Cultivation and Viral Vaccine Production.

Methods Mol Biol 2020 ;2095:141-168

Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany.

The global demand for complex biopharmaceuticals like recombinant proteins, vaccines, or viral vectors is steadily rising. To further improve process productivity and to reduce production costs, process intensification can contribute significantly. The design and optimization of perfusion processes toward very high cell densities require careful selection of strategies for optimal perfusion rate control. In this chapter, various options are discussed to guarantee high cell-specific virus yields and to achieve virus concentrations up to 10 virions/mL. This includes reactor volume exchange regimes and perfusion rate control based on process variables such as cell concentration and metabolite or by-product concentration. Strategies to achieve high cell densities by perfusion rate control and their experimental implementation are described in detail for pseudo-perfusion or small-scale perfusion bioreactor systems. Suspension cell lines such as MDCK, BHK-21, EB66, and AGE1.CR.pIX are used to exemplify production of influenza, yellow fever, Zika, and modified vaccinia Ankara virus.
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http://dx.doi.org/10.1007/978-1-0716-0191-4_9DOI Listing
February 2021

Orbitally Shaken Single-Use Bioreactor for Animal Cell Cultivation: Fed-Batch and Perfusion Mode.

Methods Mol Biol 2020 ;2095:105-123

Adolf Kühner AG, Birsfelden, Switzerland.

Increasing the cultivation volume from small to large scale can be a rather complex and challenging process when the method of aeration and mixing is different between scales. Orbitally shaken bioreactors (OSBs) utilize the same hydrodynamic principles that define the success of smaller-scale cultures, which are developed on an orbitally shaken platform, and can simplify scale-up. Here we describe the basic working principles of scale-up in terms of the volumetric oxygen transfer coefficient (ka) and mixing time and how to define these parameters experimentally. The scale-up process from an Erlenmeyer flask shaken on an orbital platform to an orbitally shaken single-use bioreactor (SB10-X, 12 L) is described in terms of both fed-batch and perfusion-based processes. The fed-batch process utilizes a recombinant variant of the mammalian cell line, Chinese hamster ovary (CHO), to express a biosimilar of a therapeutic monoclonal antibody. The perfusion-based process utilizes either an alternating tangential flow filtration (ATF) or a tangential flow filtration (TFF) system for cell retention to cultivate an avian cell line, AGE1.CR.pIX, for the propagation of influenza A virus, H1N1, in high cell density. Based on two example cell cultivations, processes outline the advantages that come with using an orbitally shaken bioreactor for scaling-up a process. The described methods are also applicable to other suspension cell lines.
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http://dx.doi.org/10.1007/978-1-0716-0191-4_7DOI Listing
February 2021

Continuous influenza virus production in a tubular bioreactor system provides stable titers and avoids the "von Magnus effect".

PLoS One 2019 5;14(11):e0224317. Epub 2019 Nov 5.

Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany.

Continuous cell culture-based influenza vaccine production could significantly reduce footprint and manufacturing costs compared to current batch processing. However, yields of influenza virus in continuous mode can be affected by oscillations in virus titers caused by periodic accumulation of defective interfering particles. The generation of such particles has also been observed previously in cascades of continuous stirred tank reactors (CSTRs) and is known as the "von Magnus effect". To improve virus yields and to avoid these oscillations, we have developed a novel continuous tubular bioreactor system for influenza A virus production. It was built using a 500 mL CSTR for cell growth linked to a 105 m long tubular plug-flow bioreactor (PFBR). Virus propagation took place only in the PFBR with a nominal residence time of 20 h and a production capacity of 0.2 mL/min. The bioreactor was first tested with suspension MDCK cells at different multiplicities of infection (MOI), and then with suspension avian AGE1.CR.pIX cells at a fixed nominal MOI of 0.02. Maximum hemagglutinin (HA) titers of 2.4 and 1.6 log10(HA units/100 μL) for suspension MDCK cells and AGE1.CR.pIX cells, respectively, were obtained. Flow cytometric analysis demonstrated that 100% infected cells with batch-like HA titers can be obtained at a MOI of at least 0.1. Stable HA and TCID50 titers over 18 days of production were confirmed using the AGE1.CR.pIX cell line, and PCR analysis demonstrated stable production of full-length genome. The contamination level of segments with deletions (potentially defective interfering particles), already present in the virus seed, was low and did not increase. Control experiments using batch and semi-continuous cultures confirmed these findings. A comparison showed that influenza virus production can be achieved with the tubular bioreactor system in about half the time with a space-time-yield up to two times higher than for typical batch cultures. In summary, a novel continuous tubular bioreactor system for cell culture-based influenza virus production was developed. One main advantage, an essentially single-passage amplification of viruses, should enable efficient production of vaccines as well as vectors for gene and cancer therapy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0224317PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6830746PMC
April 2020

Production of Defective Interfering Particles of Influenza A Virus in Parallel Continuous Cultures at Two Residence Times-Insights From qPCR Measurements and Viral Dynamics Modeling.

Front Bioeng Biotechnol 2019 18;7:275. Epub 2019 Oct 18.

Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany.

Defective interfering particles (DIPs) are a natural byproduct of influenza A virus (IAV) replication. DIPs interfere with the propagation and spread of infectious standard virus (STV), reduce virus yields by competing for viral and cellular resources, and induce antiviral responses. These properties open exciting possibilities for the development of DIP-based antivirals. Exploring options for cell culture-based DIP production, we have established a fully continuous cultivation process, where one bioreactor is used to grow cells that are fed to two bioreactors operated in parallel for virus production. This system allows head-to-head comparisons of STV and DIP replication dynamics over extended time periods. Cultivations were performed at two residence times (RT, 22 and 36 h) using MDCK suspension cells grown in a fully defined medium. For infection, we used a virus seed generated by reverse genetics containing STVs and a known DIP carrying a deletion in segment 1 (delS1(1)). Four days post infection, DIPs achieved maximum concentrations of 7.0·10 virions/mL and 8.4·10 virions/mL for RTs of 22 and 36 h, respectively. Furthermore, oscillations in virus titers with two to three maxima were found for DIP accumulation at 36 and 22 h RT, respectively. To complement the study, a basic mathematical model using simple kinetics and a reasonable number of parameters to describe DIP-propagation in continuous cultures was established. Upon fitting the model individually to each of the two data sets, oscillations in the viral dynamics and the cell population dynamics were described well. Modeling suggests that both STV inactivation and virus degradation have to be taken into account to achieve good agreement of simulations and experimental data for longer RTs. Together, the high DIP titers obtained, and the successful simulation of the experimental data showed that the combination of continuous bioreactors and mathematical models can enable studies regarding DIP dynamics over extended time periods and allow large scale manufacturing of DIP-based antivirals.
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http://dx.doi.org/10.3389/fbioe.2019.00275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6813217PMC
October 2019

Influenza A virus production in a single-use orbital shaken bioreactor with ATF or TFF perfusion systems.

Vaccine 2019 11 29;37(47):7011-7018. Epub 2019 Jun 29.

Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstr. 1, 39106 Magdeburg, Germany.

Driven by the concept of plug-and-play cell culture-based viral vaccine production using disposable bioreactors, we evaluated an orbital shaken bioreactor (OSB) for human influenza A virus production at high cell concentration. Therefore, the OSB model SB10-X was coupled to two hollow fiber-based perfusion systems, namely, tangential flow filtration (TFF) and alternating tangential flow filtration (ATF). The AGE1.CR.pIX avian suspension cells grew to 50 × 10 cells/mL in chemically defined medium, maintaining high cell viabilities with an average specific growth rate of 0.020 h (doubling time = 32 h). Maximum virus titers in the range of 3.28-3.73 log(HA units/100 µL) were achieved, corresponding to cell-specific virus yields of 1000-3500 virions/cell and productivities of 0.5-2.2 × 10 virions/L/d. This clearly demonstrates the potential of OSB operation in perfusion mode, as results achieved in a reference OSB batch cultivation were 2.64 log(HA units/100 µL), 1286 virions/cell and 1.4 × 10 virions/L/d, respectively. In summary, the SB10-X bioreactor can be operated with ATF and TFF systems, which is to our knowledge the first report regarding OSB operation in perfusion mode. Moreover, the results showed that the system is a promising cultivation system for influenza A virus vaccine production. The OSB disposable bioreactor has the potential for simplifying the scale-up from shake flasks to the large-scale bioreactor, facilitating rapid responses in the event of epidemics or pandemics.
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http://dx.doi.org/10.1016/j.vaccine.2019.06.005DOI Listing
November 2019

Semi-perfusion cultures of suspension MDCK cells enable high cell concentrations and efficient influenza A virus production.

Vaccine 2019 11 29;37(47):7003-7010. Epub 2019 Apr 29.

Bioprocess Engineering Group, Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstrasse 1, 39106 Magdeburg, Germany; Chair of Bioprocess Engineering, Otto-von-Guericke University Magdeburg, Universitaetsplatz 2, 39106 Magdeburg, Germany.

Control and prevention of rapid influenza spread among humans depend on the availability of efficient and safe seasonal and pandemic vaccines, made primarily from inactivated influenza virus particles. Current influenza virus production processes rely heavily on embryonated chicken eggs or on cell culture as substrate for virus propagation. Today's efforts towards process intensification in animal cell culture could innovate viral vaccine manufacturing using high-yield suspension cells in high cell density perfusion processes. In this work, we present a MDCK cell line adapted to grow as single cell suspension with a doubling time of less than 20 h, achieving cell concentrations over 1 × 10 cells/mL in batch mode. Influenza A virus titer obtained in batch infections were 3.6 log(HAU/100 µL) for total- and 10 virions/mL for infectious virus particles (TCID), respectively. In semi-perfusion mode concentrations up to 6 × 10 cells/mL, accumulated virus titer of 4.5 log(HAU/100 µL) and infectious titer of almost 10 virions/mL (TCID) were possible. This exceeds results reported previously for cell culture-based influenza virus propagation by far and suggests perfusion cultures as the preferred method in viral vaccine manufacturing.
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http://dx.doi.org/10.1016/j.vaccine.2019.04.054DOI Listing
November 2019

Efficient influenza A virus production in high cell density using the novel porcine suspension cell line PBG.PK2.1.

Vaccine 2019 11 17;37(47):7019-7028. Epub 2019 Apr 17.

Max Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering, Sandtorstr. 1, 39106 Magdeburg, Germany; Chair for Bioprocess Engineering, Otto-von-Guericke-University Magdeburg, Universitätsplatz 2, 39106 Magdeburg, Germany.

Seasonal and pandemic influenza respiratory infections are still a major public health issue. Vaccination is the most efficient way to prevent influenza infection. One option to produce influenza vaccines is cell-culture based virus propagation. Different host cell lines, such as MDCK, Vero, AGE1.CR or PER.C6 cells have been shown to be a good substrate for influenza virus production. With respect to the ease of scale-up, suspension cells should be preferred over adherent cells. Ideally, they should replicate different influenza virus strains with high cell-specific yields. Evaluation of new cell lines and further development of processes is of considerable interest, as this increases the number of options regarding the design of manufacturing processes, flexibility of vaccine production and efficiency. Here, PBG.PK2.1, a new mammalian cell line that was developed by ProBioGen AG (Germany) for virus production is presented. The cells derived from immortal porcine kidney cells were previously adapted to growth in suspension in a chemically-defined medium. Influenza virus production was improved after virus adaptation to PBG.PK2.1 cells and optimization of infection conditions, namely multiplicity of infection and trypsin concentration. Hemagglutinin titers up to 3.24 log(HA units/100 µL) were obtained in fed-batch mode in bioreactors (700 mL working volume). Evaluation of virus propagation in high cell density culture using a hollow-fiber based system (ATF2) demonstrated promising performance: Cell concentrations of up to 50 × 10 cells/mL with viabilities exceeding 95%, and a maximum HA titer of 3.93 log(HA units/100 µL). Analysis of glycosylation of the viral HA antigen expressed showed clear differences compared to HA produced in MDCK or Vero cell lines. With an average cell-specific productivity of 5000 virions/cell, we believe that PBG.PK2.1 cells are a very promising candidate to be considered for next-generation influenza virus vaccine production.
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http://dx.doi.org/10.1016/j.vaccine.2019.04.030DOI Listing
November 2019

High titer MVA and influenza A virus production using a hybrid fed-batch/perfusion strategy with an ATF system.

Appl Microbiol Biotechnol 2019 Apr 23;103(7):3025-3035. Epub 2019 Feb 23.

Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstr. 1, 39106, Magdeburg, Germany.

A cultivation strategy to increase the productivity of Modified Vaccinia Ankara (MVA) virus in high-cell density processes is presented. Based on an approach developed in shake flask cultures, this strategy was established in benchtop bioreactors, comprising the growth of suspension AGE1.CR.pIX cells to high cell densities in a chemically defined medium before infection with the MVA-CR19 virus strain. First, a perfusion regime was established to optimize the cell growth phase. Second, a fed-batch regime was chosen for the initial infection phase to facilitate virus uptake and cell-to-cell spreading. Afterwards, a switch to perfusion enabled the continuous supply of nutrients for the late stages of virus propagation. With maximum infectious titers of 1.0 × 10 IU/mL, this hybrid fed-batch/perfusion strategy increased product titers by almost one order of magnitude compared to conventional batch cultivations. Finally, this strategy was also applied to the production of influenza A/PR/8/34 (H1N1) virus considered for manufacturing of inactivated vaccines. Using the same culture system, a total number of 3.8 × 10 virions/mL was achieved. Overall, comparable or even higher cell-specific virus yields and volumetric productivities were obtained using the same cultivation systems as for the conventional batch cultivations. In addition, most viral particles were found in the culture supernatant, which can simplify further downstream operations, in particular for MVA viruses. Considering the current availability of well-described perfusion/cell retention technologies, the present strategy may contribute to the development of new approaches for viral vaccine production.
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http://dx.doi.org/10.1007/s00253-019-09694-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6447503PMC
April 2019

Process intensification of EB66® cell cultivations leads to high-yield yellow fever and Zika virus production.

Appl Microbiol Biotechnol 2018 Oct 8;102(20):8725-8737. Epub 2018 Aug 8.

Max Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering, Magdeburg, Sandtorstr. 1, 39106, Magdeburg, Germany.

A live-attenuated, human vaccine against mosquito-borne yellow fever virus has been available since the 1930s. The vaccine provides long-lasting immunity and consistent mass vaccination campaigns counter viral spread. However, traditional egg-based vaccine manufacturing requires about 12 months and vaccine supplies are chronically close to shortages. In particular, for urban outbreaks, vaccine demand can be covered rarely by global stockpiling. Thus, there is an urgent need for an improved vaccine production platform, ideally transferable to other flaviviruses including Zika virus. Here, we present a proof-of-concept study regarding cell culture-based yellow fever virus 17D (YFV) and wild-type Zika virus (ZIKV) production using duck embryo-derived EB66® cells. Based on comprehensive studies in shake flasks, 1-L bioreactor systems were operated with scalable hollow fiber-based tangential flow filtration (TFF) and alternating tangential flow filtration (ATF) perfusion systems for process intensification. EB66® cells grew in chemically defined medium to cell concentrations of 1.6 × 10 cells/mL. Infection studies with EB66®-adapted virus led to maximum YFV titers of 7.3 × 10 PFU/mL, which corresponds to about 10 million vaccine doses for the bioreactor harvest. For ZIKV, titers of 1.0 × 10 PFU/mL were achieved. Processes were automated successfully using a capacitance probe to control perfusion rates based on on-line measured cell concentrations. The use of cryo-bags for direct inoculation of production bioreactors facilitates pre-culture preparation contributing to improved process robustness. In conclusion, this platform is a powerful option for next generation cell culture-based flavivirus vaccine manufacturing.
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http://dx.doi.org/10.1007/s00253-018-9275-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6153634PMC
October 2018

Vaccine Technology VI: Innovative and integrated approaches in vaccine development.

Vaccine 2018 05 11;36(22):3061-3063. Epub 2018 Apr 11.

NextWaveBio, CT, USA. Electronic address:

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http://dx.doi.org/10.1016/j.vaccine.2018.03.077DOI Listing
May 2018
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