Monitoring changes in proteome during stepwise adaptation of a MDCK cell line from adherence to growth in suspension

Kluge, Sabine; Benndorf, Dirk; Genzel, Yvonne; Scharfenberg, K.; Rapp, Erdmann; Reichl, Udo

doi:10.1016/j.vaccine.2015.02.077

Cited by 19 publications

(31 citation statements)

References 59 publications

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“…For DCS, DLS, stalagmometry, and xCGE-LIF the harvested VP were clarified by depth filtration, chemically inactivated by β-propiolactone, and concentrated by tangential flow filtration (TFF). Retentates were 0.1 μm filtered to remove unwanted aggregates and particulate impurities, 0.5 mL aliquoted, frozen at −80°C, and thawed to obtain monodisperse VP samples with approximately 10 5 HAU/mL [19][20][21].…”

Section: Influenza a Virus Particle Production And Sample Preparationmentioning

confidence: 99%

Influence of the production system on the surface properties of influenza A virus particles

Hämmerling

Hennig

Serve

et al. 2017

Engineering in Life Sciences

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In this study, influenza A/Puerto Rico/8/34 H1N1 virus particles (VP) produced in adherent and suspension Madin Darby canine kidney cells were investigated with a broad analytical toolbox to obtain more information on the VP's surface properties potentially affecting their aggregation behavior. First, differences in aggregation behavior were revealed by VP size distributions obtained via differential centrifugal sedimentation and confirmed by dynamic light scattering. The VP produced in adherent cells showed increased levels of aggregation in a 20 mM NaCl 10 mM Tris‐HCl pH 7.4 low‐salt buffer. This included the formation of multimers (dimers up to pentamers), whereas VP produced in suspension cells displayed no tendency toward aggregate formation. To investigate the cause of these differences in aggregation behavior, the VP samples were compared based on their zeta potential, their surface hydrophobicity, their lipid composition, and the N‐glycosylation of their major VP surface protein hemagglutinin. The zeta potential and the hydrophobicity of the VP produced in the adherent cells was significantly decreased compared to the VP produced in the suspension cells. The lipid composition of both VP systems was approximately identical. The hemagglutinin of the VP produced in adherent cells included more of the larger N‐glycans, whereas the VP produced in suspension cells included more of the smaller N‐glycans. These results indicate that differences in the glycosylation of viral surface proteins should be monitored to characterize VP hydrophobicity and aggregation behavior, and to avoid aggregate formation and product losses in virus purification processes for vaccines and gene therapy.

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Section: Influenza a Virus Particle Production And Sample Preparationmentioning

confidence: 99%

Influence of the production system on the surface properties of influenza A virus particles

Hämmerling

Hennig

Serve

et al. 2017

Engineering in Life Sciences

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“…Basically, the size of MDCK cell aggregation was controlled by the final serum‐free media when the suspension culture adaptation completed. Different commercial serum‐free media from Sigma, Gibco, and Hyclone, or serum‐free media prepared by each research group have been reported and shown their abilities to support MDCK cells to grow in suspension culture . In this work, Ex‐cell® SFM from Sigma‐Aldrich could make MDCK grow in a single‐cell suspension culture and we speculated that it might be due to the optimized concentration of heparin and dextran sulfate in this serum‐free media.…”

Section: Discussionmentioning

confidence: 85%

“…Individual error bars deviated from multiple analysis of the HI antibody titers of each group. their abilities to support MDCK cells to grow in suspension culture [16,35,36]. In this work, Ex-cell R SFM from Sigma-Aldrich could make MDCK grow in a single-cell suspension culture and we speculated that it might be due to the optimized concentration of heparin and dextran sulfate in this serum-free media.…”

Section: Discussionmentioning

confidence: 88%

H9 subtype influenza vaccine in MDCK single‐cell suspension culture with stable expression of TMPRSS2: Generation and efficacy evaluation

Feng

Tang

et al. 2016

Engineering in Life Sciences

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Vaccination is the most effective way to protect chickens from avian influenza pandemics. Mammalian cells, especially MDCK cells, are being investigated as a good alternative to the embryonated chicken eggs for avian influenza vaccine production. In this work, we developed a new MDCK cell line, which has two main features: single‐cell suspension growth and stable expression of human TMPRSS2 protein (transmembrane protease serine S1 member 2) anchored on cells membrane, named MDCK‐Sus‐TMPRSS2. Reverse‐transcription polymerase chain reaction, western blot analysis and indirect immunofluorescence assay were employed to detect the expression of TMPRSS2 in the MDCK‐Sus‐TMPRSS2 cells. Then, H9 subtype avian influenza virus, NJ02/01 strain, was adapted in MDCK‐Sus‐TMPRSS2 cells with serial blind passages and propagated in stirred bioreactor with 9.5 log2/25 μL hemagglutination titers for inactivated avian influenza vaccine production without addition of exogenous trypsin. The MDCK‐Sus‐TMPRSS2 cell‐derived H9 subtype avian influenza vaccine could induce high titers of hemagglutiniton inhibition antibodies in specific pathogen‐free chickens and protect chickens against heterologous H9 subtype influenza virus challenge, showing lower virus shedding rate and sickness rate than in the egg‐derived vaccines group. High titers of H9 subtype‐specific antibodies above 6 log2 were maintained for 6 months in commercial chickens vaccinated by the MDCK‐Sus‐TMPRSS2 cells derived H9 subtype avian influenza vaccine and there were no negative effects on body weight increase in these chickens. Taken together, the results of this work revealed that MDCK‐Sus‐TMPRSS2 cell line could be a promising and safe substitute for embryonated chicken eggs as a host cell line for H9 subtype avian influenza virus propagation.

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“…13 Cells were infected with human influenza A/PR/8/34 (H1N1) (#3138, Robert Koch Institute (RKI), in-house generated adherent MDCKderived viral stock, TCID 50 titer of 5.17 × 10 8 infectious virions/ml, HA titer of 2.63 log 10 HA units/100 µl) with a multiplicity of infection (moi) of 0.025 and 2 × 10 −6 units trypsin (#27250-018, Gibco) per cell. 250 ml vented shaker flasks or 850 cm 2 roller bottles (both VWR) were used for all infection experiments.…”

Section: Cell Lines Cell Cultivation and Virus Infection Adherentmentioning

confidence: 99%

“…250 ml vented shaker flasks or 850 cm 2 roller bottles (both VWR) were used for all infection experiments. 12,13,14 At 72 hours post infection, supernatants were harvested and clarified for 20 min at 150 g (Avanti J-20XP, Beckmann Coulter) at room temperature (RT) to obtain clarified virus harvests (CVH).…”

Section: Cell Lines Cell Cultivation and Virus Infection Adherentmentioning

confidence: 99%

Comparison of Influenza Virus Particle Purification Using Magnetic Sulfated Cellulose Particles with an Established Centrifugation Method for Analytics

et al. 2015

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A method for the purification of influenza virus particles using novel magnetic sulfated cellulose particles is presented and compared to an established centrifugation method for analytics. Therefore, purified influenza A virus particles from adherent and suspension MDCK host cell lines were characterized on the protein level with mass spectrometry to compare the viral and residual host cell proteins. Both methods allowed one to identify all 10 influenza A virus proteins, including low-abundance proteins like the matrix protein 2 and nonstructural protein 1, with a similar impurity level of host cell proteins. Compared to the centrifugation method, use of the novel magnetic sulfated cellulose particles reduced the influenza A virus particle purification time from 3.5 h to 30 min before mass spectrometry analysis.

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Monitoring changes in proteome during stepwise adaptation of a MDCK cell line from adherence to growth in suspension

Cited by 19 publications

References 59 publications

Influence of the production system on the surface properties of influenza A virus particles

Influence of the production system on the surface properties of influenza A virus particles

H9 subtype influenza vaccine in MDCK single‐cell suspension culture with stable expression of TMPRSS2: Generation and efficacy evaluation

Comparison of Influenza Virus Particle Purification Using Magnetic Sulfated Cellulose Particles with an Established Centrifugation Method for Analytics

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