Wednesday, August 17, Mini-Review (2016) in Applied Microbiology and Biotechnology
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1 Bioreactors for high cell density and continuous multi-stage cultivations: options for process intensification in cell culture-based viral vaccine production Wednesday, Mini-Review () in Applied Microbiology and Biotechnology Felipe Tapia, Daniel Vázquez-Ramírez, Yonne Genzel, Udo Reichl
2 Overview 1. Introduction 2. Adherent or suspension cells 3. Batch vs. fed-batch 4. Perfusion system 5. Multi-stage bioreactors 6. Outlook 7. References Seite 2
3 Introduction Virus replication (lytic cycles) 1. Attachment 2. Penetration 3. Replication 4. Assembly 5. Lysis & Release Seite 3
4 Introduction DIPs - defective interfering particles spontaneously generated virus mutants The damage virus can still penetrate host cell, but require a fully virus particles existence of DIPs has been observed within nearly every class of both DNA and RNA viruses Problem by viral vaccine production => decrease maximum virus yield Seite 4
5 Introduction Key factor to increase virus production yields Cell concentration and metabolic/physiological status of the cells at time of infection Cell concentration defines the virus titer depening on status of cells Ratio of infectious particles to viable cells at the time of infection A too high number of virus particles per cell, can promote replication of defective interfering particles Residence time of virus particles within the bioreactor and time point of harvest The residence time affected the vaccine type live attenuated vaccines or inactivated vaccines depending on the application Seite 5
6 Suspension Cells Systems Hollow fiber bioreactor maximal growth is mainly limited by the total amount of nutrients and the accumulation of growth-inhibiting compounds Separate the viral particles Seite 6
7 Adherent Cells Systems Microcarrier in STR Maximum number of cells depends on the available growth surface Exchange the medium by sedimentation of carrier in STR microcarrier-beads-sem-david-m-phillips.jpg 1.8 x 10 6 cells/ml 1.1 x 10 7 cells/ml Seite 7
8 Batch vs. Fed-batch Batch: + ease of operation + process robustness Accumulation of by-products Fed-batch: + Potential to accomplish higher cell concentration + Avoid the accumulation of unwanted by-products through addition of medium Parapoxvirus ovis: 5.5 x 10 6 VP/Lh Parapoxvirus ovis: 1.1 x 10 8 VP/Lh Seite 8
9 Perfusion Systems + Potential to accomplish higher cell concentration + Avoid the accumulation of unwanted by-products through exchange of medium + Low footprint and high volumetric rates Disadvantages such as filter clog and limited scalability (spin filter) Adenovirus (HFK293): 7.8 x 10 9 IVP/ml Seite 9
10 Perfusion Systems Different production profiles and kinetics Schematic representation of the perfusion-based high cell density (HCD) production of recombinant proteins (a) and viruses (b) Seite 10
11 Multi-stage bioreactor + steady-state operation + high volumetric efficiency + lower plant turndown that enhance process yield + two-stage STR bioreactor for continuous influenza A virus production large volumes of media consumed or the extended production time by HCD production Influenza A/PR/8/34 (AGE1-CR.plX): 7.0 x 10 9 IVP/ml Seite 11
12 Multi-stage bioreactor Passage effect Increase the amount of non-occluded virus particles for up to 25 day continuous mode Three stage bioreactor enhance the passage effect with higher virus passage number an earlier drop in virus yield passage effect induced by DIPs Seite 12
13 Outlook apply knowledge for process intensification and other cell culture products automation technologies (on-line monitoring of metabolites) availability of technologies for establishing HCD under cgmp conditions continuous processes also an interesting option to batch production Limiting factor by the accumulation of DIPs => reduced product titer HCD cultivation using fed-batch or perfusion strategies, studies currently show cell concentration exceeding 10 8 cells/ml Seite 13
14 References Cortin V, Thibault J, Jacob D, Garnier A (2004) High-titer adenovirus vector production in 293S cell perfusion culture. Biotechnol Prog 20(3): Bock A, Schulze-Horsel J, Schwarzer J, Rapp E, Genzel Y, Reichl U (2011) High-density microcarrier cell cultures for influenza virus production. Biotechnol Prog 27(1): Pohlscheidt M, Langer U, Minuth T, Bodeker B, Apeler H, Horlein HD, Paulsen D, Rubsamen-Waigmann H, Henzler HJ, Reichl U (2008) Development and optimisation of a procedure for the production of Parapoxvirus ovis by large-scale microcarrier cell culture in a nonanimal, non-human and non-plant-derived medium. Vaccine 26(12): Hu YC, Wang MY, Bentley WE (1997) A tubular segmented-flow bioreactor for the infection of insect cells with recombinant baculovirus. Cytotechnology 24(2): Castilho L, Moraes A, Augusto E, Butler M (2008) Animal cell technology: from biopharmaceuticals to gene therapy. Taylor & Francis Group, New York; Abingdon England Seite 14
15 Thank you for your attention. Any questions.? Seite 15
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