Peter Neubauer Chair of Bioprocess Engineering, Technische Universität Berlin, DE berlin.de

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1 Peter Neubauer Chair of Bioprocess Engineering, Technische Universität Berlin, DE berlin.de The Biotech Challenge o Biotechnology is a key technology of the 21 st century o /3 of the worldwide industrial production derive from biological sources and biotechnological processes (market volume 300 Billion EURO) 1 o Bioprocesses are sustainable and ressource efficient o Big challenge: Bioprocess development timeline and costs 1 En Route to the Knowledge Based Bio Economy, Cologne 2007 DSM Position document on Industrial Biotechnology in Europe and The Netherlands (2004) Europabio 1

2 Potential of disposable culture systems for microbial cultures Strong interest for new bioprocesses Tight timeline from research to process Simple process strategy consistency in scale up Screening, process development Pilot scale = production scale Slow processes: Parallel medium size production vs. large scale Handling of infectious material Disposable culture systems for screening Micro 24 Pall Biolector M2p labs 2mag system 2

3 The challenges for polymer based culture sytems in the microbial area Long term stability Simple use / robustness Reapeated useability (autoclavation possible) Available sensors (robustness, range, applicability) Investment costs Technical standards, quality assurance (gas transfer rates, ph control) Biolector M2p labs 2mag system Consistent Bioprocess Development A leading principle is to start development work from the perspective of the large scale and then step down the scale, not vice versa. Consistent Bioprocess development Noorman H: An industrial perspective on bioreactor scale down: What we can learn from combined large scale bioprocess and model fluid studies. Biotechnol J 2011, 6:

4 Consistent Bioprocess Development Industrial Processes are Fed batch Processes BUT initial process development is performed as batch Industrial Processes are Inhomogenous BUT Process devlopment is performed in homogenous systems Towards a scalable fed batch process Glucose Cell mass Cumulative glucose feed Specific growth rate µ Cultivation time Specific growth rate µ Cell mass po 2 Biocatalyst Slowly or nonmetabolisable Polymer po 2 Cumulative glucose produced Glucose Cultivation time EnBase : Controlled nutrient supply by a biocatalyst allows fedbatch with internal substrate delivery 4

5 Panula Perälä et al Enzyme controlled glucose auto delivery system for high cell density cultivations in microplates and shake flasks. Microb Cell Fact 7:31 Ultrayield TM Flasks Shaking velocity, rpm Erlenmeyer flask, 10% filling volume k L a, h 1 UYF, 20% filling volume 125 ml 250 ml 500 ml 2500 ml Glazyrina et al., New Biotechnol., in revision 5

6 Production of radh in EnPresso & UltraYield Flasks Dry cell weight [g L -1 ] IPTG+Booster EnP Erl. 1.5 U/L EnP Erl. 0.6 U/L EnP UY 1.5 U/L EnP UY 3 U/L EnP UY 6 U/L TB, Erl. TB UY ZYM Erl. ZYM UY Induction, TB Induction, EnP ph Time [h] Time [h] Expression of Lactobacillus alcohol dehydrogenase in E. coli RB791 Media: EnPresso, Terrific Broth, ZYM 5052 autoinduction medium Erlenmeyer and Ultra Yield Flask, AirOtop membrane seals used in all flasks Ukkonen et al Microb Cell Fact 10: 107. Production of radh in EnPresso & UltraYield Flasks Soluble protein kda Total (soluble + insoluble) protein kda EnPresso TB ZYM TB ZYM 1. EnP Erl. 24h 1.5 U/L 2. EnP Erl. 24h 0.6 U/L 3. EnP UY 24h 1.5 U/L 4. EnP UY 24h 3 U/L 5. EnP UY 24h 6 U/L 6. TB Erl. 24h 7. TB UY 24h 8. ZYM Erl. 24h 9. ZYM UY 24h 10. TB Erl. 5h 11. TB UY 5h 12. ZYM Erl. 8h 13. ZYM UY 8h x higher volumetric activity by EnPresso Ukkonen et al Microb Cell Fact 10:

7 EnBase makes bacteria rocking o Rocking motion is widely used for cultivation of cell lines o Oxygen transfer in rocking systems is low low yields o With EnBase: Fed batch becomes possible Recombinant protein production in E. coli is feasible Simple protocol and yields same cell densities/product yields compared to other cultivation formats Glazyrina et al Glucose limited high cell density cultivation and recombinant protein production with E. coli in a rocking motion type bioreactor. Microb Cell Fact. 9:42. Small scale production in a disposable system Whole cell biocatalysis with yeast: 450 g wet cell weight per bottle Exp. OD 600 max YG standard, EF L UYF control, EnBase L UYF, EnBase + boost L UYF, EnBase, no shake 103 7

8 Part 2: Docosahexaenoic acid (DHA) Polyunsaturated fatty acid (PUFA) Human brain and eye tissue contains DHA in large quantities DHA: Lower the risk for cardiovascular diseases and Alzheimer s disease Reduce risk of neuropsychatric disorder, depression and sucide Essential for proper visual and neurogical development of infants Part 2: Docosahexaenoic acid (DHA) Crypthecodinium cohnii REM Aufnahme C. Cohnii (Parrow et al. 2006) 1:650 Cells in the growing phase 1:650 Heterotrophic marine dinoflagellate Senstitve to high shear force High oxygen demand for growth and fatty acid production High chloride concentration in the media Challenges for the process development Demand of a system characterized by low shear forces and comparably high oxygen transfer rates Materials should withstand high chlorid concentration Long term stability Cells in the stationary phase 8

9 Part 2: Docosahexaenoic acid (DHA) Consistent Bioprocess Development Deep Well plates (3mL) Tube Spin Bioreactor (100 ml) Orbital Shaker (100 L) Automated robot screening in microwell plates Conditional screening in microbioreactor system Evaluation in laboratory fermenter Consistent bioprocess development Polymerbased system to overcome corrosion problems Orbital shaken systems from small scale to large scale Shear senstive with a sufficient oxygen input Gas carrier: Perfluorodecalin (PFD) Solubility of O 2 20 times higher in PFD than in water (oxygen solubility: PFD: mm, water: 2.2 mm) Up to 40 % increaed cell density with E. coli in deep well plates with PFD in comparison to control culture Untoxic towards biological systems Chemical, biological inertness, autoclavable Pilarek et al. Microbial Cell Factories 2011, 10:50 9

10 Part 2: Polymerbased shaken bioreactors Comparision of different systems from from 15 to 150 L Wave induced shaker: e.g. CELLtainer (CELLution) Paddle driven reactor: e.g Integrity PadReactor TM (ATMI) Orbital shaker: e.g. SB200 X (Kuhner shaker) L 25 L 150 L Contact time / Cycing time Effects of insufficient mixing in bioreactors: Lin H.Y., Neubauer P Influence of controlled glucose oscillations on a recombinant fedbatch process of Escherichia coli. J. Biotechnol. 79, F const 4 min 1 min 10

11 Conclusions Polymer based disposable type of cultivations systems are widely used for microbial culture Real disposables are widely used in the screening stage Potential: Small scale cultivation Pilot production with integrated DSP Parallel production with long term use Pathogens, phages, marine / expremphilic organisms? Challenges: Gas transfer Sensor robustnes and measuring range New reactor types for cost effective production Material consistency 11