Cultivation of sensitive cell lines - Improving bioreactor performance by dynamic membrane aeration
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1 Cultivation of sensitive cell lines - Improving bioreactor performance by dynamic membrane aeration Björn Frahm, Helmut Brod Bioprocessing Summit Optimizing Cell Culture Technology, Boston,
2 Content Introduction to cell culture bioreactor development Bubble-free aeration for special applications Advantages of Dynamic Membrane Aeration (DMA) Application for continuous perfusion cultivation fundamentals & advantages Application examples Continuous perfusion culture Fed-batch Retrofitting into existing bioreactors Summary
3 Introduction to cell culture bioreactor development Fermentation - from development to production: Gene & protein Express. system Clone selection Media Lab-scale optimizat. protocol Scale-up Large-scale production High Throughput Screening Engineering Know-How - Hydrodynamics Biotechnology Know-How - Scaling laws - Genomics, Proteomics - Controls - Cell Biology, Biochemistry - Screening Technologies Fermentation Optimization
4 Cell culture bioreactors - requirements Production point of view High product titer High product quality High reproducibility Ability to cultivate sticky cells Low costs Engineering point of view High gas transfer rates Sufficient oxygen supply Removal of metabolic CO 2 Sufficient mixing chemical, thermal and biomass homogeneity Low susceptibility to bio-fouling All at minimal hydrodynamic stress for the cells
5 Cell culture bioreactors challenges Power input by agitation and / or gas sparging Growth Viability Impurities Oxygen supply CO 2 removal Mixing working range (often compromise)
6 Content Introduction to cell culture bioreactor development Bubble-free aeration for special applications Advantages of Dynamic Membrane Aeration (DMA) Application for continuous perfusion cultivation fundamentals & advantages Application examples Continuous perfusion culture Fed-batch Retrofitting into existing bioreactors Summary
7 Bubble-free aeration for certain applications Why bubble-free aeration? - aeration via gas bubbles / (micro)sparging is state of the art Certain cell lines are sensitive to bubble aeration / sparging hydrodynamic shear, flotation immediate lysis (membrane rupture) delayed lysis (apoptosis) release of host cell proteins and DNA risk of product degradation bubble rupture at free surface are susceptible to agglomeration / fouling early cultivation run termination No antifoam addition advantageous for downstream purific. Antifoam has to be cleared downstream
8 Bubble-free aeration for certain applications Why choose such sensitive cell lines? Higher titers than robust competitors Sensitivity of cell lines caused by required medium composition Patent situation In-house development
9 Bubble-free aeration for certain applications Bubble-free aeration is generally provided by membrane aeration*: Anchor stirrer Membrane tubing on stator, air / oxygen flowing through tubing, e.g. silicone Disadvantages: Oxygen limitation at high cell densities Debris and protein build-up on the aeration tubing Limited scalability Limited product output & space time yield *at scales larger than bench top
10 Economical need for more productive bioreactors Productivity ~ Scale x Intensity Process Intensity Viable Cell Density Intensify Intensify & scale-up Status quo Scale-up Bioreactor volume Process Scale
11 Content Introduction to cell culture bioreactor development Bubble-free aeration for special applications Advantages of Dynamic Membrane Aeration (DMA) Application for continuous perfusion cultivation fundamentals & advantages Application examples Continuous perfusion culture Fed-batch Retrofitting into existing bioreactors Summary
12 Advantages of Dynamic Membrane Aeration (DMA) Star-like membrane tubing set-up More surface for gas transfer Oscillatory membrane movement Better flow around tubing Much higher oxygen transport at the same low shear stress 20 L DMA cell culture run
13 Advantages of Dynamic Membrane Aeration (DMA) Debottlenecking of oxygen / gas limitations increase of gas transfer by factor > 2 (factor > 6 using thinner parallel tubing in modules) Prevents membrane fouling and cell agglomerates very advantageous for sticky cells Easy scale up DMA better than Easy & defined wrapping of membrane tubing due to corrugation and wrapping device
14 Advantages of Dynamic Membrane Aeration (DMA) DMA bioreactors at different locations: 15 L Berkeley, USA Bayer Health Care 20 L Wuppertal, Germany Bayer Health Care & Vienna, Austria University of Ghent Biocatalytic application 200 L Wuppertal, Germany Bayer Health Care The presented reactor scales are designed for continuous culture at high perfusion rates (e.g. using cell retention) Like this, a 200 L scale continuously operated can keep up in productivity with (fed-) batches in the range of 3,000 L Of course, standard (fed-)batches can also be performed
15 Content Introduction to cell culture bioreactor development Bubble-free aeration for special applications Advantages of Dynamic Membrane Aeration (DMA) Application for continuous perfusion cultivation fundamentals & advantages Application examples Summary
16 Fundamentals: (fed-)batch vs. continuous cultivation Batch Continuous V S, X, P q, S o S, X, P V Substrate S Product P Product P Cell density X Cell density X t [d] V q S X P 0 30 bioreactor volume continuous flow rate substrate concentration cell density product concentration Substrate S t [d] Continuous cultivation enables maintenance of high cell density and therefore high product output 60 90
17 Fundamentals: continuous with & without cell retention Without cell retention q, S o S, X, P With cell retention X q, S o S, X H, P harvest cell density V V retention degree R Product P Cell density X Cell density X Product P Substrate S Substrate S t [d] t [d] 90 Cell retention enables increase of cell density and therefore higher product output but dilutes product conc.
18 Comparison of space-time yields q, S o S, X, P V t P P, 0 t V S, X, P t t B B, 0 t t B B, 0 t t B B, 0 t t B B, 0 t t B B, 0 Space-time yield Y = mab produced / (reactor volume * cycle time) perfusion (fed-)batch Y Y P B P P PB t B q V t B t P tb t tp t B,0 P,0 Y Y P B X X P B t t P B tp t tb t P,0 B,0 X X P B U U P B Ui utilization (% in time) For: X P = cells ml -1 P U P = 90 % (90 d, 10 d down) 8. 8 X B = cells ml -1 U B = 77 % (10 d, 3 d down) Y Y B
19 Bioreactor sizing for three process modes example Fed-Batch V = V B t 10 d Continuous V/V B = 43 % q/v = 0.7 d -1 Residence Time t = 1.4 d Continuous with Cell Retention V/V B = 11 % q/v = 10 d -1 t 2.9 h
20 Proven perfusion capacity of cell retention systems varies strongly, but inclined-plate sedimentation is still heading the field D. Voisard et. al. Biotechn. & Bioeng. 82(2003)7 q = 3000 L/d Bayer Inclined Channel System approved in production for 180 days
21 Content Introduction to cell culture bioreactor development Bubble-free aeration for special applications Advantages of Dynamic Membrane Aeration (DMA) Application for continuous perfusion cultivation fundamentals & advantages Application examples Summary Published in Cytotechnology (2009) 59, pages 17-30: Improving bioreactor cultivation conditions for sensitive cell lines by dynamic membrane aeration Frahm B., Brod H., Langer U.
22 15 L continuous culture at Bayer Health Care, USA HKB11 cell line (human hybrid rfviii producer) Difficult to cultivate in a standard membraneaerated bioreactor (aggregation, bio-fouling) Results of comparative runs: (DMA vs. standard bioreactor) Bio-fouling strongly reduced standard reactor DMA 25 % higher cell density at one third of the power input viable viable cell cell density density X [10 7 cells ml -1 ] v [10 7 cells ml -1 ] DMA Standard reactor Standard reactor had to be terminated cultivation time [day]
23 20 L and 200 L fed-batch culture at Bayer Health Care Chinese Hamster Ovary cell line (antibody producer) Comparative runs in 20 L and 200 L scale Achieved similar cell densities and product titers (fed-batches were at a nutrient-controlled limit) proven scalability DMA can still double membrane capacity while the standard reactor is at its limit potential for process improvements potential for bigger scales Foul-safe cultivation with DMA Membrane tubing on oscillating DMA rotor arms Membrane tubing on standard reactor Cells and debris
24 Retrofitting of DMA into existing bioreactors Engineering of customized DMA rotor and engine for existing bioreactors (replaces existing aeration and stirrer) Example from Bayer Health Care, 200 L scale for antibody production
25 Summary Certain cell lines / processes require gentle bubble-free aeration / no antifoam the avoidance of cell agglomeration Current membrane aeration does not fulfill these requirements Dynamic Membrane Aeration (DMA) successfully debottlenecks the current limitations Blood clotting factor VIII (Kogenate ) DMA technology is available for retrofitting into existing bioreactors or as ready-to-use bioreactors Interdisciplinary application: Similar challenges exist for bubble-free oxidation in biocatalysis
26 Acknowledgements Bayer Health Care, Blood coagulation factor VIII production plant, USA: K. Jöris, M. Burnett, C. Zhang, K. Konstantinov et al. Bayer Health Care, Global Biological Development, Wuppertal: U. Langer, M. Wischniewski, B. Bödeker et al. Currenta, Manufacture of Special Machinery: P. Commer, Chr. Schiffczyk, A. Twyrdy, H. Drinhausen, B. Kunert, J. Gießelmann, R. Grodotzki University of Bielefeld: L. Behr University of Ghent: W. Van Hecke Competence Center Enzyme & Fermentation Technology: S. Kirchner, R. Rose Students: A. Sinthern, J. Schröter and M. Rampe
27 Modular hybrid protein production by perfusion cultivation (Bayer Schering Pharma, Berkeley, CA, USA) Thank you for your attention!
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