Scale-up & scale-down between the two. worlds of shaken and stirred bioreactors
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1 Scale-up & scale-down between the two worlds of shaken and stirred bioreactors Prof. Dr.-Ing. Jochen Büchs AVT - Biochemical Engineering, RWTH Aachen University Sammelbau Biologie, D Aachen, Germany jochen.buechs@avt.rwth-aachen.de CIB-Forum Scale-Up & Scale-Down, Monheim Traditional way of process development Clearly separated tasks performed by people with different educational background Screening Process development Production Shake flask culture systems Bench scale Production scale 2
2 Current way of process development arger number of smaller parallel bioreactors Screening Process development Production (micro bioreactors) (mini bioreactors) Micro titre plate culture systems Bench scale Production scale 3 High throughput high information content Experimental throughput Screening Process development M On-line measurement, feeding and control options 4
3 High throughput high information content Experimental throughput Screening Scale-up & Scale-down Process development M On-line measurement, feeding and control options 5 High throughput and (!) high information content feeding Experimental throughput Bioector(Pro) RAMOS FeedBeads & FeedPlates M On-line monitoring On-line measurement, feeding and control options 6
4 Important engineering parameters of a bioprocess - and m 3 -scale ) specific power consumption, (P/V ) 2) O 2 - supply, OTR max or k a It is in general only possible to keep one single 3) ventilation (stripping of CO 2, H 2 O, alcohols etc.) parameter constant during change of scale. 4) degree of mixing and homogeneity The most relevant parameter has to be found 5) hydromechanical stress (damage of for each specific fermentation system. cells, control of morphology), (P/V ) max 6) foam generation and its impact Well established methodology with the 7) dispersion of an organic liquid phase exception of some specific fields 8) suspension (homogeneity) of solids M 9) non-newtonian shear rate dependent viscosity 7 Important engineering parameters of a bioprocess - and m 3 -scale ) specific power consumption, (P/V ) 2) O 2 - supply, OTR max or k a 3) ventilation (stripping of CO 2, H 2 O, alcohols etc.) 4) degree of mixing and homogeneity 5) hydromechanical stress (damage of cells, control of morphology), (P/V ) max 6) foam generation and its impact 7) dispersion of an organic liquid phase 8) suspension (homogeneity) of solids M 9) non-newtonian shear rate dependent viscosity 8
5 Important engineering parameters of a bioprocess µ- and m-scale ) specific power consumption, (P/V ) 2) O 2 - supply, OTR max or k a 3) ventilation (stripping of CO 2, H 2 O, alcohols etc.) 4) degree of mixing and homogeneity 5) hydromechanical stress (damage of cells, control of morphology), (P/V ) max 6) foam generation and its impact 7) dispersion of an organic liquid phase 8) suspension (homogeneity) of solids 9) non-newtonian shear rate dependent viscosity 9 Quantification of specific power input (P/V ) [kw/m 3 ] based on torque measurement One flask device Multi flask measuring device Büchs et al., Biotechnol. Bioeng. 68 (2000)
6 2 Influence of viscosity on spec. power input l flask, 00 ml filling volume, 5 cm shaking diameter Spec. power input (P/V) [kw/m³] viscosity 95.6 mpas 59.3 mpas 34.3 mpas 7.4 mpas 4.4 mpas Same order of magnitude as in larger 0.8 mpas (water) e.g. stirred tank reactors!! Shaking frequency (n) [rpm] Büchs et al., Biotechnol. Bioeng. 68 (2000) Influence of viscosity on spec. power input l flask, 00 ml filling volume, 5 cm shaking diameter Spec. power input (P/V) [kw/m³] viscosity 95.6 mpas 59.3 mpas 34.3 mpas 7.4 mpas 4.4 mpas 0.8 mpas (water) Shaking frequency (n) [rpm] Büchs et al., Biotechnol. Bioeng. 68 (2000)
7 Modified power number Ne = = P P n 3 d 4 V [-] ) (P o n 3 d 4 V /3 / /3 [-] Non dimensional correlation of power input measurements in shake flasks Shaking diameter: cm, viscosity: mpa s, shaking frequency: rpm P o = 70 Re Re -0,6 +.5 Re -0,2 No geometric similarity "in phase" Betriebspkt operating "außer conditions Phase" 00 ml shaking flask 250 ml " 300 ml " 500 ml " 000 ml " 2000 ml " turbulent flow E E E E E E ,000 Reynolds number (Re = n d 2 / ) [-] Büchs et al., Biotechnol. Bioeng. 68 (2000) Peter et al., Biotechnol. Bioeng. 93 (2006) Difference between shaken and stirred bioreactor Shake flask Stirred bioreactor No significant difference in volumetric power input (P/V ) M 4
8 Important engineering parameters of a bioprocess µ- and m-scale ) specific power consumption, (P/V ) 2) O 2 - supply, OTR max or k a 3) ventilation (stripping of CO 2, H 2 O, alcohols etc.) 4) degree of mixing and homogeneity 5) hydromechanical stress (damage of cells, control of morphology), (P/V ) max 6) foam generation and its impact 7) dispersion of an organic liquid phase 8) suspension (homogeneity) of solids 9) non-newtonian shear rate dependent viscosity 5 iquid distribution in shake flasks height [cm] Height [cm] axis of rotation View: A-B sectional drawing A - B Maximal liquid height (3.07 cm) maximum liquid height (3.07 cm) Operating conditions: axis of rotation flask volume: 250 m shaking diameter: 2.5 cm shaking frequency: 200 rpm filling volume: 26 m y-axis [cm] y-axis [cm] A A x-axis [cm] Büchs et al., Bioproc. Biosystems Eng. 34 (2007), B B x-axis [cm]
9 Relevant specific mass transfer area in hydophilic shaking flasks 250 ml- flask without baffles, 0-50 ml filling volume, cm shaking diameter Calculated mass transfer area a [m 2 /m 3 ] bulk liquid surface - area film + bulk liquid surface - area Measured mass transfer area a [m 2 /m 3 ] Maier and Büchs, Biochem. Eng J. 7 (200) Influence of the flask surface properties on the OTR shaking diameter d 0 = 4.2 cm, filling volume V = 26m Disposable 20 plastic flasks (hydrophobic) have a Maximum Oxygen oxygen transfer transfer rate OTR capacity (OTR max [mmol/l/h] ) [mmol//h] lower OTR max than hydrophilic glass flasks! 5 normal hydrophilic normal hydrophilic flask flask 0 5 hydrophobic flask Maier and Büchs, Biochem. Eng J. 7 (200) Shaking frequency n (n)[/min] [rpm] 8
10 Oxygen transfer in bioreactors as function of viscosity Maximum oxygen transfer capacity (OTR max ) [mmol//h] stirred tank 25 bioreactor 250 m shake flask 20 m filling volume 350 rpm 9 % of mpa s % of mpa s Viscosity () [mpa s] Giese et al., Biotechnol. Bioeng. (204), Typical trend of OTR max in a stirred tank [percentage of OTR max, mpa s ] Difference between shaken and stirred bioreactor Shake flask Stirred bioreactor Flask wall surface property play a crucial role for OTR max. OTR max (at mpa s) slightly superior in stirred compared to unbaffled shaken bioreactor. Superior performance of shake flask at elevated viscosities. 20 M
11 Important engineering parameters of a bioprocess µ- and m-scale ) specific power consumption, (P/V ) 2) O 2 - supply, OTR max or k a 3) ventilation (stripping of CO 2, H 2 O, alcohols etc.) 4) degree of mixing and homogeneity 5) hydromechanical stress (damage of cells, control of morphology), (P/V ) max 6) foam generation and its impact 7) dispersion of an organic liquid phase 8) suspension (homogeneity) of solids 9) non-newtonian shear rate dependent viscosity 2 Oxygen mass transfer in a bubble aerated stirred tank bioreactor OTR max ~ k a ~ a b P / V u Ø g with OTR max maximum oxygen transfer capacity [mol//h] k a mass transfer coefficient [/s] (P/V ) specific power input [kw/m 3 ] u g superficial gas velocity [m/s] a, b broth and reactor dependent empirical parameters [-] 22
12 Strong correlation between oxygen mass transfer (k a) and power input (P/V ) in bubble aerated stirred tank bioreactors M Na 2 SO 4 solution k a/ u g (after Moucha et al., 995) air oxygen Specific power input (P/V ) Ø [W/m 3 ] k a: mass transfer coefficient [/s] u g : superficial gas velocity [m/s] 23 Strong correlation between oxygen mass transfer (k a) and power input (P/V ) in bubble aerated stirred tank bioreactors Cavity behind a stirrer blade M Power input is required to disperse the bubbles in the vicinity of the stirrer blades. 24
13 Is oxygen mass transfer (k a) a strict function of power input (P/V ) Ø in shake flask bioreactors? Shaking frequency (n) = const. Shaking diameter (d 0 ) = const. d (P/V ) Ø d.6 Relative filling volume (V /d 3 ) = const. k a d Shake flasks are surface aerated, not bubble aerated! d d 25 Relation between oxygen transfer and spec. power input Oxygen transfer (OTR max, k a) and spec. power input (P/V ) Ø of shake flask bioreactors are not directly related (!), in contrast to bubble aerated bioreactors like stirred tanks. OTR max ~ k a f (P/V ) Ø Oxygen transfer and power input are locally separated in shake flask bioreactors! Interfacial area for gas/liquid mass transfer Interfacial area for momentum transfer (power input and hydromechanical stress) 26
14 Important engineering parameters of a bioprocess µ- and m-scale ) specific power consumption, (P/V ) 2) O 2 - supply, OTR max or k a 3) ventilation (stripping of CO 2, H 2 O, alcohols etc.) 4) degree of mixing and homogeneity 5) hydromechanical stress (damage of cells, control of morphology), (P/V ) max 6) foam generation and its impact 7) dispersion of an organic liquid phase 8) suspension (homogeneity) of solids 9) non-newtonian shear rate dependent viscosity 27 Maximum oxygen transfer capacities in 96 well micro titre plates Maximum oxygen transfer capacity (OTR max ) [mol//h] ,000 Shaking diameter It is impossible to supply bacterial and yeast sufficiently with oxygen in 96 well MTPs! 50 mm 25 mm 2.5 mm 6 mm 3 mm 200 µl filling volume Shaking frequency (n) [rpm] Hermann et al., Biotechnol. Bioeng. 8 (2003)
15 Introduction of Baffles Sets of Geometries Reference: 48-well micro titer plate with round well geometry Alternative prototypes: constant cross sectional area (of 2 mm 2 ) per well constant filling height Increasing number of edges Rounding of edges from square to circle Rounding of edges from pentagon to circle Funke et al., Biotechnol. Bioeng. 03 (2009) Introduction of Baffles Multiform Baffles Funke et al., Biotechnol. Bioeng. 03 (2009)
16 Flower plates 48 well design with optodes at the bottom for ph and DOT measurement Commercialized by m2p-labs, Aachen Funke et al., Biotechnol. Bioeng. 03 (2009) Sufficient oxygen supply by novel MTP design E. coli K2, mineral medium with 20 g/ glucose, 200 mm MOPS, 37 C, 000 rpm Oxygen transfer rate [mmol//h] OTR max for 200 µ in normal 96 well MTP Culture time [h] Funke et al., Biotechnol. Bioeng. 03 (2009)
17 Difference between shaken and stirred bioreactor Shaken bioreactor Stirred bioreactor With carefully designed baffles shaken bioreactors can reach OTR max similar to stirred bioreactors. M 33 Important engineering parameters of a bioprocess µ- and m-scale ) specific power consumption, (P/V ) 2) O 2 - supply, OTR max or k a 3) ventilation (stripping of CO 2, H 2 O, alcohols etc.) 4) degree of mixing and homogeneity 5) hydromechanical stress (damage of cells, control of morphology), (P/V ) max 6) foam generation and its impact 7) dispersion of an organic liquid phase 8) suspension (homogeneity) of solids 9) non-newtonian shear rate dependent viscosity 34
18 Hydromechanical stress in shake flasks and stirred tanks Using the larges possible geometric element (reactor wall and bottom) for power input results in its most even introduction into the culture liquid. M (P/V) ) max (P/V) ) Ø? (P/V) ) max (P/V) ) Ø Peter et al., Biotechnol. Bioeng. 93 (2006) Hydromechanical stress in shake flasks and stirred tanks This significant difference is unavoidable and may cause major scale-up problems! M (P/V (P/V) ) max max (P/V (P/V) ) Ø Ø Same order of magnitude (P/V) (P/V max ) max (P/V) (P/V Ø ) Ø Peter et al., Biotechnol. Bioeng. 93 (2006)
19 Important engineering parameters of a bioprocess µ- and m-scale ) specific power consumption, (P/V ) 2) O 2 - supply, OTR max or k a 3) ventilation (stripping of CO 2, H 2 O, alcohols etc.) 4) degree of mixing and homogeneity 5) hydromechanical stress (damage of cells, control of morphology), (P/V ) max 6) foam generation and its impact 7) dispersion of an organic liquid phase 8) suspension (homogeneity) of solids 9) non-newtonian shear rate dependent viscosity 37 Flow curves for fermentation-broths of Streptomyces tendae at different times Viscous fermentation broths in general show non-newtonian pseudo-plastic or shear-thinning flow behaviour! Dynamic viscosity [Pa s] Shear rate 200 stirred tank, 24 C agitation rate /min [/s] 38 Time [h]
20 Shear thinning results in reduced viscosity (pseudo-plastic flow behaviour) Static sample Sample subjected to a flow field v Polymer molecules or hyphae of a filamentous microorganism The polymer molecules or hype align in parallel according to the flow field, thereby, reducing the flow resistance. 39 Flow curves for fermentation-broths of Streptomyces tendae at different times Power law (or Oswand s law) model for the viscosity of fermentation broths (valid for typical shear rates applied): K m Dynamic viscosity [Pa s] stirred tank, 24 C agitation rate /min dynamic viscosity [mpa s] K consistency factor [mpa s m ] m flow behaviour index [-] shear rate [/s] Shear rate [/s] 40 Time [h]
21 Apparent viscosity for non-newtonian fluids To calculate the apparent viscosity in a stirred tank reactor, the average shear rate ( ) as function of the operating conditions of the reactor is absolutely (!) essential. The Metzner-Otto concept has been developed for the laminar flow regime. It has also been used for the turbulent flow regime, although the justification of this approach has never been proven: A n with A However, this is not justified any more, according to the newest findings! dynamic viscosity Pa s shear stress Pa shear rate /s K consistency factor Pa s m m flow behaviour index - n agitation rate /s A coefficient - 4 Measured and calculated shear rates after the Metzner-Otto concept for a non-newtonian liquid in the turbulent flow regime Shear rates 000 calculated from heat transfer measurements (Henzler, 2007) V = 0.92 D R = 0.4 m Shear rate ( ) [/s] 00 2 V = 2.5 m 3 D R =.59 m n Metzner-Otto concept , 0 Agitation rate (n) [rpm]
22 Why is it not plausible to use the Metzner-Otto concept to calculate the shear rate for turbulent flow conditions If we assume for scale-up and -down: ) Turbulent conditions power number (Po) = const. 2) Geometric similarity (V D 3 R and d D R ) 3) Constant volumetric power input; (P/V ) = const. P V Po n V 3 d 5 n ~ D R -2/3 ~ V -2/9 Po power number - V filling volume m 3 d stirrer diameter m D R vessel diameter m P/V volumetric power input kw/m 3 n agitation rate /s 43 Operating points calculated by the Metzner-Otto concept 3 Rushton turbine (Po= 4.9) standard configuration, d/d R = 0.4, 2 kw/m 3 Xanthan fermentation broth with pseudo-plastic flow behaviour: K = 30,000 mpa s m, m = 0.8 (Galindo et al., 989) 4000 Viscosity [mpa s] m 3 50 stirred tank 9 ~ n ~ V ~ n ~ V-2-2/9 / for (P/V ) = const. These large changes of viscosity ( 00m3 / = 8.2) over the scales don t make sense at all! Shear rate [/s] 44
23 Is there an alternative to describe the apparent viscosity for non-newtonian fluids? 45 General expression for apparent viscosity of non-newtonian fluids Power concept from Henzler (985, 2007) for stirred fermenters : 2 m P V K m (with = for mass transfer correlations) Developed and proven for stirred tanks from 50 to 80 m 3 in size. This correlation could also be verified for bubble columns. K consistency factor [Pa s m ] empirical coefficient [-] m flow behaviour index [-] P power [W] V filling volume [m 3 ] dynamic viscosity [Pa s] shear rate [/s] 46
24 Scale-up and -down of viscous fermentations Shake flask culture systems Technical scale stirred tank fermentor 47 Development of a correlation for the effective shear rate in shake flasks From dimensional analysis follows for shake flasks: m P V K m V d / 3 x m It was promising to find an equation similar to that for stirred tanks and bubble columns: 2 m P V K m d flask diameter [m] K consistency factor [Pa s m ] empirical coefficient [-] m flow behaviour index [-] P power [W] V filling volume [m 3 ] x empirical factor [-] shear rate [/s] Giese et al., Chem. Eng. Sci. (submitted) 48
25 Comparison of shake flask and stirred tank Shake flasks: shake flask Viscosity in [-] stirred tank m P V K m V d relative filling volume: 5% 20% 40% / 3 x m Stirred tanks: (P/V ) = const. Flask: = 2.06, x = Stirred tank: = Flow behavior index m [-] 2 m P V K m Increasing pseudo-plastic flow behaviour 49 Comparison of shake flask and stirred tank Shake flasks: shake flask Viscosity in [-] stirred tank m P V K m V d relative filling volume: 5% 20% 40% / 3 x m Stirred tanks: Flow behavior index m [-] 2 m P V K For highly pseudo-plastic fermentation broths the apparent 0.8 viscosity will be smaller (/2) in shake flasks than in stirred tanks. Taking into account the much larger 0.7 negative impact of apparent viscosity on the gas/liquid (P/V ) = const. mass transfer 0.6 in stirred tanks, Flask: scale-up = 2.06, of x = processes from Stirred tank: = shake flasks is very challenging! m Increasing pseudo-plastic flow behaviour 50
26 Empirical process development and scale-up Screening Process development Production Shake flask culture systems Bench scale Production scale 5 Knowledge based process development and scale-up Screening Process development Production Shake flask culture systems Bench scale Production scale We are looking forward to cooperating with you. 52
27 Thank you for your attention!!! 53
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