scale-up & scale-down between the two worlds of shaken … aachen.pdf · 1.00e+01 1.00e+02...

www.avt.rwth-aachen.de

Prof. Dr.-Ing. Jochen Büchs

AVT - Biochemical Engineering, RWTH Aachen University

Sammelbau Biologie, D - 52074 Aachen, Germany

e-mail: [email protected]


Scale-up & scale-down between the two

worlds of shaken and stirred bioreactors

CLIB-Forum Scale-Up & Scale-Down, Monheim 3. 4. 2014


2

Traditional way of process development

Production scaleShake flaskculture systems

Bench scale

Screening Process development ProductionScreening Process development Production

Clearly separated tasksperformed by people with different educational background


3

Current way of process development

Production scaleBench scale

Screening Process development Production(micro bioreactors) (mini bioreactors)

Screening Process development Production

Larger number of smaller parallel bioreactors

Micro titre plateculture systems


4

Exp

erim

enta

l th

rou

gh

pu

t

On-line measurement, feeding and controloptions

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Screening

Processdevelopment

High throughput high information content


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Exp

erim

enta

l th

rou

gh

pu

t

M

Scale-up &

Scale-down

Screening

Processdevelopment


High throughput high information content


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Exp

erim

enta

l th

rou

gh

pu

t

M

High throughput and (!) high information content

On-line monitoring

feeding

BioLector(Pro)

RAMOS

FeedBeads & FeedPlates


7

M

Important engineering parameters of a bioprocess

L- and m3-scale1) specific power consumption, (P/VL)

2) O2 - supply, OTRmax or kLa

3) ventilation (stripping of CO2, H2O,

alcohols etc.)

4) degree of mixing and homogeneity

5) hydromechanical stress (damage of

cells, control of morphology), (P/VL)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

It is in general only possible to keep one single

parameter constant during change of scale.



The most relevant parameter has to be found

for each specific fermentation system.



The most relevant parameter has to be found

for each specific fermentation system.

Well established methodology with the

exception of some specific fields


8

M


L- and m3-scale1) specific power consumption, (P/VL)



alcohols etc.)








viscosity


9


µL- and mL-scale1) specific power consumption, (P/VL)



alcohols etc.)








viscosity


10

Quantification of specific power input (P/VL) [kW/m3]

based on torque measurement

Multi flask measuring device

One flask device

Büchs et al., Biotechnol. Bioeng. 68 (2000) 589-593


11

0

2

4

6

8

10

12

80 120 160 200 240 280 320

Shaking frequency (n) [rpm]

Sp

ec

. p

ow

er

inp

ut

(P/V

)

[kW

/m³]

95.6 mPas

59.3 mPas

34.3 mPas

17.4 mPas

4.4 mPas

0.8 mPas (water)

viscosity

Influence of viscosity on spec. power input1 l flask, 100 ml filling volume, 5 cm shaking diameter

Same order of magnitude as in larger

e.g. stirred tank reactors !!



12

0

2

4

6

8

10

12

80 120 160 200 240 280 320


Sp

ec

. p

ow

er

inp

ut

(P/V

)

[kW

/m³]

95.6 mPas

59.3 mPas

34.3 mPas

17.4 mPas

4.4 mPas

0.8 mPas (water)

viscosity

Influence of viscosity on spec. power input1 l flask, 100 ml filling volume, 5 cm shaking diameter




0.01

0.1

1

10

1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06

Betriebspkt "außer Phase"

100 ml shaking flask

250 ml "

300 ml "

500 ml "

1000 ml "

2000 ml "

"in phase" operating conditionsPo = 70 Re -1 + 25 Re -0,6 + 1.5 Re -0,2

101 102 103 104 105 106

101

100

10-1

10-2

Shaking diameter: 2.5 - 5 cm, viscosity: 0.8 - 200 mPa·s, shaking frequency: 80 - 400 rpm

Ne‘

=

[

-]

P

·

n3

· d

4·

VL

1/3

Reynolds number (Re = n·d2· / ) [-]

Mo

dif

ied

po

wer

nu

mb

er

(Po

= P

/ ·n

3 ·d4

·VL1/

3 )

[-]

turbulent flow

60,0

00

Non dimensional correlation of power input measurements in shake flasks

Peter et al., Biotechnol. Bioeng. 93 (2006) 1164-1176

No geometric similarity


14

Difference between shaken and stirred bioreactor

Shake flask Stirred bioreactor

M

No significant difference

in volumetric power input

(P/VL)


15





alcohols etc.)








viscosity


16

Operating conditions:

flask volume: 250 mL

shaking diameter: 2.5 cm

shaking frequency: 200 rpm

filling volume: 26 mL

Liquid distribution

in shake flasks

x-axis [cm]

y-axis [cm]

0

1

2

3

4

5

6

Height [cm]

Maximalliquid height

(3.07 cm)

-4

-3

-2

-1

0

1

2

3

4

-4 -3 -2 -1 0 1 2 3 4

axis ofrotation

A B

View: A-B

1

2

3

4

0

1

2

3

4

5

6

hei

gh

t

[cm

]

maximum liquid height

(3.07 cm)

y-ax

is

[cm

]

x-axis [cm]

axis of rotation

sectional drawing A - B

-4

-3

-2

-1A B

-4 -3 -2 -1 0 1 2 3 4

Büchs et al., Bioproc. Biosystems Eng. 34 (2007), 200–208


17

200 120010008006004000

bulk liquid surface - area

film + bulk liquid surface - area

0

200

400

600

800

1000

1200

Measured mass transfer area a [m2/m3]

250 ml- flask without baffles, 10 - 50 ml filling volume, 2.5 - 5 cm shaking diameter

Relevant specific mass transfer area in hydophilic shaking flasks

Cal

cula

ted

mas

s tr

ansf

er a

rea

a[m

2 /m

3 ]

Maier and Büchs, Biochem. Eng J. 7 (2001) 99-106


18

Influence of the flask surface properties on the OTR

shaking diameter d0 = 4.2 cm, filling volume VL = 26mL

300250200150100500

5

10

15

20

Shaking frequency n [1/min]

normal hydrophilicflask

hydrophobic flaskOxy

gen

tra

nsf

er r

ate

OT

R[m

mo

l/l/h

]

normal hydrophilicflask

Max

imu

m o

xyg

en t

ran

sfer

cap

acit

y

(OT

Rm

ax)

[m

mo

l/L/h

]

Disposable plastic flasks (hydrophobic) have a

lower OTRmax than hydrophilic glass flasks!


Maier and Büchs, Biochem. Eng J. 7 (2001) 99-106


Typ

ical

tre

nd

of

OT

Rm

ax in

a s

tirr

ed t

ank

[per

cen

tag

e o

f O

TR

ma

x,

1m

Pa

s]

Max

imu

m o

xyg

en t

ran

sfer

cap

acit

y(O

TR

max

) [

mm

ol/L

/h]

0 10 20 30 40 50 60 70 8020

25

30

35

40

45

0

20

30

35

40

100

Viscosity () [mPa·s]

Oxygen transfer in bioreactors as function of viscosity

80

60

40

250 mL shake flask20 mL filling volume

350 rpm

91 % of 1 mPa·s

4.7 % of 1 mPa·sstirred tank

bioreactor

19Giese et al., Biotechnol. Bioeng. 111 (2014), 295–308


20


M

Flask wall surface property

play a crucial role for OTRmax.

OTRmax (at 1 mPa·s) slightly

superior in stirred compared to

unbaffled shaken bioreactor.

Superior performance of shake

flask at elevated viscosities.

Shake flask Stirred bioreactor


21





alcohols etc.)








viscosity


22

with OTRmax maximum oxygen transfer capacity [mol/L/h]

kLa mass transfer coefficient [1/s]

(P/VL) specific power input [kW/m3]

ug superficial gas velocity [m/s]

a, b broth and reactor dependent empirical parameters [-]

bg

a

ØLLmax uV/P~ak~OTR

Oxygen mass transfer in a bubble aerated stirred tank bioreactorOxygen mass transfer in a bubble aerated stirred tank bioreactor


kLa: mass transfer coefficient [1/s]

ug: superficial gas velocity [m/s]

Specific power input (P/VL)Ø [W/m3]

10

1

0.1

100 1000

k La

/ ug

0.55

4

airoxygen

0.5 M Na2SO4 solution

(after Moucha et al., 1995)

Strong correlation between oxygen mass transfer (kLa) and

power input (P/VL) in bubble aerated stirred tank bioreactors

23


Cavity behind

a stirrer blade

Power input is required to disperse the

bubbles in the vicinity of the stirrer blades.

24

M

Strong correlation between oxygen mass transfer (kLa) and

power input (P/VL) in bubble aerated stirred tank bioreactors


25

Is oxygen mass transfer (kLa) a strict function of power

input (P/VL)Ø in shake flask bioreactors ?

Shaking frequency (n) = const.

Shaking diameter (d0) = const.

Relative filling volume (VL/d3) = const. kLa

d - 0.57

d

d d

(P/VL)Ø

d 1.6

Shake flasks are surface aerated, not bubble aerated !


Relation between oxygen transfer and spec. power input

Oxygen transfer (OTRmax, kLa) and spec. power input (P/VL)Ø

of shake flask bioreactors are not directly related (!), in

contrast to bubble aerated bioreactors like stirred tanks.

OTRmax ~ kLa ≠ f (P/VL)Ø

Interfacial area for momentum transfer

(power input and hydromechanical stress)

Interfacial area for gas/liquid mass transfer

Oxygen transfer and power input are locally

separated in shake flask bioreactors !

26


27





alcohols etc.)








viscosity


28

0,000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0 200 400 600 800 1000


50 mm

25 mm

12.5 mm

6 mm

3 mm

Shaking diameter

Maximum oxygen transfer capacities in 96 well micro titre plates

200 µl filling volume

Max

imu

m o

xyg

en t

ran

sfer

cap

acit

y (O

TR

max

)

[mo

l/L/h

]

It is impossible to supply

bacterial and yeast sufficiently

with oxygen in 96 well MTPs !

Hermann et al., Biotechnol. Bioeng. 81 (2003) 178-186


29

Introduction of Baffles – Sets of Geometries

Reference: 48-well micro titer plate with round well geometryAlternative prototypes: constant cross sectional area (of 112 mm2) 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. 103 (2009) 1118-1128


30

Introduction of Baffles – Multiform Baffles



31

Commercialized by

m2p-labs,

Aachen

“Flower plates”48 well design with optodes at the bottom for pH and DOT measurement



32Culture time [h]

Oxy

gen

tra

nsf

er r

ate

[m

mo

l/L/h

]

E. coli K12, mineral medium with 20 g/L glucose, 200 mM MOPS, 37°C, 1000 rpm

Sufficient oxygen supply by novel MTP design

OTRmax for 200 µL in normal 96 well MTP



33


Shaken bioreactor Stirred bioreactor

M

With carefully designed

baffles shaken bioreactors

can reach OTRmax similar to

stirred bioreactors.


34





alcohols etc.)








viscosity


35

(P/V)max

(P/V)Ø 2 - 7

M

(P/V)max

(P/V)Ø 50 - 100

M

Hydromechanical stress in shake flasks and stirred tanks

(P/VL)max

(P/VL)Ø

2 - 7?

Using the larges possible geometric element

(reactor wall and bottom) for power input results in

its most even introduction into the culture liquid.


(P/VL)max

(P/VL)Ø

50 - 100(P/VL)max

(P/VL)Ø

50 - 100


36

(P/V)max

(P/V)Ø 2 - 7

M

(P/V)max

(P/V)Ø 50 - 100

Hydromechanical stress in shake flasks and stirred tanks

This significant difference is unavoidable and

may cause major scale-up problems !

(P/VL)max

(P/VL)Ø

50 - 100(P/VL)max

(P/VL)Ø

2 - 7Same order of magnitude



37





alcohols etc.)








viscosity


Flow curves for

fermentation-broths of

Streptomyces tendae

at different times

Dyn

amic

vis

cosi

ty

[Pa·

s]

Shear rate [1/s]10 100 1000

1

0.1

0.01

0.001

200 L stirred tank, 24°C

agitation rate 400 – 800 1/min

Time[h]

38

Viscous fermentation

broths in general show

non-Newtonian

pseudo-plastic or

shear-thinning

flow behaviour !


Shear thinning results in reduced viscosity

(pseudo-plastic flow behaviour)

Static sample

vSample subjected to a flow field

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

1mK

Power law (or Oswand‘s law)

model for the viscosity of

fermentation broths (valid for

typical shear rates applied):

dynamic viscosity [mPa·s]

K consistency factor [mPa·sm]

m flow behaviour index [-]

shear rate [1/s]

Dyn

amic

vis

cosi

ty

[Pa·

s]

Shear rate [1/s]10 100 1000

1

0.1

0.01

0.001

200 L stirred tank, 24°C

agitation rate 400 – 800 1/min

Time[h]

40


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:

dynamic viscosity Pa·s

shear stress Pa

shear rate 1/s

K consistency factor Pa·sm

m flow behaviour index -

n agitation rate 1/s

A coefficient -

nA with A 11

41

However, this is not justified any

more, according to the newest

findings!


1000

Measured and calculated shear rates after the Metzner-Otto

concept for a non-Newtonian liquid in the turbulent flow regime

Shear rates calculated from heat transfer measurements (Henzler, 2007)

Agitation rate (n) [rpm]

Sh

ear

rate

()

[1/s

]

10

100

0,1 1 10101

102

VL= 0.92 L DR= 0.114 m

VL= 2.5 m3

DR= 1.59 m

0.1 1 10

Metzner-Otto concept

n


L

53

L V

dnPo

V

P

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:

1) Turbulent conditions power number (Po) = const.

2) Geometric similarity (VL DR3 and d DR)

3) Constant volumetric power input; (P/VL) = const.

Po power number -

VL filling volume m3

d stirrer diameter m

DR vessel diameter m

P/VL volumetric power input kW/m3

n agitation rate 1/s

n ~ DR-2/3 ~ VL

-2/9

43


0

500

1000

1500

2000

2500

3000

3500

4000

0 50 100 150 200 250 300

Operating points calculated by the Metzner-Otto concept3 Rushton turbine (Po= 4.9) standard configuration, d/DR = 0.4, 2 kW/m3

Xanthan fermentation broth with pseudo-plastic flow behaviour:

K = 30,000 mPa·sm, m = 0.18 (Galindo et al., 1989)

Shear rate [1/s] 44www.avt.rwth-aachen.de

Vis

cosi

ty

[mP

a·s]

100 m3 50 L 1 L stirred tank

These large changes of viscosity

(100m3/1L = 8.2) over the scales

don’t make sense at all !

~ n ~ VL-2/9

for (P/VL) = const.

9/2-LV~n~


45

Is there an alternative to describe the

apparent viscosity for non-Newtonian fluids ?


“Power concept“ from Henzler (1985, 2007) for stirred fermenters :

K consistency factor [Pa·sm]

L empirical coefficient [-]


P power [W]

VL filling volume [m3]

dynamic viscosity [Pa·s]

shear rate [1/s]

(with L = 1 for mass transfer correlations)

General expression for apparent viscosity of non-Newtonian fluids

46

1m1

L1m2

K

VPL

Developed and proven for stirred tanks from 50 L to 80 m3 in size.

This correlation could also be verified for bubble columns.


47

Scale-up and -down of viscous fermentations

Technical scalestirred tank fermentor

Shake flask

culture systems


48

1m

x3/1

L1m

1

L1m

1

d

V

K

VPL

Development of a correlation for the effective shear rate

in shake flasks

From dimensional analysis follows for shake flasks:

d flask diameter [m]




P power [W]


x empirical factor [-]

shear rate [1/s]

1m

1

L1m

2

K

VPL

It was promising to find an

equation similar to that for stirred

tanks and bubble columns:

L

x

d flask diameter [m]




P power [W]


x empirical factor [-]

shear rate [1/s]

Giese et al., Chem. Eng. Sci. (submitted)


0.2 0.4 0.6 0.8 1.00.5

0.6

0.7

0.8

0.9

1.0

Flow behavior index m [-]

5% 20% 40%

relative filling volume:

(P/VL) = const.

Flask: L = 2.06, x = - 0.331

Stirred tank: L = 1

Comparison of shake flask and stirred tank

49

1mx

3/1L

1m

1

L1m

1

d

V

K

VPL

1m1

L1m2

K

VPL

Shake flasks: Stirred tanks:

Increasing pseudo-plastic flow behaviour

shak

e fl

ask

stir

red

tan

kV

isco

sity

in

[-]


0.2 0.4 0.6 0.8 1.00.5

0.6

0.7

0.8

0.9

1.0

Flow behavior index m [-]

5% 20% 40%

relative filling volume:

(P/VL) = const.

Flask: L = 2.06, x = - 0.331

Stirred tank: L = 1

Comparison of shake flask and stirred tank

50

1mx

3/1L

1m

1

L1m

1

d

V

K

VPL

1m1

L1m2

K

VPL

Shake flasks: Stirred tanks:

Increasing pseudo-plastic flow behaviour

shak

e fl

ask

stir

red

tan

kV

isco

sity

in

[-]

For highly pseudo-plastic fermentation broths the

apparent viscosity will be smaller (1/2) in shake flasks

than in stirred tanks. Taking into account the much larger

negative impact of apparent viscosity on the gas/liquid

mass transfer in stirred tanks, scale-up of processes from

shake flasks is very challenging !


51

Empirical process development and scale-up


Bench scale



52


Bench scale


We are looking forward to cooperating with you.

Knowledge based process development and scale-up


Thank you for your

attention !!!53

scale-up & scale-down between the two worlds of shaken … aachen.pdf · 1.00e+01 1.00e+02...

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