This presentation introduces the general principles of scaling strategies with stirred single-use bioreactors, discusses key engineering parameters, and concludes with a case study of how these strategies are applied to Mobius® bioreactors from 2 liters to 2000 liters. To learn more about this topic or collaborate with our technical experts, schedule an in-person or remote visit at our M Lab™ Collaboration Centers: www.merckmillipore.com/mlab

1. Merck KGaA
Darmstadt, Germany
Lenaig Savary, Senior Biomanufacturing Engineer
Scaling Strategies with
stirred single-use
bioreactors from bench
to clinical scale
Application to Mobius® Bioreactors from 2 L to 2000 L

2. 01
02
03
Agenda
2
General Principles
Key Engineering Parameters – Scaling
Strategies
Application to the Mobius® Bioreactors
from 2 L to 2000 L

3. Scaling Strategies with Stirred Single-use Bioreactors
No Linear Factors in Scaling Up
As surface area
increases, process
volume and process
complexity also
increase.
Surface Area
Single Cell
Shake Flask Bioreactor
Complexity

4. Scale-up
General Principles
Influencing Factors
Biological Factors Chemical Factors Physical Factors
Number of generations
Mutation probability
Contamination vulnerability
pH control agents
Medium quality
Water quality
Medium sterilization
Foam formation due to
surface tension changes
Tank configuration
Aeration (O2 supply, CO2
stripping, shear stress)
Agitation (homogenization
and shear stress)
Back pressure and
hydrostatic pressure
Temperature control and
heat transfer
Reference: M. Levin. Pharmaceutical Process Scale Up. 2nd Edition Taylor & Francis 2006

5. Scale-Up and Process Transfer
General Principles
Agitation and aeration
▪ Bulk liquid mixing and shear stress
→ Agitation
▪ Oxygen transfer and pCO2 removal
→ Aeration
Parameters that can be used for scale-up include:
• Geometric similarity—impeller design
• Mixing time—Reynolds number
• Tip speed (~ shear)
• Power input per volume (P/V)
• Oxygen mass transfer coefficient (kLa)
Ensuring circulation of nutrients, dissolved gasses and removing
metabolites is important in bioreactors
The most important criteria is mixing. Mixing is more difficult as scale increases.

6. Scaling Strategies with Single-use STR
Impossible to Keep All Scale-up Criteria Constant
Mixing time
Tip speed
(shear stress)
Power input per volume (P/V)
Oxygen mass transfer
coefficient KLa
Geometric similarity
It is critical to characterize key engineering parameters
and identify a relevant scaling factor according to bioreactor design.

7. 01
02
03
Agenda
7
General Principles
Key Engineering Parameters – Scaling
Strategies
Application to the Mobius® Bioreactors
from 2 L to 2000 L

8.  Vessel height to vessel diameter (H/D)
typically between 1:1 and 2:1
 Impeller diameter to vessel
diameter (Dimp/D)
50 L 200 L
H
D
Dimp
1000 L3 L
Keeping constant ratio in dimension
of bioreactors across the scales
Geometric Similarity
Scaling Strategies with Stirred Single-use Bioreactors

9. Reynolds Number
Dimensionless number to predict flow patterns
in turbulent conditions = Re > 10,000
Why is mixing a critical parameter ?
 Even distribution of nutrients,
 Minimizing waste concentration,
 Control of pH, DO and Temperature,
 Minimizing shear stress (impeller / fluid
dynamics)
 Proper mixing is more challenging at larger
scales than smaller scales
Scaling Strategies with Stirred Single-use Bioreactors
Mixing Time and Reynolds Number
𝑹𝒆 =
𝝆𝑵𝑫 𝟐
𝝁
Where ρ = Fluid density
N = Impeller agitation Rate
D = Impeller diameter
μ = Fluid dynamic viscosity
 Based on a cylindrical tank design with centered rotating impeller
 Does not take into account impeller design
Mixing Times: compare like for like !
Mixing time is empirically determined by the
decolorization method or the conductivity
method
Laminar flow
Re < 10
Transition field
10 < re < 104
Turbulent flow
104 < Re

10. Scaling Strategies with Stirred Single-use Bioreactors
Tip Speed (m/s)
▪ Criteria for shear force is typically 2 m/s maximum.
▪ The highest shear zones in a stirred-tank bioreactor are
often described as existing within the impeller zone.
Because the outer edges of the impeller blades create
shear as they rotate through the liquid, the impeller tip
speed is often considered during bioreactor comparisons:
𝑻𝒊𝒑 𝑺𝒑𝒆𝒆𝒅 = 𝝅 ∙ 𝑫 ∙ 𝑵
Where D = Impeller diameter
N = Impeller agitation rate
0.000
0.200
0.400
0.600
0.800
1.000
1.200
1.400
1.600
1.800
20 40 60 80 100120140160180200220240260280300320340360380400
TipSpeed(m/s)
RPM
Mobius® Bioreactor Tip Speed
Calculations
3L CellReady
50L CellReady
200L CellReady
Tip speed calculation does not take into account impeller design.
3 L
50 L
200 L

11. Power Input per Volume (P/V)
Scaling Strategies with Stirred Single-use Bioreactors
𝑃𝑜
𝑉
= 𝑁 𝑃 ∙ 𝜌 ∙ 𝑁3
∙ 𝐷5
Where Po = Ungassed power input
V = Liquid working volume
Np = Impeller power number
ρ = Fluid density
N = Impeller agitation rate
D = Impeller diameter 200 L Mobius® Bioreactor with bottom-mounted impeller
Choosing an agitation rate that matches energy
dissipation rate or power per unit volume (W/m3) is
a common approach.
Impeller design, fluid density, and agitation
rate are considered in the ungassed power per unit
volume (Po/V) equation
Reference: Xing Zizhuo et al. Biotechnology and Bioengineering, Vol 103, 2009. Stephen
Kaiser et al. Computational Fluid Dynamics Technologies and Applications, InTech, 2011
Using this scaling method, agitation rate is varied to maintain
similar ungassed power per unit volume across vessels and working volumes.
40 L
200 L
Easy
calculation
of P/V
Power Input Per Volume (P/V)
 Working volume
range
 Impeller always
immersed

12. Scaling Strategies with Single-use STR
Oxygen Mass Transfer Coefficient (kLa) and Oxygen Uptake Rate
 Physical kLa are determined for various Power/Volume (P/V) and gas flow rates (vvm) using the static gassing out
method:
kLa = Oxygen Transfer Rate / DO saturation concentration = OTR/C*
 Biological kLa depends on the clone and maximum viable cell concentration :
kLa BIO = Oxygen Uptake Rate / DO saturation concentration = OURmax/C*=(QO2 max). VCCmax /C*
• A-mAb case study[4] at 15.106 cells/mL with OUR of 1.5 mmol/L.h → kLa = 7 h-1
 Typical kLa for animal cell culture below 20.106 cells/mL : 1-10 h-1
Not always known!
To prevent hypoxia (oxygen limitation), available kLa for a bioreactor design
should be superior to the biological kLa demand of cell lines in culture. kLa > kLa BIO
[4] A-mab, a case study in Bioprocess Development, CMC Biotech working group 2009
Sufficient O2/Air delivery is required not only to support cell growth, metabolism and protein production but also to
prevent excessive CO2 in the media which can impact these critical performance endpoints[3]
[3] Marks, D.M. (2003). Equipment design considerations for large scale cell culture. Cytotechnology 42:21-33

13. 01
02
03
Agenda
13
General Principles
Key Engineering Parameters – Scaling
Strategies
Application to the Mobius® Bioreactors
from 2 L to 2000 L

14. Scaling Strategies with Stirred Single-use Bioreactors
Application to the Mobius® Bioreactors from 2 L to 2000 L
Parameters that can be used for scale-up include:
✓ Geometric similarity – Impeller Design
✓ Mixing Time - Reynolds Number
✓ Tip speed (~ shear)
✓ Power input per volume (P/V)
✓ Oxygen mass transfer coefficient (kLa)

15. Ratio
Working Volume : Total Volume 0.8 0.8 0.8 0.8 0.8
Impeller Diameter : Vessel Diam. 0.6 0.3 0.3 0.3 0.3
Vessel Height : Vessel Diameter 1.8:1 2.0:1 2.0:1 2.0:1 2.0:1
Liquid Height : Vessel Diameter 1.4:1 1.7:1 1.6:1 1.6:1 1.6:1
Dual sparger (open pipe/micro or ring) YES YES YES YES YES
Min – Max Working Volume (L) 1 – 2.4 10 – 50 40 – 200 200 - 1000 400 – 2000
Mobius® Bioreactor Portfolio Overview:
Scalable from 3 L to 2000 L
2,000 L1,000 L200 L50 L3 L
5:1 turn down ratio

16. Mobius® Single-Use Bioreactor Platform
Mobius® 3 L Bioreactor as a Reliable Scale-Down Model
Probes
• Electrochemical probes
• Sterile insertion in the hood
2 spargers
• Open pipe
• Microsparger
Sampling and Additions
• Addition lines with check valves
• Weldable open pipe for flexibility
Low Shear Marine Scoping Impeller
All weldable C-Flex® tubing
Working volumes
1 L – 2.4 L

17. Mobius® Single-Use Bioreactor
Hardware: Mobius® 50 L & 200 L Bioreactor
Bioreactor Tank
• Fully Jacketed Tank
• Double doors
• Window for visibility
Mobius® SensorReady
Monitoring & Sampling Loop
2 to 8 sensors. No change in
bag design.
Vent Heater
• Primary & back-up highly
hydrophobic Aervent® XL5 filters for
added security
Impeller
• Levitating impeller
• Bottom-mounted for
large operating range
(20% to 100%)
Spargers
• Open pipe
• Microsparger or Ringsparger
(CO2 stripping & O2)
40 L
200 L
HMI
MFCs
Pumps

18. Mobius® Single-Use Bioreactor Platform
Hardware: 1000 L and 2000 L Mobius® Bioreactor
without vent filters :
2.56 m / 3.03 m
with vent filters :
3.00 m / 3.48 m
Integrated
software and
automation
Open drawer for
easy Flexware®
assembly install &
removal
Door with wide sight
glass
Visual monitoring
of the culture

19. Parameters that can be used for scale-up include:
✓ Geometric similarity – Impeller Design
✓ Mixing Time - Reynolds Number
✓ Tip speed (~ shear)
✓ Power input per volume (P/V)
✓ Oxygen mass transfer coefficient (kLa)
Scaling Strategies with Stirred Single-use Bioreactors
Application to the Mobius® Bioreactors from 2 L to 2000 L

20. 0
20
40
60
80
100
120
140
0 5 10 15 20 25
MixingTime,seconds
Power Input, W/m3
Scalability of Mixing (conductivity)
3 L
50 L
200 L
1000 L
2000 L
Scaling Strategies with Stirred Single-use Bioreactors
Mixing in Mobius® Bioreactors
 Method using conductivity
 Fast and homogeneous mixing is
achieved across the scales, at low
power input
 Importance of the internal baffle

21. Scaling Strategies with Stirred Single-use Bioreactors
Mixing in Mobius® Bioreactors: Importance of the Internal Baffle
With
Baffle
Without
Baffle
Mixing
completed

22. Parameters that can be used for scale-up include:
✓ Geometric similarity – Impeller Design
✓ Mixing Time - Reynolds Number
✓ Tip speed (~ shear)
✓ Power input per volume (P/V)
✓ Oxygen mass transfer coefficient (kLa)
Scaling Strategies with Stirred Single-use Bioreactors
Application to the Mobius® Bioreactors from 2 L to 2000 L

23. Scaling Strategies with Stirred Single-use Bioreactors
Power / Volume – Tip Speed – Reynolds Number – Mobius® Bioreactors
Bioreactor W/m3 RPM
Tip Speed
(m/s)
Re
3 L
(Np =
0.3)
1 82 0.3 8,890
10 178 0.7 19,297
20 224 0.9 24,284
50 L
(Np =
3.2)
1 61 0.3 13,397
10 131 0.7 28,771
20 165 0.9 36,238
200 L
(Np =
4.0)
1 38 0.4 23,728
10 81 0.8 50,579
20 102 1.0 63,692
1000 L
(Np = 3.5)
1 33 0.5 48,097
10 72 1.1
104,940
20 90 1.3
131,174
2000 L
(Np =
3.3)
1 33 0.6 67,177
10 70 1.2
142,497
20 87 1.5
177,104
0
20
40
60
80
100
120
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
Power/Volume(W/m^3)
Tip Speed (m/s)
3 L 50 L 200 L 1000 L 2000 L

24. Parameters that can be used for scale-up include:
✓ Geometric similarity – Impeller Design
✓ Mixing Time - Reynolds Number
✓ Tip speed (~ shear)
✓ Power input per volume (P/V)
✓ Oxygen mass transfer coefficient (kLa)
Scaling Strategies with Stirred Single-use Bioreactors
Application to the Mobius® Bioreactors from 2 L to 2000 L

25. Case Study
Oxygen Mass Transfer
Constant parameters
P/V kLa
+
P/V vvm
+
Non constant parameters
kLa
vvm
Impossible to maintain P/V, vvm and kLa constant between scales
(impact on oxygenation and pCO2)

26. 2000 L
200 L
50 L3 L
P Value < 0,05
R² within specification
KLa = f (P/V ; Vs)
ANOVA
Validation
Oxygen Mass Transfer
Case study

27. KLa or VVM?
Proof of Concept: Comparison of two scale-up strategies
Oxygen Mass Transfer
27
Case study

28. Proof of Concept: Comparison of two scale-up strategies
Oxygen Mass Transfer
28
Mobius® 3 L Bioreactor
Wv = 2,197 L
Mobius® 200 L Bioreactor
Wv = 188 L
Stirring = 180 rpm
Pug = 9,39 W/m3
Stirring = 80 rpm
Pug = 10,29 W/m3
Gas = 75 mL/min
KLa = 6,07 hr-1
Gas = 5 (7,5) L/min
KLa = 24,66 (31,8) hr-1
O2
(gas)
O2
(gas)
VVM Approach
Case study

29. Proof of Concept: Comparison of two scale-up strategies
Oxygen Mass Transfer
29
Mobius® 3 L Bioreactor
Wv = 2,1 L
Mobius® 200 L Bioreactor
Wv = 180,4 L
Stirring = 180 rpm
Pug = 9,93 W/m3
Stirring = 78 rpm
Pug = 9,94 W/m3
Gas = 75 mL/min
KLa = 6,15 hr-1
Gas = 1,64L/min
KLa = 6,24 hr-1
O2
(gas)
O2
(gas)
KLa Approach
Case study

30. Oxygen Mass Transfer
Constant KLa Constant VVM
Similar VCD & viability profile for both scale-up strategy (not shown)
Same profile for the specific productivity (not shown) with constant kLa approach (3 L=200 L)
Final titer for kLa strategy more in line with the small scale
Case study

31. Oxygen Mass Transfer
Constant kLa
(4,39 to 6,30 h-1)
Constant VVM
(kLa : 28,18 to 28,79 h-1)1
2
1 2
The scale-up strategy with constant kLa is a successful new approach
-Better DO control (SP) with the kLa strategy
-Maximum O2 injection at 1,64L/min (VS 5-7,5L/min)
Example of O2 injection at maximum VCD (PD8)
Case study

32. Oxygen Mass Transfer
Similar specific
productivity
compared to the
reference scale
Lower 02 flow needed
(1,64L/min
instead of 5 to 7,5L/min)
at 200 L
HCD CHO
High VCD
High O2
consumption
New scale-up
strategy:
Constant kLa
Successful
Proof of concept
Case study

33. Cell Culture Performances with P/V as Scaling Factor
mAb Production with CHO-S Cell Line
2000 L
3 L
200 L

34. CONCLUSION
Scaling Strategies with Stirred Single-use Bioreactors
 Scalability of the Mobius® Bioreactor Platform demonstrated from 2 L to 2000 L
 Power per volume is a relevant scaling factor combined with kLa
 Knowledge and experience are nearly as important as the scaling parameters:
You know your cells, we know the process with Mobius® Bioreactors.
Collaboration is key for success!
References :
Odeleye et al 3L Cellready fluid dynamics Chem E Science 2014
Xing, Z. (2009). Scale-up Analysis of a CHO Cell Culture Process in Large-Scale Bioreactors. Biotechnology and Bioengineering 103:733-746.
Kaiser, S.D. et al, (2011) CFD for Characterizing Standard and Single-use Stirred Cell Culture Bioreactors. In Computational Fluid Dynamics Technologies and Applications, InTech
Marks, D.M. (2003). Equipment design considerations for large scale cell culture. Cytotechnology 42:21-33.
A-mab, a case study in Bioprocess Development, CMC Biotech working group 2009

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