The document discusses scaling up a biopharmaceutical manufacturing process from laboratory scale to 2000L commercial scale in single-use bioreactors. It describes optimizing cell expansion methods, oxygen transfer modeling and scale-up using constant KLa values rather than constant VVM, in-line PID regulation, and two-stage depth filtration clarification unit design. The goal is to enable flexible, fast production of APIs through clinical development to commercialization.

1. The life science business of Merck operates
as MilliporeSigma in the U.S. and Canada.
A scale-up to 2000L,
blindfolded!
Guillaume Plane
Global Development & Marketing Manager
Cyril Rols
Process Engineer, Team Leader

2. The life science business
of Merck KGaA, Darmstadt,
Germany operates as
MilliporeSigma in the U.S.
and Canada

3. A growing need for API through Clinical Development
Research Preclinical Phase I Phase II Phase III Commercial
3
3 A scale-up to 2000L, blindfolded!

4. A growing need for API through Clinical Development
4
4 A scale-up to 2000L, blindfolded!
Process Transfer and Scale-up is a keystone of Manufacturing

5. A growing need for API through Clinical Development
5
5 A scale-up to 2000L, blindfolded!
Process Transfer and Scale-up is a keystone of Manufacturing
Process
development
lab
Clinical
manufacturing
Commercial
manufacturing
(external or
internal)
Scale-up
3L → 2000L
Single Use 3L bioreactor
Single Use 2kL bioreactor

6. A growing need for API through Clinical Development
6
6 A scale-up to 2000L, blindfolded!
Process Transfer and Scale-up is a keystone of Manufacturing
ICH Q10:
The goal of technology transfer activities is to transfer product and process knowledge between
development and manufacturing, and within or between manufacturing sites to achieve product
realization. This knowledge forms the basis for the manufacturing process, control strategy, process
validation approach, and ongoing continual improvement.
A robust process is mandatory, and its scalability makes a difference
• Flexibility and speed allow production at the right time
• Ability to tech transfer to 2000L straight forward can make a difference

7. 7
Mastering Cell Expansion
Sparging and Stirring at 2000L scale
Clarification Design

8. 8
Mastering Cell Expansion
Sparging and Stirring at 2000L scale
Clarification Design

9. Mastering Cell Expansion
9
9 A scale-up to 2000L, blindfolded!
1/ Thawing in a
1mL cryovial
2/ Expansion in
shake flasks
3/ Expansion in a
WAVE BAG
4/ Expansion in 200-L
single-use bioreactor
5/ Seeding of 2000-L
single-use bioreactor
Seeding of a 2000-L bioreactor, starting from 1mL cryovial, as fast as possible
• Use of a 200-L bioreactor at N-1
• Respect of cells nutritional needs – exponentially growth
• Gaz exchanges are also determinant

10. Mastering Cell Expansion
1
0
10 A scale-up to 2000L, blindfolded!
1 - Volumes setting in shake flasks and wave bags
To get appropriate culture conditions for each container - T°C, CO2, stirring

11. Mastering Cell Expansion
1
1
11 A scale-up to 2000L, blindfolded!
2 – Stability study at development scale
To set a maximum aging after cells thawing

12. Mastering Cell Expansion
1
2
12 A scale-up to 2000L, blindfolded!
Case study: Stability testing for a CHO Cell line

13. Mastering Cell Expansion
1
3
13 A scale-up to 2000L, blindfolded!
Case study: Stability testing for a CHO Cell line

14. Mastering Cell Expansion
1
4
14 A scale-up to 2000L, blindfolded!
Case study: Stability testing for a CHO Cell line

15. Mastering Cell Expansion
1
5
15 A scale-up to 2000L, blindfolded!
Case study: Stability testing for a CHO Cell line

16. Mastering Cell Expansion
1
6
16 A scale-up to 2000L, blindfolded!
Case study: Stability testing for a CHO Cell line

17. Mastering Cell Expansion
1
7
17 A scale-up to 2000L, blindfolded!
Case study: Stability testing for a CHO Cell line

18. Mastering Cell Expansion
1
8
18 A scale-up to 2000L, blindfolded!
Case study: Stability testing for a CHO Cell line

19. Mastering Cell Expansion
1
9
19 A scale-up to 2000L, blindfolded!
Case study: Stability testing for a CHO Cell line

20. Mastering Cell Expansion
2
0
20 A scale-up to 2000L, blindfolded!
Case study: Stability testing for a CHO Cell line

21. Mastering Cell Expansion
2
1
21 A scale-up to 2000L, blindfolded!
3 : Definition of a viable cell density target
To get enough cells reaching the seeding specifications (VCD, volume)
while keeping exponential growth state.

22. Mastering Cell Expansion
2
2
22 A scale-up to 2000L, blindfolded!
The aim of a passage is to keep cells in fresh
medium in order to favor exponential growth
The aim of an amplification is to increase total cell
quantity at a given Viable Cell Density
Slope increase =
acceleration of cell division
Slope stabilization = constant
growth speed = exponential phase

23. 23
Mastering Cell Expansion
Sparging and Stirring at 2000L scale
Clarification Design

24. Sparging and stirring at 2000L scale
2
4
24 A scale-up to 2000L, blindfolded!
Systems characteristics have to be considered
 Mixing efficiency can vary: Impeller type, position, size/tank….
 Sparging efficiency can vary: Sparger size, sparger position, bubble size…
 Fluid movement and G/L interaction can vary: Baffles type, position, interactions
Regulation systems and equipement capacities do not vary linearly

25. Sparging and stirring at 2000L scale
2
5
25 A scale-up to 2000L, blindfolded!
Tools:
• Oxygen Mass Transfer Modeling, before production
• In-line PID regulation optimization, during production
Regulation systems and equipement capacities do not vary linearly
Design-Expert® Software
Factor Coding: Actual
KLa (hr-1)
Design points above predicted value
Design points below predicted value
34.71
4.07
X1 = A: P/V
X2 = B: Vs
0.000145546
0.000291093
0.000436639
0.000582185
0.000727732
1
4.8
8.6
12.4
16.2
20
0
10
20
30
40
KLa(hr-1)
A: P/V (W/m3)
B: Vs (m/s)

26. Sparging and stirring at 2000L scale
2
6
26 A scale-up to 2000L, blindfolded!
2) Modeling
KLa = f (P/V ; Vs)
Design-Expert® Software
Factor Coding: Actual
KLa (hr-1)
Design points above predicted value
Design points below predicted value
34.71
4.07
X1 = A: P/V
X2 = B: Vs
0.000145546
0.000291093
0.000436639
0.000582185
0.000727732
1
4.8
8.6
12.4
16.2
20
0
10
20
30
40
KLa(hr-1)
A: P/V (W/m3)
B: Vs (m/s)
1) KLA measurement
3L – 50L – 200L – 2KL
2KL
200L
50L
3L

27. Sparging and stirring at 2000L scale
2
7
27 A scale-up to 2000L, blindfolded!
1 > 20 W/m3
similar for all Mobius® systems
Dependsonmobiussystem
9 points plan
Used for modeling

28. Sparging and stirring at 2000L scale
2
8
28 A scale-up to 2000L, blindfolded!
Software
ctual
above predicted value
below predicted value
0.00031
0.000568
0.000826
0.001084
0.001342
0.0016
1
4.8
8.6
12.4
16.2
20
0
5
10
15
20
25
KLa(hr-1)
A: P/V (W/M3)
B: Vs (m/s)
Design-Expert® Software
Factor Coding: Actual
KLa (hr-1)
Design points above predicted value
Design points below predicted value
34.71
4.07
X1 = A: P/V
X2 = B: Vs
0.000145546
0.000291093
0.000436639
0.000582185
0.000727732
1
4.8
8.6
12.4
16.2
20
0
10
20
30
40
KLa(hr-1)
A: P/V (W/m3)
B: Vs (m/s)
pert® Software
ding: Actual
points above predicted value
points below predicted value
V
9.17849E-005
0.00018357
0.000275355
0.000367139
0.000458924
1
4.8
8.6
12.4
16.2
20
0
5
10
15
20
25
30
KLa(hr-1)
A: P/V (W/m3)B: Vs (m/s)
Design-Expert® Software
Factor Coding: Actual
KLa (hr-1)
Design points above predicted value
Design points below predicted value
7.81
2
X1 = A: P/V
X2 = B: Vs
2.26125E-005
3.95718E-005
5.65311E-005
7.34905E-005
9.04498E-005
1
4.8
8.6
12.4
16.2
20
2
3
4
5
6
7
8
9
KLa(hr-1)
A: P/V (W/m3)B: Vs (m/s)
2KL 200L
50L 3L
(small bubbles)
%CV = 2 – 10,3%
P Value < 0,05
R² in the spécification
KLa = f (P/V ; Vs)
ANOVA
Validation

29. KLa or VVM?
Proof of Concept: Comparison of two scale-up strategies
Sparging and stirring at 2000L scale
2929 A scale-up to 2000L, blindfolded!

30. Proof of Concept: Comparison of two scale-up strategies
Sparging and stirring at 2000L scale
3030 A scale-up to 2000L, blindfolded!
Mobius® 3L Bioreactor
Wv = 2,197 L
Mobius® 200L Bioreactor
Wv = 188L
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

31. Proof of Concept: Comparison of two scale-up strategies
Sparging and stirring at 2000L scale
3131 A scale-up to 2000L, blindfolded!
Mobius® 3L Bioreactor
Wv = 2,1 L
Mobius® 200L 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

32. A scale-up to 2000L, blindfolded!
Sparging and stirring at 2000L scale
32
Constant KLa
(4,39 to 6,30 h-1)
Constant VVM
(KLa : 28,18 to 28,79 h-1)1
2
1
1
2
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)
DO
set point

33. A scale-up to 2000L, blindfolded!
Sparging and stirring at 2000L scale
33
Constant KLa Constant VVM

34. A scale-up to 2000L, blindfolded!
Sparging and stirring at 2000L scale
34
Constant KLa Constant VVM

35. A scale-up to 2000L, blindfolded!
Sparging and stirring at 2000L scale
35
Similar specific
productivity
compared to the
reference scale
Maximum 02 MFC
allowed
1,64L/min
(instead of 5 to
7,5L/min)@200L
HCD CHO
High VCD
High O2
consumption
new scale-up
strategy
Constant KLa
pCO2 control
strategy have
to be
improved
Successful
Proof of concept

36. A scale-up to 2000L, blindfolded!
Sparging and stirring at 2000L scale
36
In-line PID regulation optimization can
help, during production
Proportional-integral-derivative parameters are not directly
transposable between two software, neither between two scales

37. A scale-up to 2000L, blindfolded!
Sparging and stirring at 2000L scale
37
Proportional-integral-derivative parameters are not directly
transposable between two software, neither between two scales
The Ziegler & Nichols tuning method
Optimization of PID for oxygen regulation has to be performed in-culture with high
oxygen consumption levels: during expansion phase, before reaching maximum VCD

38. A scale-up to 2000L, blindfolded!
Sparging and stirring at 2000L scale
38
2) Seeking the ultimate gain
I and D values are first voluntary set to zero.
The P gain (Kp) is then increased until it
reaches the ultimate gain (Ku): quick, stable
and consistent oscillations.
3) Calculation methods
PID from Ku and Tu (period of oscillation)
1) Preliminary setting
Maximum oxygen flow rate for DO regulation should not be limiting, it is set at high value
Dead band is set to allow a continuous action of the three parameters

39. 39
Mastering Cell Expansion
Sparging and Stirring at 2000L scale
Clarification Design

40. A scale-up to 2000L, blindfolded!
Clarification Design – First step for DSP scale-up
40
Two stage depth-filters in line
Ex: A1HC / D0HC 1:1
0.5/0.2 filter
Ex: OPTICAP XLT30
Harvest
clarified
Harvest
End of
culture

41. A scale-up to 2000L, blindfolded!
Clarification Design – First step for DSP scale-up
41
SU 2000-L
bioreactor
Skid CL3 clarification
Storage tank

42. A scale-up to 2000L, blindfolded!
Clarification Design – First step for DSP scale-up
42
Max pressure (bar)
Volume process (L)
Filtering area (m2)
Process time (h)
Filtering capacity (L/m2)
Resistance (bar/LMH)
Resulting pressure (bar)
Resulting flux (LMH)
Resistance = f (load)
Pressure and volume monitoring during
development studies does help
Result: sizing from DEVT data with safety margin and
appropriate choice in cassette references and surfaces

43. From Cell line development to
GMP Manufacturing
Three Biodevelopment Centers
EU
NA
Successful process
development lab and GMP
Facility since 2012
Expansion to a Process
development lab in
Burlington (MA) since 2017
Expansion to a lab and a GMP
facility in Shanghai since
2017
CN
4343 A scale-up to 2000L, blindfolded!

44. © 2019 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.
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