The Biocontinuum™ Seed Train Platform

Merck KGaA
Darmstadt, Germany
Habib HORRY, Ph.D.
Asia BioForum, Fall 2020
Driving value and innovation toward the evolution of
upstream processes
The BioContinuumTM
Seed Train Platform

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

Speed
Reduce new facility build
times by 70%. Compress
production lead time by
80%.
Quality
10X robustness.
90% reduction in cost of
poor quality.
Flexibility
Reduce product change-over
time by 90%.
Cost
90% reduction in cost to
manufacture and CAPEX.
Business
Drivers
Market
Growth
Uncertainty
New Product
Classes
Cost
Pressure
Market
Trends
Process
Intensification
Process
Analytics
Software &
Automation
Key
Enablers
Single Use
* Source: BioPhorum (BPOG)
https://www.biophorum.com/category/resources/technology-roadmapping-resources/introduction/
Market Trends, Business Drivers and Key Enablers to
Drive Next Generation BioProcessing:
BioContinuumTM Seed Train Platform | Fall 20203

Faster
Going as quickly as possible to purification
Higher
Getting as much protein as possible
Stronger
Achieve the highest product quality
Less contamination, Aggregates, Charge variants,
Glycosylation patterns, Cell debris, Insoluble impurities (DNA, HCP)
Upstream Process Intensification
What does that mean concretely?

Upstream Process Intensification for mAbs
A Multitude of Strategies all based on perfusion technology
5
Batch
x
1-2ml
each
10-30 x106 vc.ml-1
N-1
or
x
Batch
N
Fed Batch
MCB/MWCB INOCULATION TRAIN
SEED TRAIN
PRODUCTION
CRD
Perfusion
CRD
50-100 x106vc.ml-1
or or
4.5ml 50-500ml bagCRD
CRD
Perfusion
N
CRD
Continuous protein harvest
• Steady State Perfusion
• Dynamic Perfusion
Single protein harvest
• High Seed Fed batch
• Ultrahigh VCD Fed-Batch
• Concentrated Fed-Batch
Production
medium
Cryo Medium
Perfused Seed Train Processes
Can be utilized with any production method
Perfusion-based Production and Harvest processes
Continuous vs. single protein harvest, High cell densities, long durations
Expansion
medium
Cell Culture Media Formulations
Enabling intensified seed and production trains
High Qp
Low CSPR
Low OUR
High Shear resistance
Perfusion Optimized Cell Line
Maximizing protein yield and quality in perfusion operations

Merck KGaA
Darmstadt, Germany
Poll Question

Evolving Guidelines for Next Generation Processes7
Poll Question
1. Where are you using (or going to use) perfusion in your process?
1) Seed Train (N-1 perfusion)
2) High Density Cell Banks
3) Production steady-state
4) Production dynamic perfusion
5) Not using / going to use perfusion at all

0
Agenda
Perfused Seed Train Process
Modeling1

Perfused Seed Train
Different Scenarios
7
14 days
10 days
Conventional
Fed-Batch
(~ 0.2 E6 vc/mL)
High Seed
Fed-Batch
(~ 5 E6 vc/mL)
Conventional
(Batch N-1)
3 days
10L
3 days
100mL
3 days
30mL
5 days
60L,200L
Compressed
Seed Train
High Seed
(N-1 only)
High Seed
(N-1 & N-2)
3 days
400mL
3 days
2L
125mL 500mL 2X500mL 2X3L 20L
200L
2000L
7 days
10L
20L
3 days
10L
20L
9 days
60L,200L
200L
7 days
10L
20L
4 days
200L
200L
Same as above
Same as above
Same as above
2000L
Each model contains a
traditional downstream!
1 vs. 6 pack bioreactors
P:S ratio: 1:1 – 2:1 – 3:1

Constant Production Titer
Single Bioreactor scenario
13.5% reduction
in CoGs
38% Throughput
increase
Decreased duration
Perfused seed train allows for
more batches per year
No protein titer increase and shorter duration
CoG’s ($/g) 116 117 101 102
Throughput (kg/yr) 90 90 125 125
Batches/yr 18 18 25 25

Single Bioreactor scenario
Increased Production Titer 22-51% reduction
in CoGs
50-200%
Throughput
increase
Depending on titer increase
No additional batches
required
Increased Protein Titer and same duration
CoG’s ($/g) 116 101 120 91 76 57
Throughput (kg/yr) 90 125 90 135 180 271
Batches/yr 18 25 18 18 18 18

6-Pack Bioreactor scenario
Increased Production Titer
7-34% reduction
in CoGs
11-39% Throughput
increase
• Less batches per year than
traditional
Increased Protein Titer and same duration
Production/Seed 1:1
CoG’s ($/g) 67 85 85 62 52 44
Throughput (kg/yr) 496 276 276 413 551 689
Batches/yr 99 55 55 55 55 55

13
Sensitivity Analysis
Media cost has moderate impact…as well
Media cost doesn’t significantly impact the overall cost of goods
CSPR .03
VVD control manual
Production: High Seed
CSPR .03
VVD control manual
Production: High Seed
Model: Perfused seed train pN-1 & pN-2

Summary
14
Perfused seed train:
 Lower cost of manufacturing
 Increase product throughput
 while keeping production bioreactor in
fed batch mode
Potential for achieving higher titers,
especially when incorporating a
dedicated media platform
If higher titers observed:
• Adding additional seed trains and production bioreactors at
1:1 ratio brings the most benefit
• CoG’s reduced 24-52%, kg/yr increase 65-230%
• Benefits even at 3:1 and 2:1, with additional benefit of
needing less batches per year
If higher titers are not observed:
• Single bioreactor scenarios still see CoG’s reduced by 13.5%
and throughput increase of 39% at shorter high seed FB
duration
• Adding additional seed trains and production bioreactors
increases throughput

Poll Question
2. What considerations do you have on implementation of N-1 perfusion? (multiple
choices)
1) Footprint of manufacturing equipment
2) Process complexity and time duration
3) Cost of media and/or consumables
4) Impact on production (titer and/or product quality)
5) Other
6) N-1 perfusion is not applicable to my process.

0
Agenda
Cellvento® 4CHO-X Expansion
Medium2

Cell Culture Media requirements for intensified upstream processing
VCD
1 2 3 4
 No adaptation to Perfusion
production
 No cell damage during
freezing and thaw
 Fast growth with minimum or
zero lag phase
 Constant growth rate and
specific productivity over
thaw, expansion, and
production
EX-CELL® Advanced
18 BioContinuumTM Seed Train Platform | Fall 2020

E xp a n sio n in E xM E x p a n s io n in E x -C E L L ® A d v a n c e d H D P e rfu s io n M e d iu m
0 5 1 0 1 5
0
2 0
4 0
6 0
8 0
5 0
6 0
7 0
8 0
9 0
1 0 0
d a y s
VCD[10
6
VC/ml]
Viability[%]
0 5 1 0 1 5
0
2 0 0
4 0 0
6 0 0
d a y s
IgG[mg/l]
N-1 N N-1 N
Cellvento® 4CHO-X Expansion Medium
Compatibility with EX-CELL® Advanced HD Perfusion Medium
 STR glass bioreactor
 CHO-S
 Working Volume: 2 L
 Conditions either adapted and
passaged to ExM or
EX-CELL® Advanced HD Perfusion
Medium
Set-Up:
 Start of N-1 bioreactor
 Simulation of inoculation
N stage bioreactor
 Bleeding of N-1 bioreactor
to starting cell density
 N-1 and N runs both operated in
perfusion using an ATF for cell
retention
N-1: CellVento® 4 CHO X or EX-CELL® Advanced HD Perfusion Medium
N: EX-CELL® Advanced HD Perfusion Medium for both conditions
+18%
Expansion in
CellVento® 4 CHO X
19 BioContinuumTM Seed Train Platform | Fall 2020

Cellvento® 4CHO-X Expansion Medium
Compatibility with Cellvento® 4CHO Fed-Batch Platform
0 5 1 0 1 5
0
5
1 0
1 5
0
5 0
1 0 0
d a y s
VCD[10
6
VC/ml]
Viability[%]
0 5 1 0 1 5
0
3 5 0
7 0 0
1 0 5 0
1 4 0 0
d a y s
IgG[mg/l]
E xp a n sio n in E xME xp a n sio n in C e llv e n to ® 4 C H O
 STR glass bioreactor
 CHOZN® GS cell line
 Working Volume: 750 ml
 Conditions either adapted
and passaged to ExM or
Cellvento® 4CHO
 Fed-Batch in Cellvento®
4CHO and Cellvento® 4Feed
Platform
 n=2 (solid and dashed line)
20
+ 20%

Cell Culture Media Design principles
Formulation Secure
supply
easy
handling
• Media prototype evaluated
for solubility
• Exchange to better soluble
raw materials
• Focus on simple hydration
• Selected raw materials in
formats with robust supply
chain
• Lean formulation to reduce
raw material broadness
• Respecting safety aspects in
product handling
• Reduced dust formation for
ease of handling with
compaction technology
• Ready for CCM EMPROVE®
program
21

Poll Question
3. What difficulties do you have on current use Cell Retention Technology? (multiple
choices)
1) Difficult to use, Complexity on operation
2) Long time for setting up of utility and/or device
3) Trouble with flushing due to large scale
4) Low scalability
5) Other
6) No problem. Everything going well.
7) Cell Retention Technology has not applied to my process yet.

0
Agenda
Cellicon™ Perfusion Solution
3

Delivering on the need
CelliconTM perfusion filter and controller
25
o High-capacity, supports high cell
densities
o Reliable and reproducible, low risk
of failure
25
Ready to Use in
Minutes
Superior Performance
Complete, Linearly
Scalable Offering
o Supplied as a complete assembly
(filter, sensors, pump)
o Dry (no flushing required)
o Gamma irradiated
o Sterile weld (PD), sterile connector
(pilot & process)
0
2
4
6
8
0 2000 4000 6000 8000
TMP(psi)
Throughput (L/m2)
190111 BRX 1 181121 BRX 2
180911 BRX 3
o Proven linear scalability from 3-50 L
o Complete offering from bench to 2000L
0
20
40
60
80
100
120
0 2 4 6
VCD(E6cells/mL)
andViability(%)
Duration (days)
Viable Cell Denisty Viability
1L
50L
200L
1000L
2000L

High Seed Fed Batch Process Development
Experimental Design
26
 Bioreactor: 3L Mobius® bioreactor, 1.5 L WV
 Inoculation Density: Control and Standard Density: 0.5e6 cells/mL, High Seed: 5e6 cells/ml
 Basal Media: EX-CELL® Advanced
 Feed Media: 50:50 Blend of EX-CELL® Advanced and Cellvento® 4Feed
 Duration: 14 day process
N-1 Perfusion
3L High Seed
FB Bioreactor
3L Standard Seed
FB Bioreactor
(0.5e6 cells/mL)
(5e6 cells/mL)
Batch
x
1-2mL
10 - 30 E6 vc/mL
WCB Inoculum train
1-2mL
10 - 30 E6 vc/mL
Control
WCB Inoculum train
Batch
x
3L Standard Seed
FB Bioreactor
(0.5 e6 cells/mL)
Bioreactor Settings:
• Temp: 36.8 ºC
• Agitation: 200 rpm
• pH: 6.90 +0.1/-0.15
• DO: 50%
• Headspace air: 50
mL/min
• Sparge: open-pipe
*Each run performed in
duplicate

High Seed Fed Batch Production Process vs. Fed-Batch
Experimental Results 3L Scale
27
0
5
10
15
20
25
30
35
- 2 4 6 8 10 12 14
VCDX1E6
Days
Viable Cell Density
0%
20%
40%
60%
80%
100%
120%
- 2 4 6 8 10 12 14
%
Days
Viability
0
1
2
3
4
5
6
- 2 4 6 8 10 12 14
g/L
Days
Titer
0
5
10
15
20
25
- 2 4 6 8 10 12 14 16 18 20
pg/cell/day
Days
Qp
3 days earlier
Titer
Increase
Within
variability range
Within
variability range

Experimental Results 3L Scale (Continued)
28
0
1
2
3
4
5
6
7
8
- 2 4 6 8 10 12 14
mM
Days
Glutamine
0
2
4
6
8
10
12
- 2 4 6 8 10 12 14
mM
Days
Glutamate
Increased
with feeds
0
2
4
6
8
10
12
14
- 2 4 6 8 10 12 14
g/L
Days
Glucose
0
1
2
3
4
5
6
- 2 4 6 8 10 12 14
mM
Days
Ammonia
Higher
VCD
Lower
viability
Unclear
drop

Experimental Results 3L Scale (Continued)
29
0
0.5
1
1.5
2
2.5
- 2 4 6 8 10 12 14
g/L
Days
Lactate
Follows VCD Shift
10
30
50
70
90
110
130
150
- 2 4 6 8 10 12 14
pCO2(mmHg)
Days
pCO2
CO2 needed for pH control
consistent with lactate trend
0
10
20
30
40
50
60
70
80
90
100
Ctrl A Ctrl B pN-1 0.5A pN-1 0.5B pN-1 5.0A pN-1 5.0B
PeakArea(%)
Size Exclusion Chromatography
consistent product
quality across conditions
0
10
20
30
40
50
60
PeakArea(%)
Charge Analysis
Correlated to lower pH resulting
from increasing lactate level

0.0
10.0
20.0
30.0
40.0
50.0
60.0
6.4
7.4
8.5
9.2
10.8
10.9(G0F-N)
11.5(G0)
11.8
12.6
12.8
13.2(G0F)
14.0(Man5)
14.6(G1a)
15.1(G1b)
15.8
16.3(G1Fa)
16.7(G1Fb)
17.3
18.2
19.0
19.7(G2F)
23.1
23.8
26.1
Area(%)
Peaks
Glycan Analysis
Experimental Results 3L Scale and Conclusion
Consistent glycan profile
observed across conditions
High Seed Fed-Batch
process developed
• CHOZN® GS cell line
• new Cellicon™ filter and controller
• new Cellvento® 4CHO-X expansion
medium
Growth, metabolism and product
quality trends were comparable to the
control Fed-Batch process
Resulted in greater cell mass
production in a shorter time, and
showed potential for significantly higher
titers to be achieved

Summary
Integrated Technologies…
BioC
Cellicon™ Perfusion Filter and
Controller offers:
• Increased ease of use
• Simplicity of use
• Reduced footprint
High Seed Fed-Batch offers
• Lower cost of manufacturing
• Increase product throughput
• While keeping production bioreactor in
fed batch mode
CellVento® 4CHO-X offers:
• State of the art cell culture media
platform for intensified seed train
• Adaptation to production not required
for perfusion and fed-batch
32
…Upstream Process Evolution Powered

33
Acknowledgements:
 Jochen Sieck
 Alison Dupont
 Jeffrey Barna
 Mona Bausch
 Aline Zimmer
 Delia Lyons
 Melanie Brandl
 Douglas Rank
 …
 and all the great collaborators
from around the world

The vibrant M, BioContinuum, Cellicon are trademarks of Merck KGaA, Darmstadt, Germany or its affiliates. All other trademarks are the property of their
respective owners. Detailed information on trademarks is available via publicly accessible resources.
© 2020 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.

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