Media and Process Development for Seed Train Intensification

The life science business of Merck KGaA,
Darmstadt, Germany operates as
MilliporeSigma in the U.S. and Canada.
Media and process development
for seed train intensification
Mona Bausch (Scientist, M. Sc.)
BioProcessing R&D
Darmstadt, Jan. 16th 2020

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

Agenda
1
2
Upstream Intensification
High Cell Density
Cryopreservation
Expansion Medium for
N-1 Expansion
3
4 Summary & Outlook

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
Market Trends, Business Drivers and Key Enablers*
to Drive Next Generation BioProcessing:
Process
Intensification
Process
Analytics
Software &
Automation
Key
Enablers
Single Use
BioContinuum™ Platform
Digital BioProcessing (incl Sensors) Single Use
* Source: BioPhorum (BPOG)
https://www.biophorum.com/category/resources/technology-roadmapping-resources/introduction/

Seed TrainInoculum Train Production
Intensified Seed Intensified Production
1. High Cell Density Cryopreservation
2. Perfused Seed Train
1. High Seed Fed-Batch
2. Concentrated Fed-Batch
3. Ultra High VCD Fed-Batch
4. Steady State Perfusion
5. Dynamic Perfusion
Several Opportunities for Intensified Upstream Processing
Media and process development for seed train intensification | Mona Bausch

Intensified Seed Train
High Cell Density Cryopreservation
Batch
1-2ml
10 - 30 E6 vc/mL
Batch
MCB/MWCB Inoculum Train
Seed Train
ProductionPerfusion
50-100 E6 vc/mL
50-500 mL bag
Perfused Seed Train

High Cell Density
Cryopreservation (HCDC)

 Cryopreservation is usually performed in cryovials of 1 mL at VCDs of
10 x 106 viable cells/mL → HD means to use higher VCDs than that, e.g.
50-100 x 106 viable cells/mL
 Generally, the advantages of HCDC increase with volume, therefore it is
most appropriate for bag application
 HCDC is ideally suited for process intermediates and not cell banking
HCDC
Our definition of high cell density cryopreservation
Cell banks (in manufacturing) have to fulfill plenty of requirements and undergo
rigorous testing, while process intermediates in process development and R&D are
relatively free of regulatory constraints. HCDC is easier to implement and
demonstrate in these process intermediates.

Comparison
Inoculation with Vial vs. Bag
[10^6
VC/mL]
Inoculation VCD 0.5
VCD - End of Batch 6
Volume [L]
Cell count for
inoculation [106 VC]
Run time (without
lag-phase) [d]
Bioreactors
n (production
bioreactor)
15000 7500000 3.5
n-1 1250 625000 6.9
n-2 105 52083 10.4
n-3 8.7 4340 13.8
n-4 0.72 361 17.3
n-5 0.06 30 20.7
n-6 0.005 2,5 24.2
Standard Vial Bag
VCD [106 VC/L] 10000 50000
Volume [L] 0.001 0.15
Cell count [106 VC] 10 7500
10.4 days
There have been reports
that high density cell banks
can significantly reduce the
time required to ramp up
cell cultures from a vial for
a manufacturing campaign
(Tao et al. 2011)

Advantages of HCDC
Decoupling of
cell expansion
and batch
production
Lower room
classification
in GMP
manufacturing
Closed
processing
No open cell culture
operation
steps in
manufacturing
Better
reproducibility in
seed train
expansion
Global distribution
from a central
expansion facility to
decentralized global
production facilities
Advantages also for R&D
and process development

Comparison
Seed Train Expansion

Simplification of high cell density cryopreservation process
Cryo
Medium
HCDC
Process
High Cell Density
Cryopreservation
Bag
Assembly

CCM required for intensified upstream processing
VCD
1 2 3 4
Medium requirements
 No adaptation to EX-CELL®
Advanced HD Perfusion
medium required
 No cell damage during
freezing and thaw
 Fast growth with minimum or
zero lag phase after thaw
 Constant growth rate and
specific productivity over
thaw, expansion and
production
EX-CELL® Advanced

Bag Assembly Prototype
Main Components
Prototype: 250 mL Bags
Filling volume: 150 mL
1
2
3
4
6
5
1 Cryo medium filling line
2 Cell suspension line/connection
to bioreactor
3 1 L waste bag for flushing of lines
4 Cryobags
5
Line for connection to bioreactor
after thaw
6
Metallic pinch pipe for sterile cutting
with the NovaSeal™ crimping tool

Filling
HCDC process
Inoculation
Connection of bag
assembly to
bioreactor
→ Cell suspension
through cooling
loop into pre-filled
freezing bags
→ dilution of
cryoprotectant
Bags can be disconnected one by one directly
after bag is filled with appropriate volumes of
freezing medium and cell suspension
→ Direct freezing and storage in -80 °C freezer
Directly add revived
cells to the bioreactor
Thawing of bag (water
bath at 37 °C, room
temperature or at 4 °C)
Connection to
bioreactor by welding
(closed connection)
Connection of freezing
medium (concentrated
cryoprotectant) bag to
the assembly

Simulated full process
Vial vs. Bag
simulated N-1 bioreactor → simulated production bioreactor
comparable
growth and titer
for both
bioreactors during
steady state
Growth and titer in simulated production bioreactor for CHO-K1
Inoculation

Expansion Medium for Seed
Train Intensification and N-1
Perfusion

Seed train expansion is usually performed in batch mode
scaling from lab to production bioreactors up to N-1
Requirements for Seed Train
• Reproducible healthy cell growth
Challenges in Seed Train
• Processing time
• Risk of contamination
Improvements
• Reduce processing type by switching to perfusion mode
• Design medium that ideally supports growth phase
Expectations
Seed Train Intensification

Seed train expansion is usually performed in the same medium that is used in production phase.
However, a difference in nutrient demand could be observed:
• Process improvements through adjusted formulation for the expansion phase
• Expanded cells can be directly transferred into the production bioreactor – no adaptation required
• Cells get optimally prepared for the production phase
• Confirmed in our inhouse platforms and expected to work with other media/feed platforms accordingly
Requirements
Expansion Medium
Expansion phase
 Focus on cell growth
Production phase
 Focus productivity per cell

CCM required for intensified upstream processing
VCD
1 2 3 4
Medium requirements
 No adaptation to EX-CELL®
Advanced HD Perfusion
medium required
 No cell damage during
freezing and thaw
 Fast growth with minimum or
zero lag phase after
 Constant growth rate and
specific productivity over
thaw, expansion, and
production
EX-CELL® Advanced

Screening for starting candidate
Cryopreservation
Inoculation
Inoculation
Inoculation
Production (Batch)Expansion (Adaption to media)
Freeze
VCD
1 2 3 4
Different Conditions
• Expansion Medium
• EX-CELL® Advanced HD Perfusion
ONLY
EX-CELL® Advanced HD Perfusion
Expansion

Simulation: Expansion → Cryopreservation → N-1 Expansion → Production
1 2 3 4
• TTP Tube experiments, 2 passage steps after thaw
• Growth and IgG concentrations monitored in simulated production batch
Indication that using a single medium for the whole production
campaign is suboptimal
N=6
ViableCell
Density(VCD)
0 1 2 3 4 5 6 7
0
5
1 0
1 5
2 0
C H O K 1
d a y s
VCD[10
6
VC/ml]
0 1 2 3 4 5 6 7
0
5
1 0
1 5
C H O D G 4 4
d a y s
VCD[10
6
VC/ml]
0 1 2 3 4 5 6 7
0
5
1 0
1 5
2 0
2 5
C H O -S
d a y s
VCD[10
6
VC/ml]
0 1 2 3 4 5 6 7
0
5
1 0
1 5
C H O Z N ® C e ll L in e
d a y s
VCD[10
6
VC/ml]
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 nE x p a n sio n in E xp a n sio n M e d iu m

N=6
Even higher titers were reached when ExM was used as a companion
medium for expansion with EX-CELL® Advanced HD Perfusion Medium
0 1 2 3 4 5 6 7
0
5 0
1 0 0
1 5 0
2 0 0
2 5 0
C H O K 1
d a y s
IgG[mg/l]
0 1 2 3 4 5 6 7
0
5 0
1 0 0
1 5 0
C H O D G 4 4
d a y s
IgG[mg/l]
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 nE x p a n sio n in E xp a n sio n M e d iu m
0 1 2 3 4 5 6 7
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
C H O -S
d a y s
IgG[mg/l]
0 1 2 3 4 5 6 7
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
C H O Z N ® C e ll L in e
d a y s
IgG[mg/l]
1 2 3 4
Simulation: Expansion → Cryopreservation → N-1 Expansion → ProductionIgG
Production
+21% +54% +26% +44%

Bioreactor Confirmation
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: ExM or EX-CELL® Advanced HD Perfusion Medium
N: EX-CELL® Advanced HD Perfusion Medium for both conditions

Expansion for
Fed-Batch Production

Simulation: N-1 Expansion → Production (Fed-Batch)
• EX-CELL® CD CHO Fusion
Production (Fed-Batch)
3 4
ExpansionDifferent Conditions
• Cellvento® 4CHO
Cellvento® 4CHO Platform
EX-CELL® Advanced Fed Batch Platform
EX-CELL® Advanced CHO Fed-Batch Medium + EX-CELL® Advanced CHO Feed 1
Cellvento® 4CHO + Cellvento® 4Feed

Compatibility with Fed-Batch Media
Cellvento® 4CHO & EX-CELL® Advanced CHO Fed Batch
Productivity
comparable or
improved when
expansion was done in
ExM

Bioreactor Confirmation
Compatibility with Cellvento® 4CHO Fed-Batch Platform
 STR glass bioreactor
 CHOZN® GS cell line
 Working Volume: 750 mL
 Conditions either adapted and
passaged to ExM or Cellvento®
4CHO Medium
 Fed-Batch in Cellvento® 4CHO
and Cellvento® 4Feed Platform
 n=2 (solid and dashed line)

Cell Culture Media Expectations
Media
design
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

Expansion Medium
high density growth at low CSPRs
• Compatible with perfusion and fed-batch
• Increase of productivity in N stage
Batch
1-2ml
10 - 30 E6 vc/mL
Batch
MCB/MWCB Inoculum Train
Seed Train
ProductionPerfusion
50-100 E6 vc/mL
50-500 mL bag
Intensified Seed Train
Bag assembly → Available on demand
Cryopreservation medium → under development

Expansion Medium
Upcoming Product Launch
Cellvento® 4CHO-X
Expansion medium
1
Cellvento® 4CHO-X Expansion Medium will launch
early 2020. The product comes in compacted media
format and supports expansion and N-1 production
phases.
2
Cellvento® 4CHO-X is compatible with our
production media for perfusion and fed-batch and
may support other media platforms as well.
3
Cellvento® 4CHO-X is designed to support high cell
growth at low CSPRs and increase the productivity at
N stage.
4
Cellvento® 4CHO-X helps our customers to save time in
processing, reduce cost and the risk of contamination
during seed train and increase their facility flexibility.

Acknowledgements
• Jochen Sieck
• Caroline Ströder
• Melanie Feigenspan
• Doris Matheis
• Christian Schultheiss
• Luis Ayala
• Jana Glawion
• Corinna Merkel
• Delia Lyons
• Dustin Davis
• Irfan Hodzic
• Tatenda Shopera
• Mike Felo
• Steve Proulx
• Marco Klatte
• Christian Martin
• Habib Horry
• Russel Jones
• Melanie Brandl
• Claudia Paul
• Kintija Dreimane
• Rifat Agkoc
• Dan Rosen
• Aurore Lahille & Team
• Dennis Binder
• Joline Bachmann
• Tamara Souris-Trautmann

© 2020 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved. The vibrant M, SAFC, EX-CELL, Cellvento, NovaSeal, Emprove, and BioContinuum 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.

Media and Process Development for Seed Train Intensification

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