Watch the interactive recording here: https://bit.ly/30FTDG0 The quest for a viable upstream process relies on generation of a cell line expressing the protein of interest. Unfortunately, the search for the best-producing clone is often compared with looking for a needle in a haystack. Making this more challenging is the pressure to get it right the first time, quickly and while mitigating risk and costs. Although a lot of efforts are made on the clonal selection, there is often few to none optimization done on the expression cassette, including promoter and enhancer selection, or signal peptide. The statistical approach on how many clones should be screened to get to a good producer is often overlooked as well. We combined a new generation of promoters and enhancers to improve strategies on pool and mini pool screening with both CHO-K1 and our own CHOZN® GS which helped deliver high-producing clones in an accelerated timeline. In addition, we are able to begin process development in parallel with cell line development, further reducing timelines. In this webinar, you will learn: * How the strategy approach can help reducing the overall timeline of cell line generation * How we have expanded our platform by designing a completely new vector/cell/process template * How we have worked on promoters, enhancers, pool/mini-pool approach as well as on timelines from DNA to clone

1. Merck KGaA
Darmstadt, Germany
Sébastien Ribault, PhD
Accelerate
Delivery of
High Producing
Cell Lines

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

3. Experience
32 years in PD,
24 in GMP
250+ biologics
70+ Unique GMP
Drug Substance
batches released since
2012
Built our own Single
Use PD/GMP facility in
<1 year
Tech expertise
We are CDMO as well as a
developer & manufacturer
of process technologies
100% Scale up success rate
90% Originator, 10% Biosimilar
65% Fed batch 35% Perfusion
Project Manager
Our experience and expertise ensure quality and efficiency
3

4. Global resources Locally delivered
Boston, US
Shanghai, China
Bordeaux, France GMP
Wisconsin
St Louis
Glasgow, UK
Rockville
Singapore
Biodevelopment
and production
Antibody-drug conjugate
Product characterization,
Biosafety testing
Cell line development
4

5. Pre-clinical Phase I Phase II Phase III Commercial
Cell line development
Any mammalian cells
Analytical development
Process development
Templated or customized
GMP Clinical Manufacturing
Single-Use 50 to 2,000 L scale
Facility design, equipment commissioning
Process validation, scale up, TT
Cell banking
Characterization testing Lot Release testing
We are your process development and manufacturing
partner
5

6. Pre-clinical Phase I Phase II Phase III Commercial
Cell line development
Any mammalian cells
Cell banking
We are your process development and manufacturing
partner
DNA
Construction
Pre MCB Generation Clone Testing MCB
MCB Generation
Characterization
Genetic
Stability Study
Single Cell Cloning and CharacterizationMinipool strategy
Expression
Vector
Construction
4 weeks 13 weeks 13 weeks 13 weeks 27 weeks
✓ Codon
optimization
✓ Cell thawing and
expansion of
Parental cell line
600MP 100MP 20MP 20MP 1000 clones 100 clones 20 clones 20MP
✓ 300 vials
✓ Full characterization
✓ Spin tube
✓ In duplicate
✓ Eq.to 60 PDL
6

7. Parental Cell
Line
Media and
Feeds
Expression
Vector
Cell Line
Traceability
Protocols
7
CHOZN® GS-/- platform: depleted of GS activity
✓ Original approach of ZN finger technology
✓ No drug selection amplification needed
✓ High clone stability in production processes
CHOZN® GS-/- Program
High, Stable Expression of Therapeutic Proteins
Implemented in our Biodevelopment centers as a generic platform

8. Platform
improvments

9. Mini-pool approach for more robustness
9
n=50
n=12
Mini-pool approach associated to 3 screening steps
✓ Reduced the diversity within the transfected pool
✓ Increase the chance to get best performers (min 60MP max 600MP)
Confidence about our process
Longer process and weak predictivity for 96-well plate assay compensated by:
✓ High number of candidate by screening steps
✓ New: threshold establishment at 96-well plate=> Go/No Go at early stage
Mini-pool approach
600 clones 100 clones 20 clones
9

10. Average Titer
Data compilation based
on 21 MABs CLD project
14
24
62
Lead Pool by titer level (%)
<0,5g/L
0,5<Titer<1g/L
>1g/L
14
71
14
Lead Clone by titer level (%)
<2g/L
2<Titer<4g/L
>4g/L
Robustness: 85% of clone > 2g/L whose 14% > 4g/L
Different projects : standard CLD or coupled with signal peptide screening,
cell type screening, humanization, etc…
12 or 14-days Fed batch assays. 12 or 14-days Fed batch assays.
10

11. Ensure systematic success?

12. 4 weeks 11 weeks 13 weeks
Expression vector Stable pool generation Single Cell Cloning
4 weeks 2 weeks 7 weeks
Expression vector Stable pool generation Single Cell Cloning
Automation
Process Development
FAST
TRACK
Process Development
12

13. Automation
 Mab difficult to produce: more plates, while maintaining processing capabilities
 Fast Track: high yield, acceptable reliability
 Speed up cell line construction
High
throughput
technologies
IgG
quantification
Cell sorter
Cell seeder
Multi-culture
system
Cell counter
✓ Incoming assays at 96-well plate stage
✓ Saving time: 13 weeks
✓ Manually impossible
✓ Predictivity improved
✓ Mandatory for HT process
✓ Saving time
✓ Predictivity improved
✓ Set up on going
13

14. Automation
IncubatorCell Imager
Automatized-
Arm
▪ Semi-automatized process: cell growth monitoring and cell cloning step
▪ Remaining manual handling steps: cell seeding, medium change, productivity assessment
14

15. How to reduce timelines ? Fast track process
Process Development
✓ Customer request
✓ Minimize risk: low cell seeding of
mini-pool
✓ More likely similar cell behavior of
mini-pool vs clone
15,000
cells/well n=50
n=12
5,000
cells/well
4 weeks 13 weeks 13 weeks
Expression vector Stable pool generation Single Cell Cloning
Enables to start process development before the end of CLD
15

16. Improvment using molecular tools
Single expression vector for LC and HC integration improved for the cell line
 Screening of endogenous regulatory elements of CHO cells: IL33, DRC7, Ras, H2AJ promoters
 Task force to identify sources of improvements :
cell
protein
vector
Targeted integration
Enhancement of crucial metabolic pathways
Improved media and feeds
16

17. New Cell Line development platform
Goals :
• Platform Development for recombinant proteins production at lower cost
• IP
Constructs :
CHOZN-K1
17

18. Objectives: Screening of promoter and enhancer candidates – transient expression/GFP
• 3 CMV enhancers were combined with 7 promoters
• 4 promoters are specific to CHO cell and are temperature inducible
Positive control Negative control
75% of cells remaining
25% of cells with highest GFP expression level
0
0,5
1
1,5
2
2,5
3
3,5
4
4,5
5
GFPexpressionlevel(log)
cell repartition within GFP level expression
Positive response to temperature shift
 Selection of two endogenous promoters responding positively to temperature shift: mRAS and mH2AJ
 Selection of the best viral promoter and enhancer, murine and human origin: IE1
→ Generation of 16 constructs for expression of a Mab
→ Combination of promoters to assess HC and LC expression modulation
Cell repartition within GFP level expression
25% of cells with highest GFP expression level
75% of cells remaining
New Cell Line development platform
hCMV hCMV
hIE1 hIE1
mIE1 mIE1
hCMV Ras
hIE1 Ras
mIE1 Ras
hIE1 H2AJ
mIE1 H2AJ
enhancerpromoter
18

19. hCMV hCMV
hIE1 hIE1
mIE1 mIE1
hCMV Ras
hIE1 Ras
mIE1 Ras
hIE1 H2AJ
mIE1 H2AJ
Selection of
combinations
for both
expression
cassettes
enhancer promoter
PL62 hIE1 hIE1 HC mIE1 mIE1 LC
PL63 mIE1 mIE1 HC hIE1 hIE1 LC
PL64 hIE1 hIE1 HC mIE1 Ras LC
PL65 mIE1 Ras HC hIE1 hIE1 LC
PL66 hIE1 hIE1 HC mIE1 H2AJ LC
PL67 mIE1 H2AJ HC hIE1 hIE1 LC
PL68 mIE1 H2AJ HC hIE1 Ras LC
PL69 hCMV Ras HC mIE1 H2AJ LC
PL70 mIE1 H2AJ HC hCMV Ras LC
PL71 hIE1 H2AJ HC mIE1 mIE1 LC
PL72 mIE1 mIE1 HC hIE1 H2AJ LC
PL73 mIE1 mIE1 HC hCMV Ras LC
PL74 hCMV Ras HC mIE1 mIE1 LC
PL75 mIE1 Ras HC mIE1 Ras LC
PUROMYCINER 1ST CASSETTE 2ND CASSETTE
Objectives: Screening and selection of the top promoting cassette combination– transient expression/Mab
New Cell Line development platform
19

20. Objectives: Screening and selection of the top promoting cassette combination – transient expression/Mab
• Assessment in transient expression
• Analysis of Mab expression by cell cytometry (Guava) and by Mab titration (Octet) at 2 time points ( 2 & 6 days)
 The top 4 constructs are confirmed by Guava analysis except for PL67 & PL71
 The best performance (PL75) is obtained with RAS promoter driving both HC and LC expression
 The two challengers combine mIE1 and hIE1
 PL65 combines RAS and IE1
 Expression is improved when a stronger promoter drives LC expression
Negative control
0
1
2
3
4
5
6
0
1
2
3
4
5
6
7
8
9
10
PL75 PL62 PL63 PL65 PL70 PL64 PL68 PL74 PL69 PL66 PL67 PL71 PL72 PL73 PL38 NE
Qp(pg/cell/day)
Titers(mg/L)
Mab expression 48h post electroporation
New Cell Line development platform
20

21. Objective 1: Assessment of LC and HC positioning in regards of puromycin resistance gene – stable expression Mab
• Assessment by generation of mini-pool using two different MAbs – an easy and a difficult to express
• Test of 16 constructs (8 constructs/molecule)
Objective 2: Endogenous promoter optimization
• Generation of truncated promoter by deletion of 5’ terminal
• Selection based on GFP expression
New Cell Line development platform
21

22. Screening using 6 days batch culture in 96-well plate at 37°C (results for one of the Mabs)
– stable expression of Mab
0
5
10
15
20
Titre(mg/L)
S210*vHC_vLC
0
5
10
15
20
Titre(mg/L)
S210*eHC_vLC
0
5
10
15
20
Titre(mg/L)
S210*eHC_eLC
0
5
10
15
20
Titre(mg/L)
S210*əHC_eLC
0
5
10
15
20
Titre(mg/L)
S210*vLC_vHC
0
5
10
15
20
Titre(mg/L)
S210*vLC_eHC
0
5
10
15
20
Titre(mg/L)
S210*eLC_eHC
0
5
10
15
20
Titre(mg/L)
S210*eLC_əHC
➢ 80 mini-pools/construct : viral promoter allowed best producers mini-pools
➢ Expression level differed transient expression & stable expression
➢ Very low range of titers for constructs harboring endogenous promoters => eliminated
➢ Selection of 15-20 best mini-pools/construct => 240 mini-pools
New Cell Line development platform
22

23. ➢ Endogenous promoters combinations below viral promoters:
0,70g/L with temperature shift (non optimum process) for S210 Mab
Candidate
New cell line
mini-pool
Competitive CHO
Mab 1 1,7 g/L
Mini-pool:1,51 g/L
Clone: 2,4 g/L
Mab 2 0,6 g/L Pool : 0,076 g/L
✓ Viral promoter based vector
✓ Endogenous promoter vector
➢ Best construct: phIE1*HC_pmIE1*LC
New Cell Line development platform
23

24. Case studies

25. Case Study: Screening of leads and signal peptide
Cell line development services
Lead 1
Lead 2
Lead 3
Signal peptide 1
Signal peptide 2
Signal peptide 3
Signal peptide 1
Signal peptide 2
Signal peptide 1
Signal peptide 2
From 7 constructs, selection of best
signal peptide for each lead
Final selection of best lead for cloning
0
500
1000
1500
Titers(mg/L)
SP1 SP1 SP2 SP1 SP2
Lead 1 Lead 2 Lead 3
0
10
20
30
40
50
Mini-poolTiter(mg/L)
Lead2-SP1 Lead2-SP2 Lead3-SP1
Lead3-SP2 Lead1-SP1 Lead1-SP2
0
5
10
15
20
Mini-poolTiter
(mg/L)
Lead2-SP1
Lead2-SP2
Lead3-SP1
Lead3-SP2
Lead1-SP1
Lead1-SP2
Lead1-SP3
3rd MP screening in TPP
(8 MP/lead
14days-Fed-Batch
assay)
1st MP screening in 96-
well plate
(240MP/construct
7days-Batch assay)
25

26. Case Study: how to increase the titer of a difficult to express MAb ?
Our proposal: Signal peptide screening:
 Titer too low
 Alternative : super-transfection of previous mini-pool & gene amplification using MSX
0
5
10
15
20
25
30
Titer(mg/L)
Titer profile at 96-well stage
single transfection super transfection
0
100
200
300
400
500
600
700
800
A B C D E F G H I J K L M N O P Q R S T
Titer(mg/L)
Final titer of Stable pool generation
Super transfection Single transfection
A 4 fold increase of the titer at MP stage after super transfection of MP
26

27. oftware
ual
bove predicted value
elow 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)
rt® Software
g: Actual
oints above predicted value
oints below predicted value
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
%CV = 2 – 10,3%
P Value < 0,05
R² in the spécification
KLa = f (P/V ; Vs)
ANOVA
Validation
Sparging and stirring at 2000L scale
Scale-up strategy
27

28. 2
8
SU scale up process performance
0
10
20
30
40
50
0 2 4 6 8 10 12 14 16
VCD(x106cells/mL)
Production Days
VCD
3L (DEVT) 2KL (PROD) 200L (PILOT)
0 2 4 6 8 10 12 14 16
Biacore(g/L)
Production Days
TITER
3L (DEVT) 2KL (PROD) 200L (PILOT)
28

29. Title of Presentation | DD.MM.YYYY29
Conclusions

30. 30
THANKS
BioReliance® End-to-End Solutions Team
DEVTLS
LAHILLE Aurore
RAYMOND Céline
CORBIN Cyrielle
DEVTLS-BDS Team
AMOURIQ Marine
DREAN Raphaelle
FRANCOIS Charlène
GRINDES Lucie
JUSTINE Clémence
PHILIPPAT Amandine

31. Merck, BioReliance®, CHOZN and the vibrant M 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.
© 2018 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.
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