Straight to the Point: Reaching Clinical Stage Development with a CHOZN® Cell Line

The life science business of Merck KGaA, Darmstadt, Germany
operates as MilliporeSigma in the U.S. and Canada.
Reaching Clinical Stage
Development with a
CHOZN® GS -/- Cell Line
Murielle Vergès & Guillaume Plane
BioReliance® End-to-End Solutions
29 August 2019, Martillac

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

Agenda
Introduction
The CHOZN® GS-/- program
1
2
3 Case study

Reaching Clinical Stage Development with a CHOZN® GS -/- Cell Line| 29.08.2019
From DNA to Clinic
4
A path paved with pitfalls

A path paved with pitfalls
From DNA to Clinic
5
Clonality
Yield
Speed
Compliance
Quality
Robustness

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

8
➢ CHOZN® GS-/- cell line: depleted of GS activity
✓ Original approach of ZN finger technology
✓ No drug selection amplification needed
✓ High clone stability in production processes
Why adopting CHOZN® GS-/- Program ?
High & Stable Expression of Therapeutic Proteins
Parental Cell
Line
Media and
Feeds
Expression
Vector
Cell Line
Traceability
Protocols
Implemented in our Biodevelopment centers as a generic platform

Why adopting CHOZN® GS-/- Program ?
Its characteristics
0
10
20
30
40
50
60
CHO M CHO S CHO ZN GS-/- CHOK1
Qp(pq/Cell/day)
Specific productivity
The CHOZN® GS-/- can rival the more popular cell lines
✓ Convenient VCD range
✓ High performance
✓ High viability during all production process
✓ Good stability
➢ Clones CHOZN® GS -/- behavior

Case study:
From cell line development to
clinical phase in fast-track mode

Fast-track mode
Plug & Play Upstream Development Process
USP development
Round 1 Round 2 Reproducibility
Pre clinical production
Cell line development
MCB generation
Stability study
➢ Principle
✓ Reduce time of the study as possible as with a measured risk
✓ Customer request
✓ Need robust process

Mini pools generation: Screening of leads and signal peptide
Cell line development part
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 /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 spin tube (8 MP/lead, 14days-Fed-Batch assay)
➢ 1st MP screening in 96-well plate (240MP/construct, 7days-Batch assay)

Single cell cloning & clone selection
Cell line development part
➢ From cloning of best mini pools in 96WP….
0
10
20
30
40
50
60
Titers(mg/L)
21D11 23F08 24F08
0
5
10
15
20
25
30
0.0
0.5
1.0
1.5
2.0
FinalQp(pg/cell/day)
FinalTiter(g/L)
Clone performance: 1,6 g/L
Selected clones does not come
from minipools chosen for PD
➢ ….To selection of the best clone in spin tube

14 Reaching Clinical Stage Development with a CHOZN® GS -/- Cell Line| 29.08.2019
MCB
Days
PDL18 :
1.55 g/L
PDL39 :
1.37 g/L
PDL62 :
1.24 g/L
PDL86 :
1.23 g/L
Bio expansion Bioreactor ExCB
Clone stability confirmed
Clone stability study duration longer than the current process (86 PDL vs 58 PDL)
Stability study
Clone characterization

Master Cell Bank Generation
Viable cell number (106)
FEM 1 FEM 2 FEM 3
Day 0 8,3 7,3 8,1
Day 4 87,1 88,4 80,6
➢ Thawing of 3 vials from MCB – Expansion during 4 days
Acceptance criteria regarding MCB generation
 Viability >70% upon thawing
 By day 4 post thawing, number of cells must
double
Viability upon thawing
Vial 1 Vial 2 Vial 3
Day 0 97 97 98
Recovery
Acceptance criteria of recovery met

Round I : feed screening & high cell seeding density
Upstream process development part
➢ VCD ➢ Viability ➢ Final productivity
The CHOZN® cell line is proposed with its media platform
but
depending on clone performance we can change media and feed
Convenient VCD range ( ~10 to ~25.106 VC/mL) & high viability during production
Mini-pool performance >1,4g/L
0
5
10
15
20
25
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
VCD(x106VC/mL)
Production Days
80%
85%
90%
95%
100%
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
viability(%)
Production Days
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
4 5 6 7 8 9 10 11 12 13 14
Titer(g/L)
Production Days

Round II : T°c shift, feeding schedule, new supplement assays
Positive impact of T°C shift.
Gain of 2 days of production
Gain of 20% of final titer: 1,7g/L
60%
65%
70%
75%
80%
85%
90%
95%
100%
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Viability(%)
Production Days
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
4 5 6 7 8 9 10 11 12 13 14 15 16
Titerinharvest(g/L)
Production Days
➢ Viability ➢ Final productivity

18
Reproducibility study: consistency & robustess
➢ Consistency : selected condition x3
➢ Robustness: T°C shift and T°C shift date variations
=> Strongly advised in standard projects BUT
mandatory in fast track program
CLD and PD processes are robust enough to compensate the difference
between mini pools and clone
Clone Performance: 2,7g/L
The repro. study allowed to adapt the
process to the clone behavior
0.0
0.5
1.0
1.5
2.0
2.5
3.0
4 5 6 7 8 9 10 11 12 13 14 15 16
Titerinharvest(g/L)
Production Days
0
5
10
15
20
25
4 5 6 7 8 9 10 11 12 13 14 15 16
Qp(pg/cellperday)
Production Days
➢ Final productivity
➢ Specific productivity

200L scale production in SUB
Pre-clinical batch
Clone performance 3,5 g/L
Additional gain of productivity at higher scale
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4 5 6 7 8 9 10 11 12 13 14 15 16
Titerinharvest(g/L)
Production Days
0
2
4
6
8
10
12
14
16
18
20
4 5 6 7 8 9 10 11 12 13 14 15 16
SpecificProductivity
(pg/cellperday)
Production Days
➢ Final productivity ➢ Specific productivity

20 Reaching Clinical Stage Development with a CHOZN® GS -/- Cell Line| 29.08.201920
200L scale production is more favorable for this clone probably due to geometry of
equipment
Evolution of the productivity during PD
➢ Steady increase of productivity level from PD to pre-
clinical batch
✓ Mini pools /clone change
✓ Scale change
1.4
1.7
2.7
3.5
0
1
2
3
4
Round I Round II Repro. Pre clin. batch
titre(g/L)
Productivity
Mini pool Clone

Pre formulation study
Downstream process development part
Step 1
BUFFER
SCREENING
DSF
Combo 2/3
EXCIPIENT
SCREENING
DoE (screening
design)
FORMULATION
COMPOSITION
ADJUSTMENT
DoE (surface
response)
Study performed in parallel with process development

Drug Substance characterization
Downstream process development part
PA-Capture
Acidic Virus
Inactivation
Cation Exchange
Chromatography
Anion Exchange
Chromatography
Nanofiltration
Ultrafiltration /
Diafiltration
Development starts during Round 2
DSP Steps overview
➢ Convenient VCD peak & high viability
maintained=> weak HCP/ High purity
➢ Global yield : 80%.
✓ Targeted purity reached early
✓ Optimal condition for viral elimination.
✓ Simplified process
537
387
60
<2.4 <0.6
167
29.0
<1.8 <1.3 <0.4
0
100
200
300
400
500
600
Post Capture
(Mean value)
Post Depth
filtration
Pool
Post CEX
Pool
Post AEX
Pool
DS Clarified
harvest
HCP evolution - ppm DNA evolution - pg/mg
265000
➢ HCP and DNA Evolution

23
Project history
Dec.
Jan.
2019
Feb Mar Apr May June July
Pre-for. study
USP development
Round 2 reproducibility
Pre clinic. prod
Cell line development Stability study
DSP development
Prod for pre. F. study Round 1
Round 2Round 1
Round 3
Reproducibility
MCB gene.
Pre-clinical and phase I done

Work plan
GMP production
Process scale
up • Scale up protocol reviewed by QA
GMP
700L scale
production
SUB
• ExCB characterization
• Viral validation : 2 virus, 3 steps
• Bulk harvest testing (sterility / virus)
DS Release
• CoA (CQAs)
• Intermediate reference standard
• Tech transfer report provision
Q3 2019
Q4 2019
Q1 2020
24

Conclusion: Why to chose CHOZN® GS-/- Program ?
High titer/qp
high viability
High potential
for CHOZN® GS-/-
Weak HCP
High purity
yield
The CHOZN® GS-/- can rival
with more popular cell lines

Thanks
BioReliance® End-to-End Solutions team
CLD team
Clémence Justine
Amandine Philippat
USP team
Margaux Paillet
Myriam Eyquard
Elodie Airola
DSP team
Arthur Leclercq
Guillaume Godonnier
Caroline Pennacino

Any questions?
© 2019 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved. The vibrant M, CHOZN and BioReliance 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 on publicly assessible resources.

Straight to the Point: Reaching Clinical Stage Development with a CHOZN® Cell Line

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Straight to the Point: Reaching Clinical Stage Development with a CHOZN® Cell Line