PepTalk January 15th, 2019

A SMART Platform for Scalable Biotherapeutic Development:

cDNA to 200L Single-use Stirred-tank Bioreactor – a Case Study and Workflow

Presenter: Divya Goel, MBS - VP of Business Development, Celltheon

CELLTHEON SMARTTM
 AGENDA
1. Current challenges in bioprocessing
2. Celltheon SMARTTM Platform addresses industry challenges
3. Overcoming obstacles in scale-up using the Allegro STR Single-Use Bioreactors
4. Case Study: Celltheon SMARTTM Platform integrates Allegro STR SUBs for easy scale-up

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 CHALLENGES IN BIOPROCESSING
CURRENT INDUSTRY NEEDS CELLTHEON SMARTTM PLATFORM SOLUTION

LOWER COST OF MANUFACTURING OPTIMIZED PLATFORM

FASTER TIME TO MARKET cDNA to RCB IN 16 WEEKS
HIGH TITERS STABLE POOLS: 1.5g/L; SINGLE CLONES: 5g/L
PROCESS SCALABILITY TECH TRANSFER PLATFORM TO 200L
CRITICAL QUALITY ATTRIBUTES CQAs MATCHING BETWEEN 3L AND 200L STR
CELL LINE STABILITY >95% OF CLONES STABLE FOR >120 GENERATIONS
DIFFICULT TO EXPRESS PROTEINS HIGH QUALITY, HIGH EXPRESSION OF DIFFICULT TO
EXPRESS PROTEINS: scFv-Fc, Bispecifics, Extracellular Proteins
HIGH ASSURANCE OF CLONALITY HIGH RESOLUTION IMAGING: SOLENTIM CELL METRIC

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 CELLTHEON SMARTTM PLATFORM

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 CELLTHEON SMARTTM EXPRESSION
PLATFORM
Modified CHOK-1 cells
for expression of
Celltheon
Reduced lactate and
recombinant protein SUPERCELLSTM
ammonia production for
therapeutics –
improved product quality
Patent pending
(i.e: low aggregation, etc.)

Celltheon
pCTTM
suPRETM Elements (Post SMARTTM
Vectors Bioprocessing
Transcriptional Regulatory
Elements) Patented

CT StabilizerTM (synthetic Platform fed-batch

MAR) Patent pending process for reduced time
and high titers

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 CELLTHEON SMARTTM PLATFORM:
SUPERCELLTM OVERVIEW
ENGINEERED CHOK-1 DERIVED CELLS TO IMPROVE QUALITY ATTRIBUTES

Screening/selection
- High % monomer
High Product Purity
- Extremely low aggregation

- 18H doubling time

Easy Scaleup Combined Strategy
- No clumping
Directed Evolution +
- Qp: 60pg/cell/day CHOK-1 Cell
High Cell Specific - 15x106 cells/mL peak VCD Engineering
Productivity
- High titers Parent- derived from CHOK-1
(full documentation)
Optimized for Next - Low oxygen consumption
Generation Culture - Low glucose consumption
Platforms* - Low Ammonia, low lactate
* KEY ATTRIBUTES FOR PERFUSION

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 CELLTHEON SMARTTM PLATFORM:
CT-CHOTM SUPERCELLS
Celltheon CT-CHO cells have 18-20hr doubling time with no clumping

Doubling Time of Different Clones

24

Doubling Time (Hours)

20
16
12
8
4
0
Unique Stable Clones

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CELLTHEON SMARTTM PLATFORM:
SUPERCELLTM METABOLIC PROFILE OPTIMAL FOR CQAs
SUPERCELLTM Lactate Profile: 13 Unique Clones
SUPERCELLTM Ammonia Profile: 13 Unique Clones
2 4.5
1.8
4
1.6
3.5
1.4
3

Ammonia (mM)
Lactate (g/L)

1.2
1 2.5

0.8 2

0.6 1.5
0.4 1
0.2 0.5
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Time (Days)
Time (Days)

Maximum lactate levels of 2.0g/L Maximum ammonia levels of <6mM

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 CELLTHEON SMARTTM PLATFORM:
suPRETM ELEMENTS
2-4x increase in titer over industry standard WPRE

Key points: Transient Expression in CHO of

suPRE Elements (n=3)
• suPRE™ Elements are a family of chimeric
2500.0
post-transcriptional regulatory elements
optimized for high expression
2000.0
• High functionality in different cell types
• Useful in transient or stable production
• Applicable in many biotherapeutics: 1500.0

Titer (mg/L)
• mRNA
• CRISPR 1000.0

• RNAi
• Bioproduction 500.0

• Gene Editing
• Cell therapy 0.0
No PRE WPRE suPRE 1 suPRE 2 suPRE 3

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 CT STABILIZERTM INCREASES NUMBER OF HIGH EXPRESSING
CLONES RESULTING IN OVERALL HIGHER EXPRESSION

Key points:
• CT STABILIZER™ is a 1.8kb synthetic MAR element
• Engineered for ease of cloning
• Engineered for high stability
• Engineered for high Qp
• Engineered for high expression across all clones

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 CELLTHEON SMARTTM PLATFORM:
ALLOWS EFFICIENT CLONE SELECTION

❑ Limiting dilution of stable pools in 96 well plates

❑ Imaging (Solentim Cell Metric) for clonality and 96-well screen by ELISA

❑ No added FACS sorting of high expressing clones

❑ No generation of mini pools and selection of high expressing colonies
❑ No automated colony picking and selection of high expressing clones

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 CT STABILIZERTM RESULTS IN MAB CLONES
WITH HIGH Qps OF ~40-60pcd
Specific Productivity (Qp) of Top Clones in Fed-batch
60
Qp (pg/cell/day) 50
40
30
20
10
0
Top Stable Clones
Key points:
• CT STABILIZER™ is a 1.8kb synthetic MAR element
• Engineered for ease of cloning
• Engineered for high stability
• Engineered for high Qp
• Engineered for high expression across all clones

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CELLTHEON SMARTTM PLATFORM: RESULTS IN CLONES WITH
HIGH GENERATIONAL STABILITY >120 GENERATIONS

Top 4 Clone Stability for 100-120 generations

100%
Normalized Titer

80%
60%
40%
20%
0%
37 64 90 104 27 67 98 117 32 72 101 120 24 64 96 114
Generations

Molecule #1 Molecule #2
Clone 1 Clone 2 Clone 3 Clone 4

Key points:
• CT STABILIZER™ is a 1.8kb synthetic MAR element
• Engineered for ease of cloning
• Engineered for high stability
• Engineered for high Qp
• Engineered for high expression across all clones

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CELLTHEON SMARTTM PLATFORM INCLUDING
CT STABILIZER™ AND SUPERCELLS™ RESULT IN HIGH
EXPRESSION OF DIFFICULT TO EXPRESS PROTEINS
Difficult to Express Proteins in Celltheon SMART TM Platform Pools
1600
1400
1200
1000
Titer mg/L

800
600
400
200
0
Fc-Fusion Antibody Bispecific Human Fab Recombinant Growth Factor Human mAb
protein Fusion protein antibody protein
Celltheon SMART Expression Platform

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 CELLTHEON SMARTTM WORKFLOW

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 CHALLENGES IN SCALE-UP ADDRESSED BY THE
PALL ALLEGRO STR SINGLE-USE BIOREACTORS

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 CHALLENGES IN PRODUCTION AND
SCALE-UP IN BIOREACTORS
MIXING EFFICIENCY SPARGER DESIGN VESSEL GEOMETRY POWER INPUT

Key Challenges:
• Limitations in power input and impeller design (low P/V) result in low cell growth and productivity*
• CO2 stripping is not efficient at larger scales due to sparger design and small gas SA : volume ratio
• Suboptimal mass transfer with large macrospargers / inefficient mixing (kLa is not scalable)

*Key limitation in many commercial SUBs- difficult for scaling up high density cultures (i.e: high-density
fed-batch/ perfusion)

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 HIGHLIGHTS OF PALL ALLEGRO STR
Pall’s Allegro STR single-use bioreactors are engineered with:
• Unique square design and baffling- optimizes mixing and higher
gas surface area : volume ratio for CO2 stripping
• Direct drive, elephant ear impeller that ensures high P/V
• Ring sparger efficiently strips CO2 while maintaining high kLa
• Easy to assemble: 10 minutes from unboxing to inflation
• Fool-proof assembly with guided instructions and sensors to ensure proper set-up
• Excellent technical training and support team

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 KEY FEATURES OF ALLEGRO STR

Probe Tray
Direct-drive, Baffled
elephant ear housing
impeller

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 CASE STUDY:
cDNA TO SCALE-UP IN PALL ALLEGRO 200L STR

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STABLE POOLS TO CLONES IN FED BATCH RESULTS IN
EXPRESSION OF 3-5gm/L
Molecule #1 Without CT Stabilizer Molecule #2 With CT Stabilizer
Fed-Batch Pool Titer Molecule #1 Fed-Batch Pool Titer Molecule #2
2000 2000
Fed Batch

Titer (mg/L)
Titer (mg/L)
Pool
1000 1000
Screening

0 0
STABLE POOLS STABLE POOLS
Batch Clone Titers Molecule #1 Batch Clone Titer Molecule #2
2000 2000
Titer (mg/L)

Titer mg/L
Batch 1000 1000
Clone
Screening
0 0
STABLE CLONES STABLE CLONES

Fed-Batch Clone Titers Molecule #1 Fed-Batch Clone Titers Molecule #2

5000 5000
4000 4000
Titer (mg/L)

Fed Batch

Titer (mg/L)
3000 3000
Clone
2000 2000
Screening
1000 1000
0 0
FED-BATCH SCREENING Pool FED-BATCH SCREENING Pool
titer Titer

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 EXPERIMENTAL DESIGN
Objective: To compare scalability criteria between 3L bench-scale STR, 50L
Cylindrical STR and Allegro 200L STR (Cuboidal).

Methods: The fed-batch process defined in the 3L bench-scale STR was

scaled up in the 50L and 200L STR bioreactors using conventionally scalable
parameters (P/V, tip speed and kLa).

Modifications: The 50L STR was limited in maximum power input thus, by the
end of the culture, the P/V was lower than the 3L and 200L STRs. kLa was
maintained constant between all 3 scale bioreactors.

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RESULTS: CELL GROWTH AND PRODUCTIVITY
OF A DIFFICULT TO EXPRESS ANTIBODY
200L vs 50L vs 3L Fed-batch Bioreactor: 200L vs 50L vs 3L Fed-batch Bioreactor:
VCD and Viability VCD and Titer
14 100% 14 2000
VCD x10e6 cells/mL

VCD x10e6 cells/mL

12 12
80%
1500

Viability (%)
10 10

Titer (mg/L)
8 60% 8
1000
6 40% 6
4 4
20% 500
2 2
0 0% 0 0
0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16
Time (Days) Time (Days)

200L STR Fed-Batch 3L STR Fed-batch 50L STR Fed-batch 200L STR Fed-Batch 3L STR Fed-batch 50L STR Fed-batch

1. Peak VCD 10-12 x 106 cells/mL is attained in all fed-batch processes

2. Relative titer is comparable in 200L STR to 3L and 50L scale model
3. Lactate peaks at ~2g/L and is then consumed and is consistent across all three scales
4. Ammonia peaks at <6mM and is consistent across all three scales

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RESULTS: PRODUCT QUALITY 3L vs 200L STR
N-Glycan Analysis Size Exclusion Analysis
3LBioreactor Purified protein from the
200L STR
3L STR and 200L STR were
analyzed for CQAs (critical
quality attributes):
• N-glycan
• Size-Exclusion
(aggregation)
• Charge Variant
Charge Variant Analysis • CDC Bioactivity
3L Bioreactor
200L STR
The 3L process was
reproducible in the Allegro
STR 200L with respect to
product quality, cell growth
and productivity.

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 CONCLUSION
✓ The Celltheon SMART™ Platform yields homogenously, high-expressing clones for Mabs (4-5g/L), with high specific
productivity (30-60pg/cell/day) and >95% of clones are stable for >120 generations
✓ The CT Stabilizer™ and suPRE™ Elements have been used to optimize expression and cell line stability
✓ Celltheon’s SUPERCELLS™ have been engineered for improved metabolic profiles (low ammonia and lactate), no
clumping and high expression of difficult to express proteins
✓ The Celltheon SMART™ Platform has been applied to over 60 molecules with consistent reproducible results

✓ Pall’s Allegro STR Single-Use Bioreactors feature a novel, square design with baffles, optimized impeller and ring
sparger resulting in higher kLa and P/V, eliminating limitations in scale-up from bench to production scale
✓ Scale up of a clone from 3L to 200L PALL STR using the Celltheon SMART™ scalable platform resulted in reproducible
cell growth profiles, productivity, and product quality.

• Integrated, scalable fed-batch process reduces timelines to 6 months – ready for tech transfer
• Allegro STR SUBs: scalable production equipment eliminate limitations in production
• The Celltheon SMART™ Platform has been adopted and licensed for several programs

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 ACKNOWLEDGEMENTS
CELLTHEON PALL BIOTECH
• Amita Goel, CEO • Matt Niloff
• Nicy Varghese • Scott Westerhout
• Nirmala Kuppalu • Donna Sinclair
• Ruchika Srivastava • Joe Capone
• Beant Kaur • Ian Sellick
• Nikhil Goel • Andrew Laskowski
• Todd Lundeen
• Leticia Reyes-Regis
• Bart Berger
• Marco Sanchez
• Davone Platz
• Elias Khoury

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 POSTER PRESENTATION

Please visit us at the Pall Biotech Booth

to review our poster presentation. We
will be answering additional questions
regarding the Celltheon SMARTTM
Platform and the Allegro STR single-use
bioreactors.

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 CONTACT US AT
info@celltheon.com
T : +1 ​510-306-2355

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