Simplicity is the key
Continuous purification of
monoclonal antibodies
L. Landric-Burtin
Head of Downstream Processing Development, France
Integrated Continuous Biomanufacturing Conference,
Barcelona, 2013
mAbs manufacturing processes
have reached a good level of
maturity
Experience
Cell line platforms
High titers in cell culture
Purification platforms
Single use
QbD
Capacity ≥ Demand
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But our manufacturing
processes are still..
COMPLEX !
LONG !
EXPENSIVE !
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COMPLEX.
Equilibration buffer #1
OPERATOR INTERVENTION
Wash buffer #1
Elution buffer #1
HOLD TIME
pH, concentration
adjustment, sampling
OPEN PHASE
Sampling &
OPEN PHASE
Storage
OPERATOR INTERVENTION
Equilibration buffer #2
Wash buffer #2
Elution buffer #2
HOLD TIME
OPEN PHASE
Sampling &
Storage
Bulk Harvest
0.2 µm
filtration
Protein A
Wash buffer #3
pH, conductivity
adjustment
Chrom2
Low pH & Neutralization
OPERATOR INTERVENTIONEquilibration buffer #3
OPEN PHASE
0.2 µm
filtration
Chrom3
OPERATOR INTERVENTION
HOLD TIME
Sampling &
Storage OPEN PHASE
Nanofiltration
OPERATOR INTERVENTION
HOLD TIME
Sampling &
OPEN PHASE
Storage
TFF & storage
≥ 7 different
buffers
Contaminations,
Human errors
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7 consecutives, discontinued steps (12 incl. storage)
LONG, DISCONTINUED.
Processing time (hr)
Around 50 hr to complete
the purification process
« n » step cannot start
before completion of
« n-1 »
Interruption between steps
for concentration, pH or
conductivity adjustement and
storage.
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EXPENSIVE.
• ≥ 100 €/gr
• Chromatography resin = 30 to 75 % of DSP
costs.
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COMPLEX, LONG,
EXPENSIVE...
Any solution ?
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SIMPLIFY, SIMPLIFY,
SIMPLIFY.
→ SIMPLICITY is key to get a quick,
reliable and cheap process.
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Simplicity
&
Purification of Mabs ?
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Let’s go back to the essentials
Equilibration buffer #1
Wash buffer #1
Elution buffer #1
pH, concentration
adjustment, sampling
Equilibration buffer #2
Wash buffer #2
Elution buffer #2
Sampling &
Storage
Sampling &
Storage
Bulk Harvest
0.2 µm
filtration
Protein A
0.2 µm
filtration
Chrom2
Low pH & Neutralization
Equilibration buffer #3
Wash buffer #3
Sampling &
Storage
pH, conductivity
adjustment
Chrom3
Sampling &
Storage
Nanofiltration
TFF & storage
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Simplicity means NO
inter-step ADJUSTMENT.
→We have selected resins that afford no
inter-step adjustment.
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Simplicity means FEW
BUFFERS.
→We have imagined a buffer strategy
composed of only 3 components for the
entire process.
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Simplicity means FEW
BUFFERS.
→4 buffers in total, ensuring pH and
conductivity compatibility between all
steps.
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How simplicity looks
like.
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SIMPLE.
Bulk Harvest
Equilibration buffer #1
Wash buffer #1
Elution buffer #1
Equilibration buffer #1
Elution buffer #2
Wash buffer #1
Sampling &
Storage
Low pH
Protein A
Mixed
mode
AEX
Nanofiltration
TFF & storage
• 4 different buffers
• 6 steps (7 incl. storage), 4 being continuous.
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Can a simplified process make the job ?
Clarified
supernatant
Protein A
Mixed-mode
AEX
High Molecular
Weight species
8.7 %
1.0 %
0.2 %
0.2 %
Host Cell Proteins
2.2E+05 ppm
800 ppm
40 ppm
5 ppm
Host Cell DNA
3E+06 ppb
2E+04 ppb
10 ppb
< 0.1 ppb
Endotoxins < 0.05 EU/mL along the process
Overall yield ≥ 90 %
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Isoelectric point
Can a simplified process be a platform ?
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Project 2
Yield : 89%
Project 3
Yield : 92%
Project 1
Yield : 93%
Project 4
Yield : 94%
Project 5
Yield : 86%
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→ Successfully evaluated on 5 different projects
without optimization.
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Simplicity
advantages
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SIMPLIFIED PROCESS GAINS
→ reduced our process time:
2X higher per-hour productivity.
→ reduced number of buffers:
30 % cost saving on buffers.
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SIMPLIFIED PROCESS GAINS
→ Continuous manufacturing with no
human handling.
•
•
•
•
Reduced risk of contamination
Reduced sampling plan
Reduced storage needs
Possibility to run the process in a
multicycle mode.
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SIMPLE.
Bulk Harvest
Equilibration buffer #1
Wash buffer #1
Elution buffer #1
Equilibration buffer #1
Elution buffer #2
Wash buffer #1
Sampling &
Storage
Low pH
Protein A
Mixed
mode
AEX
Nanofiltration
TFF & storage
• 4 different buffers
• 6 steps (7 incl. storage), 4 being continuous.
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SIMPLE, SMALL and AUTOMATED.
Bulk Harvest
Equilibration buffer #1
Wash buffer #1
Elution buffer #1
Equilibration buffer #2
Elution buffer #2
Sampling &
Storage
Wash buffer #1
Run X
Run 3
Run 2
Run 1
Low pH
Protein A
Mixed
mode
AEX
Nanofiltration
TFF & storage
→We have automated the process to run it in
multi cycles with small columns.
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Batch Mode
ASAP process gains.
Continuous
Multi-cycling
• 66% of resin volume
saved for an
equivalent batch
duration.
•
Up to 97% for > duration.
• Perfect application for
disposable columns.
• No packing
• Full use of resin lifetime
• No storage
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Batch Mode
SIMPLE, SMALL and CHEAP.
• Smaller systems and
facilities.
Continuous
Multi-cycling
• Limited staff
requested.
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SIMPLE, SMALL and PRODUCTIVE
14.1 L resin, 4 cycles prot A, (24 hr* to 72h duration)
1 m3 Bulk Harvest
Productivity : 2.7 g/L/hr*
(*):with shift work
1.2 L resin - 47 cycles - 71 hrs duration
Productivity : 10.5 g/L/hr
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SIMPLE and ADAPTATIVE
→ Adapt your column size to your requirements
Processing time
50 hr
X hr
High product needs,
facility constraints:
→ Big columns to
process rapidely
one batch
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Batch cost
<< 100€/g
~100€/g
Cost constraint:
→ Small columns to decrease resin
costs and optimize resin lifetime
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CASE STUDY:
Continuous purification at lab scale
50 or 100 mL
resin volume
70 L of
buffers
43 L of
supernatant
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CASE STUDY:
Continuous purification at lab scale
● 43 L of supernatant fully purified
continuously in 69h :
● 45 runs performed automatically.
● 67g of purified mAb collected
● 93% yield
X3 24 ml min Charge 40min X9 (1012)001:10_UV1Col1
X3 24 ml min Charge 40min X9 (1012)001:10_UV1Col2
X3 24 ml min Charge 40min X9 (1012)001:10_UV1Col3
X3 24 ml min Charge 40min X9 (1012)001:10_Logbook
mAU
mAU
X3 24 ml min Charge 40min X9 (1012)001:10_UV1Col1
X3 24 ml min Charge 40min X9 (1012)001:10_UV1Col2
X3 24 ml min Charge 40min X9 (1012)001:10_UV1Col3
X3 24 ml min Charge 40min X9 (1012)001:10_Logbook
mAU
mAU
3000
2500
3500
3000
2500
3000
2000
2500
2000
2500
2000
1500
2000
1500
1500
1000
1500
1000
1000
1000
500
500
500
500
0
0
0
0
0
500
1000
1500
100
2000
200
2500
300
3000
400
3500
min
0
4000
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min
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SCALE UP and GMP compliance
evaluation (On-going)
● 500 L batch
● Final target : > 1Kg of purified mAb
● Column < 10L
● < 10 runs with a final duration of 16h
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CONCLUSION
→If you seek for quick, reliable and cheap
process, get the superfluous off your process.
→ Starting from this perspective, we have
designed
- a simplified purification platform,
- able to run continuously,
- with 4 fold increase in productivity
- significant gain on resin cost.
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and excellent staff
Simplicity is the key
Continuous purification of
monoclonal antibodies
Special thanks to:
Benoit MOTHES
Didier DUTHE
Céline HEMET
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