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Simplicity is the key Continuous purification of
monoclonal antibodies
L. Landric-BurtinHead of Downstream Processing Development, FranceIntegrated Continuous Biomanufacturing Conference, Barcelona, 2013
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mAbs manufacturing processes have reached a good level of maturity
Capacity ≥ Demand
Cell line platforms
Single use
High titers in cell culturePurification platforms
Experience
QbD
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But our manufacturing processes are still..
COMPLEX ! LONG ! EXPENSIVE !
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Protein A Chrom2
Chrom3
Equilibration buffer #2Wash buffer #2
Elution buffer #2
Bulk Harvest
Low pH & Neutralization
pH, concentration adjustment, sampling
0.2 µm filtration
0.2 µm filtration
Nanofiltration
COMPLEX.
≥ 7 different buffers7 consecutives, discontinued steps (12 incl. storage)
Sampling & Storage
pH, conductivity adjustment
Equilibration buffer #1Wash buffer #1
Elution buffer #1
Sampling & Storage
Sampling & Storage
TFF & storage
Equilibration buffer #3Wash buffer #3
Sampling & Storage
OPEN PHASEHOLD TIME
OPEN PHASEHOLD TIME
OPERATOR INTERVENTION
OPEN PHASE
OPEN PHASE
HOLD TIME HOLD TIMEOPEN PHASEOPEN PHASE
OPERATOR INTERVENTION
OPERATOR INTERVENTION OPERATOR INTERVENTION
Contaminations, Human errors
OPERATOR INTERVENTION
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LONG, DISCONTINUED.
Processing time (hr)
« n » step cannot start before completion of « n-1 »
Around 50 hr to complete the purification process
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
Protein A Chrom2
Chrom3
Equilibration buffer #2Wash buffer #2
Elution buffer #2
Bulk Harvest
Low pH & Neutralization
pH, concentration adjustment, sampling
0.2 µm filtration 0.2 µm
filtration
Nanofiltration
Sampling & Storage
pH, conductivity adjustment
Equilibration buffer #1Wash buffer #1
Elution buffer #1
Sampling & Storage
Sampling & Storage
TFF & storage
Equilibration buffer #3Wash buffer #3
Sampling & 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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Protein A Mixed mode
AEX
Equilibration buffer #1Elution buffer #2
Nanofiltration
SIMPLE.
• 4 different buffers• 6 steps (7 incl. storage), 4 being continuous.
Equilibration buffer #1Wash buffer #1
Elution buffer #1
TFF & storage
Wash buffer #1 Sampling & Storage
Bulk Harvest
Low pH
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Can a simplified process make the job ?
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Clarified supernatant
High Molecular Weight species 8.7 %
Host Cell Proteins 2.2E+05 ppm
Host Cell DNA 3E+06 ppb
Overall yield ≥ 90 %
Endotoxins < 0.05 EU/mL along the process
Protein A
1.0 %
800 ppm
2E+04 ppb
Mixed-mode
0.2 %
40 ppm
10 ppb
AEX
0.2 %
5 ppm
< 0.1 ppb
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Can a simplified process be a platform ?
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9 8 7 6 5
Project 1Yield : 93%
Project 4Yield : 94%
Project 3Yield : 92%
Project 2Yield : 89%
Project 5Yield : 86%
→ Successfully evaluated on 5 different projects without optimization.
Isoe
lect
ric p
oint
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Simplicity advantages
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→ reduced our process time: 2X higher per-hour productivity.
→ reduced number of buffers:30 % cost saving on buffers.
SIMPLIFIED PROCESS GAINS
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→ 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.
SIMPLIFIED PROCESS GAINS
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Protein A Mixed mode
AEX
Equilibration buffer #1Elution buffer #2
Nanofiltration
SIMPLE.
• 4 different buffers• 6 steps (7 incl. storage), 4 being continuous.
Equilibration buffer #1Wash buffer #1
Elution buffer #1
TFF & storage
Wash buffer #1 Sampling & Storage
Bulk Harvest
Low pH
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→We have automated the process to run it in multi cycles with small columns.
SIMPLE, SMALL and AUTOMATED.
Protein A Mixed mode AEX
Equilibration buffer #2
Elution buffer #2
Nanofiltration
Equilibration buffer #1Wash buffer #1
Elution buffer #1
TFF & storage
Wash buffer #1Sampling &
Storage
Bulk Harvest
Run 3Run 2
Run 1
Run X
Low pH
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• 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
Continuous Multi-cycling
Bat
ch M
ode
ASAP process gains.
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Bat
ch M
ode
ContinuousMulti-cycling
SIMPLE, SMALL and CHEAP.
• Smaller systems and facilities.
• Limited staff requested.
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SIMPLE, SMALL and PRODUCTIVE
1 m3 Bulk Harvest
14.1 L resin, 4 cycles prot A, (24 hr* to 72h duration)
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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→ Adapt your column size to your requirementsSIMPLE and ADAPTATIVE
Processing time
Batch cost0
50 hr
<< 100€/g ~100€/g
X hr
High product needs, facility constraints: → Big columns to process rapidely one batch Cost constraint:
→ Small columns to decrease resin costs and optimize resin lifetime
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CASE STUDY:Continuous purification at lab scale
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50 or 100 mL resin volume
70 L of buffers
43 L of supernatant
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●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
0
500
1000
1500
2000
2500
3000
mAU
0
500
1000
1500
2000
2500
3000
3500
mAU
0 500 1000 1500 2000 2500 3000 3500 4000 min
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
0
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1000
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2000
2500
mAU
0
500
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2500
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mAU
0 100 200 300 400 min
CASE STUDY:Continuous purification at lab scale
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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
Special thanks to:Benoit MOTHESDidier DUTHECéline HEMET
Simplicity is the key Continuous purification of
monoclonal antibodies
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