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

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

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

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

500

1000

1500

2000

2500

mAU

0

500

1000

1500

2000

2500

3000

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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