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 TIME

OPEN 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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