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 Capacity ≥ Demand Cell line platforms Single use High titers in cell culture Purification platforms Experience QbD 2

But our manufacturing processes are still.. COMPLEX ! LONG ! EXPENSIVE ! 3

Protein AChrom2 Chrom3 Equilibration buffer #2 Wash buffer #2 Elution buffer #2 Bulk Harvest Low pH & Neutralization pH, concentration adjustment, sampling 0.2 µm filtration Nanofiltration COMPLEX. ≥ 7 different buffers 7 consecutives, discontinued steps (12 incl. storage) Sampling & Storage pH, conductivity adjustment Equilibration buffer #1 Wash buffer #1 Elution buffer #1 Sampling & Storage TFF & storage Equilibration buffer #3 Wash buffer #3 Sampling & Storage OPEN PHASE HOLD TIME OPEN PHASE HOLD TIME OPERATOR INTERVENTION OPEN PHASE HOLD TIME OPEN PHASE 4 OPERATOR INTERVENTION Contaminations, Human errors OPERATOR INTERVENTION

LONG, DISCONTINUED. Processing time (hr) « n » step cannot start before completion of « n-1 » 5 Around 50 hr to complete the purification process Interruption between steps for concentration, pH or conductivity adjustement and storage.

EXPENSIVE. ≥ 100 €/gr Chromatography resin = 30 to 75 % of DSP costs. 6

COMPLEX, LONG, EXPENSIVE... Any solution ? 7

SIMPLIFY, SIMPLIFY, SIMPLIFY. → SIMPLICITY is key to get a quick, reliable and cheap process. 8

Simplicity & Purification of Mabs ? 9

Let’s go back to the essentials Protein AChrom2 Chrom3 Equilibration buffer #2 Wash buffer #2 Elution buffer #2 Bulk Harvest Low pH & Neutralization pH, concentration adjustment, sampling 0.2 µm filtration Nanofiltration Sampling & Storage pH, conductivity adjustment Equilibration buffer #1 Wash buffer #1 Elution buffer #1 Sampling & Storage TFF & storage Equilibration buffer #3 Wash buffer #3 Sampling & Storage 10

Simplicity means NO inter-step ADJUSTMENT. →We have selected resins that afford no inter-step adjustment. 11

Simplicity means FEW BUFFERS. →We have imagined a buffer strategy composed of only 3 components for the entire process. 12

Simplicity means FEW BUFFERS. →4 buffers in total, ensuring pH and conductivity compatibility between all steps. 13

How simplicity looks like. 14

Protein A Mixed mode AEX Equilibration buffer #1 Elution buffer #2 Nanofiltration SIMPLE. 4 different buffers 6 steps (7 incl. storage), 4 being continuous. Equilibration buffer #1 Wash buffer #1 Elution buffer #1 TFF & storage Wash buffer #1 Sampling & Storage Bulk Harvest Low pH 15

Can a simplified process make the job ? | 16 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 ? | Project 1 Yield : 93% Project 4 Yield : 94% Project 3 Yield : 92% Project 2 Yield : 89% Project 5 Yield : 86% → Successfully evaluated on 5 different projects without optimization. Isoelectric point

Simplicity advantages 18

→ reduced our process time: 2X higher per-hour productivity. → reduced number of buffers: 30 % cost saving on buffers. SIMPLIFIED PROCESS GAINS 19

→ 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 20

Protein A Mixed mode AEX Equilibration buffer #1 Elution buffer #2 Nanofiltration SIMPLE. 4 different buffers 6 steps (7 incl. storage), 4 being continuous. Equilibration buffer #1 Wash buffer #1 Elution buffer #1 TFF & storage Wash buffer #1 Sampling & Storage Bulk Harvest Low pH 21

→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 #1 Wash buffer #1 Elution buffer #1 TFF & storage Wash buffer #1 Sampling & Storage Bulk Harvest Run 3 Run 2 Run 1 Run X Low pH 22

| 23 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 Batch Mode ASAP process gains.

| 24 Batch Mode Continuous Multi-cycling SIMPLE, SMALL and CHEAP. Smaller systems and facilities. Limited staff requested.

| 25 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

→ Adapt your column size to your requirements SIMPLE and ADAPTATIVE Processing time Batch cost 0 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 26

CASE STUDY: Continuous purification at lab scale | or 100 mL resin volume 70 L of buffers 43 L of supernatant

| 28 ● 43 L of supernatant fully purified continuously in 69h : ● 45 runs performed automatically. ● 67g of purified mAb collected ● 93% yield CASE STUDY: Continuous purification at lab scale

| 29 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

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. 30

and excellent staff Special thanks to: Benoit MOTHES Didier DUTHE Céline HEMET 31 Simplicity is the key Continuous purification of monoclonal antibodies

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