New Approaches in Continuous BioManufacturing: Continuous XD® cell cultures (around 100 mln cells/mL) coupled to the Rhobust® EBA integrated clarification and purification technology Gerben Zijlstra, PhD Sr. Scientist DSM Biologics, The Netherlands

Outline Introduction DSM Biologics DSM XD® Technology RHOBUST® Expanded Bed Adsorption (EBA) Technology Continuous XD® coupled to RHOBUST® EBA Principle Continuous XD® examples Rhobust® EBA on continuous XD® harvest Concluding Remarks

DSM Biologics: Who are we? Part of DSM: A Leading Global Life Sciences & Material Sciences Company Active in 50 countries & 5 continents at over 200 locations 2012 revenue > € 9 billion ~23,000 employees DSM Biologics Manufacturing Locations The Netherlands and Australia DSM Biologics Services Contract manufacturing for mammalian cell culture: From development to commercial manufacturing cGMP for all clinical phases & market supply Regulatory support Global reach Proprietary Process Technologies: XD® - intensifying upstream process technology RHOBUST® - direct capture technology

XD® Technology Very high-density mammalian processes Increased bioreactor output & yield per volume 5 – 15 fold High & Consistent product quality Reduced capital expenditure requirements Lower scale-up risk XD® is a registered trademark of DSM

Cell Culture Mode of Manufacturing XD®: Proprietary Process Intensification Cell Culture Modes Cell Culture Mode of Manufacturing Fed Batch Feed concentrate Build up Metabolites Osmo increase Changing environment Reducing cell viabilities Concentrated Harvest batch identification XD® Medium Feed Wash out Metabolites No Osmo increase Constant environment High cell viabilities Concentrated Harvest batch identification Perfusion Medium Feed Wash out Metabolites No Osmo increase Constant environment High cell viabilities Dilute harvest Large harvest Fed Batch Feed concentrate Build up Metabolites Osmo increase Changing environment Reducing cell viabilities Concentrated Harvest batch identification Fed Batch Feed concentrate Build up Metabolites Osmo increase Changing environment Reducing cell viabilities Concentrated Harvest batch identification XD® Medium Feed Wash out Metabolites No Osmo increase Constant environment High cell viabilities Concentrated Harvest batch identification XD® Medium Feed Wash out Metabolites No Osmo increase Constant environment High cell viabilities Concentrated Harvest batch identification Perfusion Medium Feed Wash out Metabolites No Osmo increase Constant environment High cell viabilities Dilute harvest Large harvest Perfusion Medium Feed Wash out Metabolites No Osmo increase Constant environment High cell viabilities Dilute harvest Large harvest XD® Process XD® Process

XD® : Process Intensification Scale-up - PER.C6®; Viable cell count

XD® scale up Scale-up - PER.C6®; Product IgG

XD®: Process Intensification Bioreactor Set-up

Preferred retention system: TFF Fully disposable, simple operation, robust, flexible filter choice

DSM RHOBUST® Technology Direct product capture Reduced unit operations From 3 to 1 Higher yields Proven scalability Reduced labor cost & process time Suitable for recombinant proteins, antibodies, vaccines Tungsten carbide incorporated in the agarose bead

RHOBUST® Direct Capture Technology

RHOBUST® in Action with the XD® Harvest Equilibration Cell wash out XD® Harvest ~150x106 cell/mL First cell breakthrough Wash RHOBUST® 1 step! Complete cell breakthrough Elution Post-Protein A intermediate

RHOBUST® experiments with XD® Harvest Results Fed-batch and classical Protein-A packed bed vs. XD® and RHOBUST® Protein-A: Yield (%) Purity (%) HCP (ug/mg Mab) DNA (ng/mg Mab) Protein A (ppm) Fed-batch, clarification, Protein-A Packed Bed > 85 > 95 1 – 15 (n=15) 23-49 (n=2) 9-12 XD®, Protein-A RHOBUST® > 90 0.3 - 23 (n=19) 3.7-121 (n=6) 4-14 With high cell viabilities ~10% higher yield HCP, DNA & res.Protein A After column in normal range

Outline Introduction DSM Biologics DSM XD® Technology RHOBUST® Expanded Bed Adsorption (EBA) Technology Continuous XD® coupled to RHOBUST® EBA Principle Continuous XD ® examples Rhobust® EBA on continuous XD® harvest Concluding Remarks

Continuous XD® - Rhobust® principle Tungsten carbide incorporated in the agarose bead 2-8°C Product/Cell Bleed Elution buffer Product eluate Low pH treatment Waste Medium XD® Bioreactor Cell/Product Bleed Rhobust® EBA+Low pH Low pH treated EBA bulk Concentrated Product Bleed Diluted Waste Stream

Continuous XD® example 1: Myeloma - IgG Continuous XD® cultures with Myeloma cells producing highly potent IgG at around 60 mln cells/mL.

Continuous XD® example 1: Myeloma - IgG The Qp (slope of cumulative titer) was constant at maximum Qp.

Continuous XD® example 2: CHO - IgG Continuous XD® cultures with CHO cells producing Biosimilar IgG at around 100 mln cells/mL.

Continuous XD® example 2: CHO - IgG The IgG titer in the product was bleed around 2.5 g/L in production phase.

Continuous XD® example 4: PER.C6® – IgG Continuous XD® with IgG producing PER.C6® cells at around 100 mln cells/mL. IgG titer in the bleed 4.5 - 5 g/L

Rhobust® EBA example 2: IgG An continuous XD® bioreactor processed with eight Rhobust® EBA runs (6 cell bleeds and 2 final bioreactor harvest loads). Product recovery, averaging at 93% was substantially higher compared to the combination of dead-end filtration and fixed bed Protein-A.

Rhobust® EBA example 2: IgG Residual DNA and HCP were within normal ranges and comparable to packed bed values. Aggregate levels and relative potency were relatively constant throughout the run.

Concluding remarks Advantages Continuous XD® technology coupled to Rhobust® EBA USP (Continuous XD® cell culture): Functional advantages: Robust, stable performance: Stable growth rate by Cell bleed Very high Productivity: Very high cell density x Maximum Qp No product loss: Bleed = Product! Constant Quality: High viability & Constant environment Operational advantages: Concentrated product flow: Harvest holding point possible DSP (Rhobust® EBA Clarification / Capture): Very high Recovery: Single unit operation, Concentrated product flow Very high Purity: Optimized Rhobust® EBA Easy to use, no column packing, air bubbles and precipitates no problem One step clarification and capture

Acknowledgements: R&D Scientist team DSM-B GMP Process Technologist team Gerben Zijlstra Imre Akkerman Olaf Mol Maria Perlasca Dick Smit Mark Dressen Jurjen de Jong Harriet van der Molen Piet den Boer Mark Doeven Erik kremer Henk van Urk Jaco van der Merwe R&D Director GMP OPS Manager Fritjof Linz Esther Heuberger All Technicians involved in this work

Thank you Gerben.Zijlstra@DSM.com

Outline Introduction DSM Biologics DSM XD® Technology RHOBUST® Expanded Bed Adsorption (EBA) Technology Continuous XD® coupled to RHOBUST® EBA Principle Continuous XD ® examples Rhobust® EBA on continuous XD® harvest Concluding Remarks

Proprietary Technologies vs. Classical Concept Optimize individual Unit Operations Process Intensification & Process Integration

XD®: Process Intensification Bioreactor Set-up Concentrated Product Bleed

XD®: Process Intensification Scaled-up in different 50 L Single Use Bioreactors

Scale-up: 200 L XD® Results 200 L XD® run with CHO cell line performed in PD with 2L satellite run under equal conditions 200 L XD® run performed in standard Sartorius STR bioreactor (only increased size side ports) Successful scale-up to equal cell densities (130 mln cells/mL in 200 L) No oxygen or other limitation observed Titer similar to satellite run > 10 g/L Specific productivity the same

Outline Introduction DSM Biologics DSM XD® Technology RHOBUST® Expanded Bed Adsorption (EBA) Technology Continuous XD® coupled to RHOBUST® EBA Principle Continuous XD ® examples Rhobust® EBA on continuous XD® harvest Concluding Remarks

Rhobust®: In action Elution of concentrated product

RHOBUST® GMP column (30 cm diameter) and MabDirect ProteinA adsorbent GMP EBA column 30cm diameter 20cm - 60cm settled bed EBA level monitoring and control (ultrasound) Low pressure system RFD (variable speed) Disposable flow path UV, flow, conductivity, pressure, temperature, pH using AktaReady Operational MabDirect ProteinA RSF available

Rhobust® EBA on Cont. XD® example 1 Cell density: 60-90 million cells/mL (viability >80%) Antibody titer: 1.3 g/L Comparison: MabDirect proteinA EBA vs. clarification & protein A packed bed chromatography Load ratio: Approx. 22 mg IgG/mL settled bed (both protein A resins) Process Overall yield (%) Purity HP-SEC Buffer use (CV) Process time lab-scale1 (hours) Manufacturing– scale, estimated2 Rhobust® EBA 82 99.6 67 6.8 Clarification & ProteinA packed bed 70 99.4 118 12.5 18.5 1 Clarification and chromatographic process (pH treatment, filtration and filling not included) 2 Clarification manufacturing scale will take 6-8 hours (includes dilution, pre-rinse, filtration, post-rinse)

Concluding remarks Advantages RHOBUST® technology Operational: Easy to use, no column packing Can deal with air bubbles and precipitated material One step clarification and capture No separate clarification  8-hour time reduction of process time Suitable for other high viscosity feed streams (incl. microbial and yeast). Development and Scale-up Reduced-scale model available (1 cm and 2 cm column + AKTA Explorer) Scalable concept: Pilot scale unit (10 – 60 cm columns + Rhobust Flex or AKTA Ready) Fully automated GMP unit (10 – 60 cm colums + AKTA Ready) 30cm-diameter EBA column with floating piston EBA level monitoring and control (ultrasound) Disposable flow path and in process monitoring in place (AKTA Ready) Resins and ligands: Available Resins: MabDirect ProteinA, MabDirect MIMO, FastLine SP Large ligand library (Incl): IMAC, Q, DEAE

Continuous XD® example 3: PER.C6® – Rec. Continuous XD® culture with PER.C6® cells producing recombinant protein (> 300 kD). Discrete intermittent product bleeds were taken for subsequent DSP

Principle of the Kremer Method A one step flow-through intermediate purification and polishing procedure, which can optionally be followed by virus filtration. Benefits: One unit operation for 2 chromatography steps Standard dual pump chromatography systems required Product in flow-through, impurities bind ( small resin volumes) No intermediate storage Good removal of aggregates and HCPs The Kremer method™ has successfully been applied to post Rhobust® intermediate yielding very similar purity as classical DSP.

Rhobust® EBA from Cont. XD® example 1 500 1000 1500 2000 2500 3000 3500 mAU 2.0 4.0 6.0 8.0 10.0 Volume (mL) F F3 F4 F5 F6 F7 OD 280 pH Fractions F3- Load F4 - Wash 1 F5 – Wash 2 F6 - Elution F7 - Strip F8 - Cleaning F8