New Approaches in Continuous BioManufacturing :

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, PhDSr. ScientistDSM Biologics, The Netherlands

• 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

Outline

• 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

DSM Biologics: Who are we?

• 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

XD® Technology

Perfusion Medium FeedWash out MetabolitesNo Osmo increaseConstant environmentHigh cell viabilitiesDilute harvestLarge harvest

Fed Batch Feed concentrateBuild up MetabolitesOsmo increaseChanging environmentReducing cell viabilitiesConcentrated Harvestbatch identification

Medium FeedWash out MetabolitesNo Osmo increaseConstant environmentHigh cell viabilitiesConcentrated Harvestbatch identification

Cell Culture Modes

Process

XD®: Proprietary Process Intensification

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

0 1 2 3 4 5 6 7 8 9 10 11 12 131

10002L XD reference 50L XD run #1 50L XD run #2

Time (day)

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

5 6 7 8 9 10 11 12 13 141

17.0520.6

2L XD reference 50L XD run #1 50L XD run #2

Time (day)

XD®: Process Intensification Bioreactor Set-up

Preferred retention system: TFF

Fully disposable, simple operation, robust, flexible filter choice

• 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

DSM RHOBUST® Technology

RHOBUST® Direct Capture Technology

XD® Harvest~150x106 cell/mL

Post-Protein Aintermediate

Equilibration

First cell breakthrough

Elution

Cell wash out

Complete cell breakthrough

RHOBUST® 1 step!

RHOBUST® in Action with the XD® Harvest

RHOBUST® experiments with XD®

HarvestResults 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 > 95 0.3 - 23

(n=19)3.7-121

(n=6) 4-14

With high cell viabilities~10% higher yield

HCP, DNA & res.Protein AAfter column in normal range

• Continuous XD® coupled to RHOBUST® EBA– Principle– Continuous XD ® examples– Rhobust® EBA on continuous XD® harvest

Outline

Continuous XD® - Rhobust® principle

Tungsten carbide incorporated in the agarose bead2-8°C

Product/Cell Bleed

Elution buffer

Product eluateLow pH treatment

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 remarksAdvantages 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):• Functional advantages:

– Very high Recovery: Single unit operation, Concentrated product flow– Very high Purity: Optimized Rhobust® EBA

• Operational advantages:– Easy to use, no column packing, air bubbles and precipitates no problem– One step clarification and capture

R&D Scientist team DSM-B GMP Process Technologist teamGerben Zijlstra Imre AkkermanOlaf Mol Maria PerlascaDick Smit Mark DressenJurjen de Jong Harriet van der MolenPiet den BoerMark DoevenErik kremerHenk van UrkJaco van der Merwe

R&D Director GMP OPS ManagerFritjof Linz Esther Heuberger

All Technicians involved in this work

Acknowledgements:

Thank you

Gerben.Zijlstra@DSM.com

Outline

Proprietary Technologies vs. Classical Concept

Optimize individual Unit Operations

Process Intensification & Process Integration

XD®: Process Intensification Bioreactor Set-up

Concentrated Product Bleed

XD®: Process IntensificationScaled-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

Biomass corrected titer (Nephelometer)

0,00 100,00 200,00 300,00 400,00 500,00 600,00 700,00 800,00 900,00 1000,00

200L XD

2L P23037

Outline

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

Process Overall yield

PurityHP-SEC

Buffer use

Process timelab-scale1

(hours)

Process timeManufacturing–

scale, estimated2

(hours)Rhobust® EBA 82 99.6 67 6.8 6.8

Clarification &ProteinA packed bed

70 99.4 118 12.5 18.5

Cell density: 60-90 million cells/mL (viability >80%)Antibody titer: 1.3 g/LComparison: MabDirect proteinA EBA vs. clarification & protein A

packed bed chromatographyLoad ratio: Approx. 22 mg IgG/mL settled bed (both

protein A resins)

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 remarksAdvantages 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

OD 280 pHFractions

F3- LoadF4 - Wash 1F5 – Wash 2F6 - Elution F7 - StripF8 - Cleaning

500 1000 1500 Volume (mL)F F3 F4 F5 F6 F7 F8

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