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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
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• 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
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• 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?
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• 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
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Perfusion Medium FeedWash out MetabolitesNo Osmo increaseConstant environmentHigh cell viabilitiesDilute harvestLarge harvest
Fed Batch Feed concentrateBuild up MetabolitesOsmo increaseChanging environmentReducing cell viabilitiesConcentrated Harvestbatch identification
XD®
Medium FeedWash out MetabolitesNo Osmo increaseConstant environmentHigh cell viabilitiesConcentrated Harvestbatch identification
Cell Culture Modes
XD®
Process
XD®: Proprietary Process Intensification
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XD® : Process Intensification
Scale-up - PER.C6®; Viable cell count
0 1 2 3 4 5 6 7 8 9 10 11 12 131
10
100
10002L XD reference 50L XD run #1 50L XD run #2
Time (day)
VC
D (
*10
E6
ce
lls/m
L)
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XD® scale upScale-up - PER.C6®; Product IgG
5 6 7 8 9 10 11 12 13 141
10
100
17.0520.6
2L XD reference 50L XD run #1 50L XD run #2
Time (day)
IgG
(g
/L)
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XD®: Process Intensification Bioreactor Set-up
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Preferred retention system: TFF
Page 9 Fully disposable, simple operation, robust, flexible filter choice
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• 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
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RHOBUST® Direct Capture Technology
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XD® Harvest~150x106 cell/mL
Post-Protein Aintermediate
Equilibration
First cell breakthrough
Elution
Wash
Cell wash out
Complete cell breakthrough
RHOBUST ® 1 step!
RHOBUST® in Action with the XD® Harvest
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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
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• 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
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Continuous XD® - Rhobust® principle
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Tungsten carbide incorporated in the agarose bead
2-8°C Product/Cel
l Bleed
Elution buffer
Product eluateLow pH treatment
Waste
Waste
Medium
XD® Bioreactor Cell/Product Bleed Rhobust® EBA+Low pH Low pH treated EBA bulk
Concentrated Product Bleed
Diluted Waste Stream
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Continuous XD® example 1: Myeloma - IgG
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Continuous XD® cultures with Myeloma cells producing highly potent IgG at around 60 mln cells/mL.
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Continuous XD® example 1: Myeloma - IgG
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The Qp (slope of cumulative titer) was constant at maximum Qp.
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Continuous XD® example 2: CHO - IgG
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Continuous XD® cultures with CHO cells producing Biosimilar IgG at around 100 mln cells/mL.
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Continuous XD® example 2: CHO - IgG
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The IgG titer in the product was bleed around 2.5 g/L in production phase.
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Continuous XD® example 4: PER.C6® – IgG
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Continuous XD® with IgG producing PER.C6® cells at around 100 mln cells/mL.IgG titer in the bleed 4.5 - 5 g/L
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Rhobust® EBA example 2: IgG
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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.
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Rhobust® EBA example 2: IgG
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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.
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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
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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:
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• 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
26
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Proprietary Technologies vs. Classical Concept
Optimize individual Unit Operations
Process Intensification & Process Integration
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XD®: Process Intensification Bioreactor Set-up
Concentrated Product Bleed
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XD®: Process IntensificationScaled-up in different 50 L Single Use Bioreactors
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Scale-up: 200 L XD® Results
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• 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
2,00
4,00
6,00
8,00
10,00
12,00
14,00
0,00 100,00 200,00 300,00 400,00 500,00 600,00 700,00 800,00 900,00 1000,00
IVC
Tit
er
(g/L
)
200L XD
2L P23037
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• 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
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Rhobust®: In action
Elution of concentrated product
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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
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Rhobust® EBA on Cont. XD® example 1
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Process Overall yield
(%)
PurityHP-SEC
(%)
Buffer use
(CV)
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)
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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
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Continuous XD® example 3: PER.C6® – Rec.
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Continuous XD® culture with PER.C6® cells producing recombinant protein (> 300 kD). Discrete intermittent product bleeds were taken for subsequent DSP
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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.
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Rhobust® EBA from Cont. XD® example 1
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OD 280 pHFractions
F3- LoadF4 - Wash 1F5 – Wash 2F6 - Elution F7 - StripF8 - Cleaning
0
500
1000
1500
2000
2500
3000
3500
mAU
2.0
4.0
6.0
8.0
10.0
500 1000 1500 Volume (mL)F F3 F4 F5 F6 F7 F8