new approaches in continuous biomanufacturing :
DESCRIPTION
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. - PowerPoint PPT PresentationTRANSCRIPT
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
2
Page 3
• 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?
Page 4
• 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)
VCD
(*10
E6 c
ells
/mL)
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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
)
Page 8
XD®: Process Intensification Bioreactor Set-up
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
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
• 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
14
Continuous XD® - Rhobust® principle
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Tungsten carbide incorporated in the agarose bead2-8°C
Product/Cell 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
Continuous XD® example 1: Myeloma - IgG
Page 16
Continuous XD® cultures with Myeloma cells producing highly potent IgG at around 60 mln cells/mL.
Continuous XD® example 1: Myeloma - IgG
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The Qp (slope of cumulative titer) was constant at maximum Qp.
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.
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.
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
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.
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.
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
23
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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:
• 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
Page 27
Proprietary Technologies vs. Classical Concept
Optimize individual Unit Operations
Process Intensification & Process Integration
Page 28
XD®: Process Intensification Bioreactor Set-up
Concentrated Product Bleed
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XD®: Process IntensificationScaled-up in different 50 L Single Use Bioreactors
Scale-up: 200 L XD® Results
Page 30
• 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
Tite
r (g/
L)
200L XD
2L P23037
• 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
31
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Rhobust®: In action
Elution of concentrated product
33
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
34
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)
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
35
Continuous XD® example 3: PER.C6® – Rec.
Page 36
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.
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Rhobust® EBA from Cont. XD® example 1
38
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