continuous antibody capture with protein a countercurrent tangential chromatography: a new...
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Continuous Antibody Capture with Protein A Countercurrent Tangential
Chromatography: A New Column-Free Approach for Antibody
Purification
Andrew L. ZydneyDepartment Head and Walter L. Robb Family Chair
Department of Chemical EngineeringThe Pennsylvania State University
Presented at the ECI Conference on Integrated Continuous Biomanufacturing
Castelldefels, Spain, October 21, 2013
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• Significant potential opportunities– Reduced capital costs / facility requirements– Higher productivity – Easier scale-up
• Major technology developments in place– Perfusion bioreactors– In-line filters
• Critical challenge is chromatography
Continuous Bioprocessing
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• Multi-column periodic counter-current chromatography (PCC) – GE Healthcare
• Simulated moving bed chromatography (SMB) – Semba, Tarpon, Contichrom
• Sequential multi-column chromatography (SMCC) – Novasep
• These approaches typically do not provide truly steady-state operation, potentially leading to variability in product quality
Chromatography Options
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Example: SMB
New Developments in Simulated Moving Bed ChromatographySeidel-Morgenstern, Kessler, and Kaspereit Chemical Engineering Technology, 31: 826 (2008)
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• Develop and demonstrate a new technology that can provide truly continuous protein purification using available chromatography resins, e.g., Protein A
• Design criteria:– Comparable yield and purity to columns– High productivity (10x packed columns)
– Single use capability (no stainless steel)
Objectives
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Countercurrent Tangential Chromatography - CTC
Chromatographic resin (beads) flows as a slurry through a series of static mixers and hollow fiber membrane modules
All operations (binding, washing, elution, stripping, equilibration) performed directly on the slurry
Countercurrent staging used to reduce buffer and resin requirements, increase product yield and purity
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Continuous CTC System
BindingBinding WashingWashing ElutionElution StrippingStripping Regnera-tion
Regnera-tion
SlurryTank
SlurryTank
FeedTankFeedTank
Waste
Waste
Waste Waste
ProductTank
ProductTank
True moving bed Conveyor like processResin slurry moves counter-currently to buffer in each step
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Chromatographic “Stage”
Provides residence time needed for equilibration in binding and elution steps
Excellent radial mixing with minimal pressure drop
Provides complete separation between resin particles and fluid phase
High single pass conversion with low pressure losses
+
Koflo static mixer
Spectrum hollow fiber module
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Centrate Feed
Wash 1 Buffer
Wash 2 Buffer
Elution Buffer
Stripping Buffer
Equilibra-tion
Buffer
Continuous CTC System
Resin Tank
UF
Step
Binding
Wash 1
Wash 2
Elution
Stripping
Equilibration
Binding Permeate
Wash 1 Permeate
Product Tank
UF Permeate
Wash 2 Permeate
Stripping Permeate
Equilibra-tion
Permeate
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Countercurrent Staging - Elution
1st stage
2nd stage
Washing
pH 3ElutionBuffer
Purified mAb
Resin Slurry from Wash
Resin Slurry to Strip
Stage 1 Stage 2
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1st stage
2nd stage
Washing
pH 3ElutionBuffer
Purified mAb
Resin Slurry from Wash
Resin Slurry to Strip
Stage 1 Stage 2
Countercurrent Staging - Elution
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1st stage
2nd stage
Washing
pH 3ElutionBuffer
Purified mAb
Resin Slurry from Wash
Resin Slurry to Strip
Stage 1 Stage 2
Countercurrent Staging - Elution
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1st stage
2nd stage
Washing
pH 3ElutionBuffer
Purified mAb
Resin Slurry from Wash
Resin Slurry to Strip
Stage 1 Stage 2
Countercurrent Staging - Elution
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1st stage
2nd stage
Washing
pH 3ElutionBuffer
Purified mAb
Resin Slurry from Wash
Resin Slurry to Strip
Stage 1 Stage 2
Countercurrent Staging - Elution
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Tangential flow filter
Static mixer
Static mixer
Tangential flow filter
Tangential flow filter
Static mixer
Concentrated slurry with
bound product
Concentrated resin slurry
Elution Buffer
Product
P
P
R
P R
R
Example: 3-Stage Elution Step
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Number of
stages
Experimental yield
Theoretical yield
1 78 ± 2% 77%
2 94 ± 2% 94%
3 98 ± 1% 98%
qp = permeate flow rate qr = retentate flow rate n = number of stages
Effect of Staging – Elution Step
Results for qp / qr = 0.75
From Shinkazh et al., Biotech. Bioeng, 108: 582 (2011)
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Experimental System
• Clarified cell culture fluid (Fujifilm Diosynth)– Monoclonal antibody product
• POROS® MabCapture A resin – Life Technologies– 45 µm diameter particles, Protein A ligand
• MidiCros® hollow fiber modules - Spectrum Lab– 0.5 µm PES membranes, 1 mm ID, 200 cm2 area
• Static mixers – Koflo Corportation– 29 cm length, 1 cm ID
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Critical Filtrate Flux
Feed: 10% slurry, 100 mL/min
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Critical Filtrate Flux
Feed: 10% slurry, 100 mL/min • Critical flux corresponds to 80% conversion using 10% slurry
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Critical Filtrate Flux
Feed: 10% slurry, 100 mL/min • Critical flux corresponds to 80% conversion using 10% slurry
• System design:– 7.5% slurry– 75% conversion
• Extra safety limit enables stable operation for long times
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CTC Process using Protein A
Operation Numbe
r of stages
Buffer pHMixed
pH
Binding 2 -- 7.4 7.6
Wash 1 3 20 mM Na2HPO4 + 0.5 M NaCl 7.1 7.5
Wash 2 3 20 mM Na2HPO4 7.2 7.0
Elution 3 40 mM Citrate 3.2 3.3
Strip 2 10 mM HCl + 0.1 M NaCl 2.0 2.5
Equilibration 2 20 mM Na2HPO4 8.1 7.0
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Multiple Runs
RunmAb(g/L)
mAb Load
RunTime
Feed Flow Rate
(L/hr)
mAb Load per
Resin
1 – feasibility 1.2 16 g 3 hr 4.5 190 g/L
2 – long time 0.72 8 g 24 hr 0.45 470 g/L
3 – high titer 4.5 8 g 4 hr 0.45 230 g/L
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Run 1 - Pressure Profiles
Elapsed Time, t (hr)
Pre
ssu
re,
P (
psi
g)
0
2
4
6
8
10
12
0 0.5 1.0 1.5 2.0 2.5
• Stable operation
• Pressure <10 psi
• Laminar flow
• All plastic tubing and connectors
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Run 1- mAb Purification
Elution
PuremAb
CCCF
SEC Profiles
Elution Time, t (min)
Ab
sorb
ance
• >95% yield
• >98% purity
• Productivity of 63 g mAb/L resin/hr (10x packed column)
• No detectable protein aggregates
• No detectable changes in resin
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Run 1 - mAb Purification
SampleHost Cell Protein
(ppm)
Clarified Harvest 675,000
CCTC System 1,200
Packed Column 2,800
• Host cell protein measured relative to mAb via ELISA• HCP level in CCTC system 2x lower than packed column• Yield >95%, purity >98%• Similar levels of high MW to purified (reference) mAb
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Run 2 – Product Profile
• Steady-state with respect to product concentration and impurity profile
• Long time operation possible
• For t > 12 hr hollow fiber modules had to be replaced due to bacterial growth
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Run 2 – HCP levels
• HCP level remains constant throughout 24 hr run
• >95% purity
• Productivity of 19 g mAb/L resin/hr (reduced due to low titer feed)
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Run 3 - mAb Purification
SampleHost Cell
Protein (ppm)
2 hr 310
3 hr 345
4 hr 382
• Very low HCP level due to use of high titer feed (4.5 g/L) spiked with purified mAb
• >99% purity
• Productivity of 52 g mAb/ L resin / hr
• 2.6 cycles / hr for resin• HCP measured via ELISA
relative to mAb
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Advantages of CCTC System
• Continuous operation with high productivity– All resin used at all times– Steady-state operation with respect to product
concentration and impurity profiles
• No columns / packing– Reduced labor costs and validation– Greater flexibility in multi-product facilities
• Disposable flow path if desired– Potential for single-use systems– Ideal for production of clinical batches
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Future Opportunities
• Use of smaller resin particles– Much better mass transfer less residence time
needed in binding and elution steps
– Lower hold-up volume greater productivity
– No issues with pressure drop for slurry flow
• Direct integration with perfusion bioreactor– Opportunity for continuous steady-state processing
– Dramatic improvements in overall productivity
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Summary
• Countercurrent tangential chromatography (CCTC) for mAb purification– Continuous and steady-state operation demonstrated
for 24 hr
– Purity and yield comparable to packed column
– Countercurrent staging reduces resin requirements while increasing product yield and purity
– Low pressure operation opportunities for disposable single-use flow path
– Modular design for enhanced flexibility
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Acknowledgements• Oleg Shinkazh
– Founder and President, Chromatan
• Boris Napadensky– VP of Engineering, Chromatan
• Achyuta Teella– Senior Scientist at Chromatan, Post-doc at Penn State
• Travis Tran– Associate Scientist, Chromatan
• Gary Brookhart– Senior Research Scientist, Fujifilm Diosynth
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Funding / Support