Continuous Antibody Capture with Protein A Countercurrent Tangential
Chromatography: A New Column-Free Approach for Antibody
Purification
Andrew L. Zydney Department Head and Walter L. Robb Family Chair
Department of Chemical Engineering The Pennsylvania State University
Presented at the ECI Conference on Integrated Continuous Biomanufacturing
Castelldefels, Spain, October 21, 2013
• 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
• 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
Example: SMB
New Developments in Simulated Moving Bed Chromatography Seidel-Morgenstern, Kessler, and Kaspereit Chemical Engineering Technology, 31: 826 (2008)
• 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
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
Continuous CTC System
Binding Washing Elution Stripping Regnera-tion
Slurry Tank
Feed Tank
Waste
Waste
Waste Waste
Product Tank
True moving bed Conveyor like process Resin slurry moves counter-currently to buffer in each step
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
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
Countercurrent Staging - Elution
1st stage
2nd stage
Washing
pH 3 Elution Buffer
Purified mAb
Resin Slurry from Wash
Resin Slurry to Strip
Stage 1 Stage 2
1st stage
2nd stage
Washing
pH 3 Elution Buffer
Purified mAb
Resin Slurry from Wash
Resin Slurry to Strip
Stage 1 Stage 2
Countercurrent Staging - Elution
1st stage
2nd stage
Washing
pH 3 Elution Buffer
Purified mAb
Resin Slurry from Wash
Resin Slurry to Strip
Stage 1 Stage 2
Countercurrent Staging - Elution
1st stage
2nd stage
Washing
pH 3 Elution Buffer
Purified mAb
Resin Slurry from Wash
Resin Slurry to Strip
Stage 1 Stage 2
Countercurrent Staging - Elution
1st stage
2nd stage
Washing
pH 3 Elution Buffer
Purified mAb
Resin Slurry from Wash
Resin Slurry to Strip
Stage 1 Stage 2
Countercurrent Staging - Elution
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
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)
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
Critical Filtrate Flux
170
200
230
260
290
0.6
0.8
1.0
1.2
1.4
0 500 1000 1500 2000
Filtrate Flux, Jv (L m
-2 hr -1)
Tran
smem
bran
e Pr
essu
re, T
MP
(psi
)
Time, t (s)
TMP
Flux
Feed: 10% slurry, 100 mL/min
Critical Filtrate Flux
170
200
230
260
290
0.6
0.8
1.0
1.2
1.4
0 500 1000 1500 2000
Filtrate Flux, Jv (L m
-2 hr -1)
Tran
smem
bran
e Pr
essu
re, T
MP
(psi
)
Time, t (s)
TMP
Flux
Critical Flux
Feed: 10% slurry, 100 mL/min • Critical flux corresponds to 80% conversion using 10% slurry
Critical Filtrate Flux
170
200
230
260
290
0.6
0.8
1.0
1.2
1.4
0 500 1000 1500 2000
Filtrate Flux, Jv (L m
-2 hr -1)
Tran
smem
bran
e Pr
essu
re, T
MP
(psi
)
Time, t (s)
TMP
Flux
Critical Flux
Operating 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
CTC Process using Protein A
Operation Numbe
r of stages
Buffer pH Mixed 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
Multiple Runs
Run mAb (g/L)
mAb Load
Run Time
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
Run 1 - Pressure Profiles
Elapsed Time, t (hr)
Pres
sure
, P (p
sig)
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
Run 1- mAb Purification
Elution Pure mAb CCCF
SEC Profiles
Elution Time, t (min)
Abs
orba
nce
• >95% yield • >98% purity • Productivity of
63 g mAb/L resin/hr (10x packed column)
• No detectable protein aggregates
• No detectable changes in resin
Run 1 - mAb Purification
Sample Host 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
Run 2 – Product Profile
0
0.2
0.4
0.6
0.8
1
0 2 4 6 8 10 12
UV
- E
lutio
n
Time (hours)
• 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
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)
Run 3 - mAb Purification
Sample Host 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
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
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
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
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
Funding / Support