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
Continuous Bioprocessing
• 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
Chromatography Options
• 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
Example: SMB
New Developments in Simulated Moving Bed Chromatography
Seidel-Morgenstern, Kessler, and Kaspereit
Chemical Engineering Technology, 31: 826 (2008)
Objectives
• 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)
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
Slurry
Tank
Binding
Washing
Elution
Stripping
Regnera
-tion
Waste
Waste
Waste
Feed
Tank
Waste
Product
Tank
 True moving bed  Conveyor like process
 Resin slurry moves counter-currently to buffer in each step
Chromatographic “Stage”
Koflo static mixer
 Provides residence time
needed for equilibration in
binding and elution steps
 Excellent radial mixing with
+
Spectrum hollow fiber module
minimal pressure drop
 Provides complete separation
between resin particles and
fluid phase
 High single pass conversion
with low pressure losses
Continuous CTC System
Centrate
Feed
Equilibra
-tion
Permeate
UF Step
Stripping
Permeate
Equilibra
-tion
Buffer
Equilibration
Wash 2
Permeate
Stripping
Buffer
Stripping
Elution
Wash 1
Permeate
Elution
Buffer
Wash 2
Buffer
Wash 2
Binding
Permeate
Wash 1
Binding
Resin
Tank
Wash 1
Buffer
Product
Tank
UF
Permeate
Countercurrent Staging - Elution
Stage 1
Resin Slurry
from Wash
Stage 2
Resin Slurry
to Strip
Washing
2nd
stage
1st
stage
pH 3
Elution
Buffer
Purified
mAb
Countercurrent Staging - Elution
Stage 1
Resin Slurry
from Wash
Stage 2
Resin Slurry
to Strip
Washing
2nd
stage
1st
stage
pH 3
Elution
Buffer
Purified
mAb
Countercurrent Staging - Elution
Stage 1
Resin Slurry
from Wash
Stage 2
Resin Slurry
to Strip
Washing
2nd
stage
1st
stage
pH 3
Elution
Buffer
Purified
mAb
Countercurrent Staging - Elution
Stage 1
Resin Slurry
from Wash
Stage 2
Resin Slurry
to Strip
Washing
2nd
stage
1st
stage
pH 3
Elution
Buffer
Purified
mAb
Countercurrent Staging - Elution
Stage 1
Resin Slurry
from Wash
Stage 2
Resin Slurry
to Strip
Washing
2nd
stage
1st
stage
pH 3
Elution
Buffer
Purified
mAb
Example: 3-Stage Elution Step
Concentrated
slurry with
bound product
Elution Buffer
Static
mixer
Tangential
flow filter
P
Product
P
R
Tangential
flow filter
Static
mixer
R
P
Static
mixer
Tangential
flow filter
R
Concentrated
resin slurry
Effect of Staging – 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
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
Feed: 10% slurry, 100 mL/min
1.4
290
TMP
Flux
1.2
260
1.0
230
0.8
200
0.6
170
0
500
1000
Time, t (s)
1500
2000
Filtrate Flux, Jv (L m-2 hr-1)
Transmembrane Pressure, TMP (psi)
Critical Filtrate Flux
Feed: 10% slurry, 100 mL/min
1.4
290
TMP
Flux
1.2
260
Critical Flux
1.0
230
0.8
200
0.6
170
0
500
1000
Time, t (s)
1500
2000
Filtrate Flux, Jv (L m-2 hr-1)
Transmembrane Pressure, TMP (psi)
Critical Filtrate Flux
• Critical flux
corresponds to
80% conversion
using 10% slurry
Feed: 10% slurry, 100 mL/min
1.4
290
TMP
Flux
1.2
260
Critical Flux
1.0
230
Operating Flux
0.8
200
0.6
170
0
500
1000
Time, t (s)
1500
2000
Filtrate Flux, Jv (L m-2 hr-1)
Transmembrane Pressure, TMP (psi)
Critical Filtrate Flux
• 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
Feed Flow
mAb
Rate
Load per
(L/hr)
Resin
Run
mAb
(g/L)
mAb
Load
Run
Time
1 – feasibility
1.2
16 g
3 hr
4.5
190 g/L
2 – long time
0.72
8g
24 hr
0.45
470 g/L
3 – high titer
4.5
8g
4 hr
0.45
230 g/L
Pressure, P (psig)
Run 1 - Pressure Profiles
12
• Stable operation
10
• Pressure <10 psi
• Laminar flow
8
• All plastic
tubing and
connectors
6
4
2
0
0
0.5
1.0
1.5
2.0
Elapsed Time, t (hr)
2.5
Run 1- mAb Purification
Absorbance
SEC Profiles
Elution
Pure
mAb
CCCF
• >95% yield
• >98% purity
• Productivity of
63 g mAb/L resin/hr
(10x packed
column)
• No detectable
protein aggregates
• No detectable
changes in resin
Elution Time, t (min)
Run 1 - mAb Purification
Sample
Clarified Harvest
•
•
•
•
Host Cell Protein
(ppm)
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
1
• Steady-state with
respect to product
concentration and
impurity profile
UV - Elution
0.8
0.6
• Long time
operation possible
0.4
• For t > 12 hr
hollow fiber
modules had to be
replaced due to
bacterial growth
0.2
0
0
2
4
6
8
Time (hours)
10
12
Run 2 – HCP levels
HCP Concentration (ppm)
10000
• HCP level remains
constant throughout
24 hr run
8000
• >95% purity
6000
• Productivity of
19 g mAb/L resin/hr
(reduced due to low
titer feed)
4000
2000
0
0
4
8
12
16
Time, t (hr)
20
24
Run 3 - mAb Purification
• Very low HCP level
due to use of high
titer feed (4.5 g/L)
spiked with purified
mAb
Sample
Host Cell
Protein (ppm)
2 hr
310
3 hr
345
• >99% purity
382
• Productivity of 52
g mAb/ L resin / hr
4 hr
• HCP measured via ELISA
relative to mAb
• 2.6 cycles / hr for
resin
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