Performing Production Scale
Chromatographic Separations in
Pre-Packed Disposable Columns
Dana C. Pentia, William J. Wilde, James R. Peyser
Repligen Corporation, Waltham, MA, USA
Top Cap
(3)
Inlet
Outlet
(1)
(1)
Flow Distributor
w/ 12 µm Mesh
(1)
Column body
(1)
Side-guard
(3)
Return Line
(2)
Legend:
Product Contact Material:
1 = Polypropylene
2 = Platinum Cured Silicone
Non-product Contact Materials:
3 = Acrylonitrile Butadiene Styrene (ABS) copolymer
Pressure-gradient
optimized flow field
Anti-jet funnel
Flow Distributor
w/ 12 µm Mesh
(1)
Bottom Cap
(3)
Summary
• Repligen’s OPUS® (Open Platform User Specified) Pre-Packed Disposable Columns with internal diameters
from 1.2 to 30 cm, and column heights from 5 cm and up, offer a scalable solution for the purification of biological
products.
• Design of the flow distributor permits uniform flow distribution for all column sizes. Consistency of purification is
maintained for various scales of the columns, making OPUS columns ideal for upscaling and downscaling
purification processes. Column characteristics are not compromised during transportation.
• The ability to use a single pre-packed column for a multi-cycle campaign is addressed by extensive re-usability
tests that include purification of a biological molecule followed by column sanitization. Cleaning of small
molecules, bioburden and endotoxins is investigated quantitatively.
Packing Performance
Multi-cycle Performance of a 20 cm ID OPUS Column
Method:
• Column packing quality testing for different diameters OPUS columns pre-packed with Sepharose® 6 FF resin
• Separation resolution of molecular weight markers on different sizes of OPUS columns
Method:
Results:
• Extensive use test: re-circulate the same OPUS column with high salt buffer for 2 weeks
2959
1.2
20 cm
3295
1.2
2.5 cm
2.6
6.1
1.1
8 cm
2.3
6.1
1.2
20 cm
2.8
7.1
1.2
BSA (66x103 Da)
Acetone
(58 Da)
Conclusions:
• Consistency of resolution factors and column properties show optimum flow distribution
• Chromatographic performance is demonstrated and maintained over a range of OPUS column diameters
SDS-PAGE of CaptivA PriMab purification
1.2
1
0.8
0.6
0.4
Flow Through
Load
Ladder
1.4
Asymmetry
Plates/m
5000
4500
4000
3500
3000
2500
2000
1500
1000
500
0
1.2 2.5 8 20 1.2 2.5
8 20
IDs
IgG
BSA
0
2.5 cm
8 cm
40
20
1
Conditions
Plate Count
plates/m
Asymmetry
Pre-run
2815
1.3
Post-run
3512
1.2
2
3
4
5
Yield
6
7
8
9
Cycle number
10
Purity (HPLC)
11 12
Pressure drop
Overlay of 2 additional cycles of purification
2
Column quality during extensive use test
Column Volumes
of buffer
Plate count
(Plates/m)
1.8
1.6
Asymmetry
0
2820
1.1
95
2890
1.0
355
2885
1.2
470
3535
1.1
985
3113
1.3
1225
2840
1.3
1.4
1.2
1
0.8
0.6
0.4
0.2
0
20
30
40
50
60
70
80
Volume (L)
Conclusions:
• Column packing quality attributes and consistency of purification are maintained throughout the study
0.2
1.2 cm
60
Column quality testing pre and post-run
Method:
• Quality testing of different diameters OPUS columns packed with an affinity media
• Affinity separation of hIgG from BSA on OPUS columns with diameters of 1.2, 2.5, 8, 20 cm, packed at 20 cm
bed height with CaptivA™ PriMab™ (Sepharose® 4FF backbone)
Results:
Column properties for different sizes CaptivA PriMab OPUS columns
80
0
Separation Performance
Eluates
100
extensive-use test
8 cm
0.9
Sanitization
1.2
4.6
Elution
3465
2.2
Wash
2.5 cm
1.2 cm
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
120
Load
1.1
Blue Dextran (2x106 Da)
mAU
3010
Resolution
Resolution Blue
Resolution
Dextran/BSA Dextran/Acetone BSA/Acetone
Percentage (Yield, Purity)
1.2 cm
Column
ID
Elution
Asymmetry
@ 100 cm/h
Quantitative results for 10 cycles of purification followed by
additional 2 cycles after the extensive re-use test
Overlay of 10 chromatograms
Wash
Plates/m
@ 100cm/h
Results:
Load
Column
ID
Chromatogram for separation of
molecular weight markers
Sanitization
Quality of OPUS columns pre-packed
Separation resolutions for various sizes of OPUS columns
with Sepharose® 6FF
• Purification of a recombinant protein from filtered cell lysate on an OPUS 20 x 20 cm column packed with SP
Sepharose®; for 10 cycles, plus 2 additional cycles after extensive re-use test
bar
• Disposable and single-use technologies have become standard in many of the world’s leading biopharmaceutical
companies. Faster product changeover, favorable economics, and improved safety have driven this paradigm
shift. Until now, however, there has not been a broadly applicable solution for disposable column
chromatography.
• Performance characteristics are maintained for simulated equivalent flow of >100 process cycle
20 cm
Column size
Plates/m pre-run
Plates/m post-run
Asymmetry pre-run
Asymmetry post-run
Cleaning of Small Molecules, Bioburden, and Endotoxin
Transportation Qualification
Method:
• A 20 x 20 cm OPUS column packed with SP Sepharose® was shipped 6000 miles by truck and air.
Results:
• Column and packaging found undamaged and intact
20 x 20 cm OPUS column quality testing pre and post transportation
Conditions
Pre-ship @ 100 cm/h
Plate Count
plates/m
2815
Asymmetry
1.3
Post-ship, down-flow @ 100 cm/h
2820
1.1
Post-ship, up-flow @ 100 cm/h
2850
1.2
Conclusions:
• Packaging withstood the rigors of a commercial shipping environment
• Chromatographic performance maintained after shipping
Note: An International Safe Transit Association’s (ISTA) test was also performed; OPUS column packaging passed the test, and the column
quality was not compromised (data not shown)
Method:
• Colorimetric measurement of phosphate reduction on a 20 x 20 cm OPUS column packed with Sepharose®
6FF washed with water
• Sanitization with 1M sodium hydroxide of a 20 x 20 cm OPUS column packed with Sepharose® 6FF, loaded
with E. coli, followed by bioburden and endotoxin quantitative measurement
Phosphate detection in the column effluent
Results:
1.0E+01
Bioburden and endotoxin test results before and after
sanitization of contaminated OPUS column
Sample
Pre-inoculation
water rinse
Flow-through after
E. coli incubation
Post-sanitization
water rinse
CFU/mL @ 2 CFU/mL @ 4 Endotoxin
days
days
(EU/mL)
0
0
< 0.25
9x106
9x106
> 0.25
0
0
< 0.25
PO4 load
Re-circulation
Wash
1.0E+00
PO4 concentration (M)
Conclusions:
• Demonstrated scalability of OPUS columns, quality attributes are maintained for different column diameters
• Chromatographic separation remains unchanged across the OPUS platform
1.0E-01
1.0E-02
1.0E-03
1.0E-04
1.0E-05
1.0E-06
Below LOQ
1.0E-07
-7
-6
-5
-4
-3
-2
-1
0
1
2
3
4
5
Column Volume
Conclusions:
• Phosphate is reduced to undetectable levels after two column volumes of water rinse; more than 6 log
reduction
• Endotoxin and bioburden can be effectively removed from OPUS columns with the appropriate sanitization
protocol
Conclusions
• Pre-packed disposable columns of the OPUS platform are ideal for the purification of biological molecules due to the well-engineered design which
delivers consistent chromatographic performance, robust packed bed stability for commercial shipping, and industry standard product contact materials
• Consistency in chromatographic performance at different column sizes makes OPUS column platform ideal for development and scale-up of purification
processes
• Reliable cleaning and sanitization, along with demonstrated reusability make pre-packed disposable columns suitable for production scale
manufacturing purifications in single-use or multi-cycle processes
Acknowledgements: Fletcher Malcom – for editorial assistance
Adam Nelson – for packing the OPUS columns
Sepharose is a registered trademark of GEHC
OPUS and CaptivA PriMab are registered trademarks of Repligen Corporation
Company information:
Repligen Bioprocessing
41 Seyon Street
Waltham, MA 02453
www.repligen.com/bioprocessing
+1-781-215-0111