Pilot Scale Experiments to Improve Chromatography Column Packing Procedures
Tony Roberts, Carl Richey, Patrick Asplund, Venkata Bangla of Cook Pharmica LLC, 1300 S. Patterson Dr. Bloomington, IN, 47402
Drew Williams, Rose Hulman Institute of Technology, Terre Haute, IN 47803
RESULTS
0.060
1.80
ABSTRACT
1.60
0.050
1.40
1.20
1.00
0.030
0.80
Table 1: Summary of Integrity Test Results from Varied Packing Methods with 3 Resins
0.020
0.60
HETP
Asymmetry
Packing Method
MATERIALS and METHODS
Resin
HETP
No. Plates/Meter Asymmetry
Asymmetry
0.040
HETP
Large scale liquid chromatography is the dominant purification technique used in the production of recombinant proteins.
The quality of the column packing is a key parameter and its integrity impacts the purification performance. A ten cm
diameter Index column (GE Healthcare) was packed with three different types of resin and three packing methods were
executed for each resin. After each pack, the column was integrity tested by the Height Equivalent of Theoretical Plate
method (HETP). The integrity test results showed that in most cases, packing by axial compression resulted in an
increased number of theoretical plates. HETP integrity testing was also performed at varied linear flow rates for each
column pack. A decreased flow rate was correlated with increased number of theoretical plates. Consistent methodology
using the same residence time on the column is recommended for comparison of column integrity test results with similar
column scales (diameter and bed heights) between projects.
The column integrity test results for the resins, each packed by three different methods are summarized
in Table 1. The axial compression method provided the highest number of plates per meter and
asymmetry values closer to 1.0, for each respective method. It was noted that the worst asymmetry for
the Poros resin was obtained with the column pack after settling the resin by gravity. This observation
was in agreement with the manufacturer’s information to avoid gravity settling during packing.
0.40
0.010
0.20
Flow Pack
Phenyl Sepharose FF
0.0463
2162
1.11
0.000
Gravity Settled
Phenyl Sepharose FF
0.0276
3626
1.23
0.00
0
50
100
150
200
250
300
Velocity (cm/h)
Axial Compression
Phenyl Sepharose FF
0.0255
3918
1.10
Column: Index 10 cm diameter (GE Healthcare)
Flow Pack
Poros 50 HQ
0.0127
7888
1.89
Chromatography Resins: Phenyl Sepharose Fast Flow and MabSelect SuRe (GE Healthcare),
Poros 50 HQ (Life Technologies)
Gravity Settled
Poros 50 HQ
0.0123
8115
3.00
Axial Compression
Poros 50 HQ
0.0074
13444
0.95
Flow Pack
MabSelect SuRe
0.0289
3456
1.51
Chromatography System: ÄKTA Pilot (GE Healthcare)
Gravity Settled
MabSelect SuRe
0.0597
1674
1.47
Solutions: 0.1 M NaCl (equilibration for integrity testing)
Axial Compression
MabSelect SuRe
0.0272
3675
1.35
Packed Resin Bed Height: 20 ± 2 cm
1 M NaCl (2% of column vol. injection pulse for integrity testing)
Integrity test results from each column after packing by axial compression are shown in Figures 2 - 4. A
general trend of increased HETP with increasing flow rate was observed with each resin. The magnitude
of change was significantly less with the Poros HQ resin.
Flow Packing Method
Each resin slurry (~60-70 % by volume in deionized water) was poured into the column.
Water was flowed through the top column adaptor to remove air.
The top adaptor was immediately placed into the top of the
column, ensuring no air was trapped beneath the adaptor.
The top adaptor was locked in place and water flow started at 400 mL/minute.
Flow was stopped after three column volumes.
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Figure 4: MabSelect SuRe Integrity Test Results at Varied Linear Velocity
Table 2 shows the yield and purity summary from the three MabSelect SuRe affinity
chromatographic runs. The integrity test results shown were performed just prior to the process
step, at a linear flow rate of 100 cm/hr. It was noted that the column packed by axial compression
yielded a higher HETP than obtained in the integrity test from an earlier date. The yields and
purity were similar from the three runs. The column with the lowest HETP was correlated with a
higher product concentration and smaller elution volume. All results were considered satisfactory.
Table 2: Analyses of Neutralized MabSelect SuRe Eluate from Three Chromatographic Runs
1.80
1.60
Column Packing Method
1.40
Flow
0.050
HETP of
Column
Pack prior
to run
Concentration
(mg/mL)
SEC-HPLC
Monomer
(%)
CHO HCP
Conc.
(ng/mg)
Step Yield
(%)
Elution
volume
(column
volume)
0.0289
16.60
99.6
668
94.7
1.14
0.0468
12.41
99.7
558
92.9
1.50
0.0597
12.14
99.6
668
93.3
1.54
Figure 1: Index 10 cm Diameter Column
0.040
Axial Compression
1.20
1.00
0.030
0.80
0.020
Axial Compression
Removal of the adaptor and slurry of the resin was performed as for the above method. After re-slurry of the resin with a
paddle, the adaptor was lowered into the top of the column, using water and a peristaltic pump. The resin bed height was
compressed to 22 cm.
0.60
Gravity Settled
Asymmetry
HETP
Gravity Settled Method
The column adaptor was pushed to the top by reverse flow with 0.1 M NaCl, then removed. The resin was re-slurried in
the column with a paddle and allowed to gravity settle overnight. The top adaptor was then placed into the top of the
column, ensuring no air was trapped beneath the adaptor and locked in place. The adaptor was lowered into the top of
the column by axial compression, using water and a peristaltic pump. The resin bed height compressed to the same
height as obtained with the previous flow pack (22 cm).
0.40
0.010
DISCUSSION
HETP
0.20
Asymmetry
After each method of column packing, the integrity test was performed at a linear flow rate of 100 cm/hour.
0.000
Column Integrity Test
1.0 M NaCl (2% CV) was introduced into the column close to the column inlet
The solution was flowed through the column with 0.1 M NaCl
The broadening of the conductivity pulse was analyzed using the evaluation mode of the Unicorn software
The repeatability of HETP and As data over the life of the packed column gives assurance that the packed bed
remains stable and the column continues to be suitable for use
0.00
0
10
20
30
40
50
60
70
80
90
100
Velocity (cm/h)
Figure 2: Phenyl Sepharose FF Integrity Test Results at Varied Linear Velocity
0.060
HETP = L/N (cm/plate)
N/(m) = N/L(cm) * (100 cm/m)
h = HETP/dp = normalized
VR
1.60
0.050
1.40
Sample Injection
h10% a b
h1/2
Wh
0.040
1.20
Volume (L) or Time (min)
HETP
1.00
Asymmetry (As) = b/a
The integrity test results from varied linear velocity indicated a general positive correlation with flow rate and HETP.
A similar trend was obtained with bench scale chromatography columns (data not shown). Prior to column packing,
the percentage of the resin slurry is measured and the compression factor is also included in the calculation for the
volume of slurry required to packed the column to the target height. These practices are used at Cook Pharmica,
prior to column packing by axial compression or flow.
0.030
0.80
For each of the columns packed by axial compression, integrity testing was repeated at varied linear flow rates. HETP
and asymmetry values were determined for each respective elution. The results were plotted in Excel.
MabSelect SuRe Affinity Capture of Monoclonal IgG
After column packing by the three methods, clarified harvest from Chinese Hamster Ovary cell culture was
chromatographed on the column equilibrated in 20 mM sodium citrate pH 6.0. After loading and a 4 CV wash with
equilibration buffer, the IgG was eluted with 20 mM sodium citrate pH 3.6. The linear flow rate was 300 cm/hr. for all
steps. The elution pool was neutralized, then sampled and analyzed for yield and purity. After each use, the column was
cleaned with 0.1 M NaOH, 1 M NaCl. The column was stored in 2% benzyl alcohol.
HETP
0.020
Asymmetry
0.60
0.40
0.010
0.20
0.000
0.00
0
50
100
150
200
250
300
Velocity (cm/h)
Figure 3: Poros 50 HQ Integrity Test Results at Varied Linear Velocity
Asymmetry
N = (µf/)2 = 5.54(VR/Wh) 2
= ratio between retention time and peak variance
= signal to noise ratio
1.80
The packing study using 3 different resins showed axial packing compression provided better integrity test results
relative to the flow and gravity packing methods with an Index 10 cm diameter column. The design of other columns
may not allow the ability of packing by axial compression. Flow packing is also a commonly used method. The
resin manufacturer’s recommendation as well as the particular column design should be considered for packing of
the column. In this study, the gravity settling method resulted in a relatively poor asymmetry integrity test result for
the Poros 50 HQ. This observation was consistent with the literature from Life Technologies, which does not
recommend gravity settling of the Poros resin.
CONCLUSIONS
The axial compression packing method is preferred for Index chromatography columns. Typically a satisfactory
pack/integrity test result can be obtained using axial compression in a single attempt. When comparing integrity
test results between projects with columns of similar diameter and bed height, consistent methods using the same
residence time on the column is recommended.