Christopher Sucato , Mario DiPaola and Malcolm G. Pluskal .

Quantitation and sample preparation of recombinant proteins for glycosylation analysis, including sialylation levels,
®
®
using a POROS CaptureSelect IgG Fc affinity column.
Christopher Sucato1, Mario DiPaola1 and Malcolm G. Pluskal2. Blue Stream Laboratories, 763 Concord Ave, Cambridge, MA 021381 and Life Technologies, 35 Wiggins Ave, Bedford , MA 017302.
standard preparations were serially diluted in Buffer A (50 mM Na
Phosphate pH 7.0 + 150 mM NaCl ) from a 50 mg/mL stock and analyzed on a
POROS® CaptureSelect® IgG Fc analytical column (2.1mmD x 30 mmL, 0.1 mL) on
an Agilent® 1200 HPLC system. The column was equilibrated in Buffer A: at 1.5
mL/min flow rate. Retained material was eluted with a step gradient of Buffer B: 12
mM HCl pH 1.9 + 150 mM NaCl and the resulting peaks integrated with the Agilent ®
HPLC analysis software. The data in Figure 1 is the mean peak area of four 20 µL
replicate injections monitored at 280 and 215 nm. Linear curve fit data was forced
through zero, since all values had been background corrected. %CV was estimated
and found to be <1% at each data point.
CHO supernatant was obtained from cGMP-banked CHO-S® cells. The cells were
thawed in CD FortiCHO™ Medium (Life Technologies) supplemented with 8 mM Lglutamine (FC + gln). Cells were passaged 7 times prior to seeding: On day 0,
3x105 viable cells/mL were seeded in 120 mL of FC + gln media in duplicate. On
day 4, both flasks were fed 6 g/L glucose: (Sigma-Aldrich). On day 7, conditioned
medium/supernatant was then harvested by centrifugation (100 x g for 5 min) and
the resulting supernatant sterile-filtered (0.2 µm). Media was evaluated +/- addition
of an anti-clumping agent (ACA).
Collection of flow through peaks was carried out at the outlet of the UV detector.
Dilute samples were re-concentrated using centrifugal ultrafiltration (UF) devices
with a 50 k molecular weight cut off membrane (Microcon® YM-50, EMD Millipore,
Billerica, MA). Note: > 95% of added Enbrel® was recovered as a retentate with
these UF membranes.
.13
19
4e
+0
06
3000
2
3
Ar
12000
2000
6000
4000
Wash
1500
1000
Elute
Panel B: Expanded scale of data shown in Panel A
500
35.416
A
rea
:2
62
.84
4
4000
8000
2000
29.680
A
re a
:2
20
47
.3
6000
10000
Load
2500
y = 11292x
R² = 0.9992
Absorbance at 280 nm
y = 724.43x
R² = 0.9983
Peak area @ 215nM (mAu)
Peak area @ 280 nm (mAU)
8000
2000
0
0
0
0.0
2.0
4.0
6.0
Enbrel® Units (mg/mL)
8.0
10.0
0
0.0
0.2
0.4
0.6
Enbrel® Units (mg/mL)
0.8
Figure 2. Quantitation of Enbrel® added
to a CHO cell supernatant
0.138
Panel A, CHO + 1mg/mL
M
450
1
2
3
Elute
2.690
110 kDa
80 kDa
Wash
250
Load
LU
60 kDa
50 kDa
150
40 kDa
100
3.205
1.005
30 kDa
0
0
0.5
1
1.5
2
2.5
3
3.5
min
Panel B, CHO blank
20 kDa
MWD1 A, Sig=280,16 Ref=360,100 (C:\CHEM32\...ATA\MALCOLM\MGP4132012_RUN 1 2012-04-13 10-55-41\001-0101.D)
0.131
mAU
15 kDa
400
30
9.5
retention
time
(min)
17.4
25
Fluorescence intensity (FU)
200
25
6.0
16.6
25.2
7.9
0
0.5
1
1.5
2
2.5
3.208
3
3.5
min
Elution time (min)
Enbrel® was added to a CHO cell derived supernatant
to a final concentration of 1 mg/mL. A 20 µL sample
(Panel A) was then analyzed on a POROS®
CaptureSelect® IgG-Fc column as described in Methods.
A sample of CHO supernatant with no added Enbrel® was
run in parallel and is shown in Panel B.
Samples of CHO cell supernatant and flow through (FT)
fractions were precipitated with 3 volumes of cold (-20oC)
acetone for 30 min. in the cold and the precipitate collected at
14,000 x g for 10 min. After removal of the supernatant and air
drying the following samples; M - MWt. Standards (Novex®
Sharp unstained protein standard), 1- CHO supernatant + 1
mg/mL Enbrel®, 2- FT from CHO - ACA , 3-FT from CHO+ ACA
and 4 - FT from a Buffer A + 1 mg/mL Enbrel® l sample, were
resuspended in SDS-sample buffer + 50 mM DTT and heated to
95oC for 3 min. Samples (10 µL volume) were then analyzed on
a 12% pre-cast NuPAGE® SDS-PAGE gel with a MOPS running
buffer and stained with colloidal Coomassie blue (SimplyBlue™
Safe Stain from Life Technologies) and destained in water.
Table 1. Recovery and Precision of Enbrel® assay in a CHO cell supernatant
Calculated Enbrel®
Concentration
(mg/mL)
Sample
CHO – ACA + 1 mg/mL Enbrel®
CHO + ACA + 1 mg/mL
Buffer A + 1 mg/mL
Enbrel®
Enbrel®
Enbrel®
1.05
1.04
1.04
Table 3. Analytical Ultracentrifuge (AUC) Data on Eluted Fractions and Enbrel®
control
%CV (n)
+/- 1.04% (4)
+/- 1.26% (4)
+/- 3.8% (4)
A sample of
was added to CHO cell supernatant +/- ACA to a final concentration of 1.0 mg/mL. This crude
sample was then injected (20 µL) onto the POROS® CaptureSelect® IgG-Fc analytical column and the eluted peak
area was estimated and the concentration of analyte read off the standard curve for A280 as shown in Figure 1,
panel A above. The above values represent four replicate injections of the same sample.
Relative
abundance
(%)
SEDFIT
(S)
Data in
sec x
10-13
Glycan structures
15.1
9.5
24.7
15
10.3
6.0
10
20
30
5.5
11.2
6.7
16.6, 17.4
32.9
% Total
Approx MWt.
(kDa)
% Total
67
<0.1
4.8/4.9
130
98.9
150
96.6
8.0/7.8
280
0.7
300
2.7
11.5
480
0.1
15.6/14.3
750
0.3
750
0.6
40
Prior to analysis by AUC, the Enbrel® fraction post-column was adjusted with 1 N NaOH to a pH of 7.4 from a
starting value of approximately 4.0. The Enbrel® sample at 50 mg/mL in formulation matrix was then diluted to
0.5 mg/mL by 100-fold dilution with 1 x PBS pH 7.4. Enbrel® samples were loaded into the sample channels
of AUC cells having quartz windows and 12-mm double-sector Epon® centerpieces. Buffer blank (1 x PBS) was
loaded into the corresponding reference channel of each cell. The centrifugation was carried out at 20oC and
45000 rpm. Radial scans of the concentration profile were collected sequentially by absorbance at 280 nm,
until full sedimentation was reached. The resulting data sets were analyzed using the program SEDFIT with
a continuous c(s) distribution model, yielding best-fit distributions for the number of sedimenting species and
the effective molecular weights.
16.3
min
N-acetyl glucosamine
Retention time (min)
Galactose
Mannose
Approximately 100 g of the eluted peak fraction from Figure 4 was treated with
PNGase F overnight, following vendors recommended digestion conditions at 37 C.
Fucose
The released glycans were harvested by ethanol extraction. The precipitated protein
N-acetyl Neuraminic acid
from the ethanol extraction procedure was saved for further analysis by ESI-ToF MS
(see Figure 6), while the extracted glycans were brought to dryness and then derivatized with 2-aminobenzamide
(2-AB) in the presence of sodium cyanoborohydride. After clean-up for removal of excess dye, the derivatized
glycans were chromatographed by HILIC-chromatography using a NH2P-50 2D column (Shodex™) at a flow rate of
0.5 mL/min and a linear gradient with mobile phase A, containing 2% acetic acid and 1%THF in acetonitrile, and
mobile phase B, containing 5% acetic acid, 3% triethylamine and 1%THF in water. Detection was by fluorescence
with excitation at 320nm and emission at 430nm. Peaks of interest were analyzed using an Agilent® ion-trap mass
spectrometer (6300 series). Putative glycan structures are summarized in Table 2.
Table 3. Sialic acid content of the eluted peak
Sialic
Acid (SA)
content
Approx MWt.
(kDa)
Enbrel® Control sample
22.4
10.3
10
Eluted Peak Fraction
2.9
11.2
300
0
Approximately 100 µg of N-linked deglycosylated elution peak protein (see Figure 5) was precipitated with ice-cold
ethanol and recovered by centrifugation and then resuspended with about 20 µL of a solution of 0.1% formic acid/50%
water/49.9% acetonitrile. A 5 µL aliquot of the resuspended protein was injected and analyzed by ESI-ToF mass
spectroscopy (Agilent®, 6200 Series).
1.1
24.7, 25.2
100
min
7.9
20
5
200
35
Figure 5. Analysis of PNGase
Table 2. Released Glycan structures
released N-linked glycans by
HILIC
– Ion Trap MS
Peak
FLD1 A, Ex=320, Em=430 (N_LINK_GLYC_10APR12\N_LINK_GLYC_10APR12 2012-04-10 19-27-41\001-0101.D)
260 kDa
160 kDa
300
20
4
400
350
15
Panel A: POROS® CaptureSelect® IgG Fc analytical column on a Agilent® 1200 HPLC system, operated in
preparative mode (loading via the system pump). A 7mL sample of CHO cell supernatant with Enbrel® added to a
final concentration of 0.1 mg/mL was loaded onto a CaptureSelect® IgG-FC column (2.1mmD x 30mmL) at a flow
rate of 0.5 mL/min. After loading, the column was washed with approximately 5 mL of PBS (pH 7.2) at a flow rate
of 0.5 mL min. A sample was taken (FT) at the end of the loading to assess for break-though of the target analyte
on an SDS-PAGE gel. Note: at this load (700 µg) some Enbrel® l was seen in the FT fraction. At slightly lower
loads (500 µg) no breakthrough was seen. Elution was achieved with PBS adjusted to a pH of 2.9 at a flow rate
of 0.5 mL/min. Fractions were collected across the resulting peak and subjected to SDS-PAGE analysis as
described in Figure 3, without acetone precipitation. Panel B: Gel analysis of samples; 1 - Load sample, 2- FT
fraction (see panel A) and 3 - eluted fraction.
Figure 3. Analysis of Flow-through
fractions by SDS-PAGE
Enbrel®
10
Elution time (min)
MWD1 A, Sig=280,16 Ref=360,100 (MALCOLM\MGP372012_RUN 1 2012-03-15 13-33-54\006-0401.D)
mAU
5
1.0
POROS® CaptureSelect® IgG Fc analytical column on a Agilent® 1200 HPLC system. 20 µL injections of a range
of Enbrel® concentrations from 0.02 to 10 mg/mL (dilution range from 1/5 to 1/2500) in 50 mM Na Phosphate pH
7.0 + 150 mM NaCl were analyzed and peaks eluting monitored at 280 and 215 nm.
2.702
Enbrel®
Panel B, range from 0.02 to 1.0 mg/mL at 215 nm
1
FT sample
Panel A: Max-Ent deconvolution results of the ESI-ToF MS spectrum obtained from N-linked deglycosylated
and reduced Enbrel®
ea
:3
Panel A, range from 0.5 to10.0 mg/mL at 280 nm
Absorbance at 280 nm
MATERIALS AND METHODS
14.204
VWD1 A, Wavelength=280 nm (PO290312\010-0101.D)
mAU
Figure 6. ESI-ToF Mass Spectrometry Analysis of Eluted Peak Fraction
Panel B: SDS-PAGE analysis of
eluted fraction
Panel A: Chromatogram
Figure 1. Enbrel® Standard Curve
50
POROS® CaptureSelect® affinity resins, are packed as high-flow, high-performance
columns, designed to work on HPLC systems. These analytical columns can be
used to quantitate analytes that are recognized with high affinity by the immobilized
ligand. The POROS® CaptureSelect® IgG Fc ligand was used to quantitate the level
of a TNF-Fc fusion protein Enbrel® , spiked into a sample of CHO cell culture derived
supernatant. In addition to quantitation with these columns, small scale preparative
purification was demonstrated as an interface to other orthogonal analysis
methodologies, such as N-glycan/sialylation content and analytical
ultracentrifugation (AUC) to look at molecular aggregation states.
Figure 4. Preparative isolation of dilute analyte
0.047
INTRODUCTION
RESULTS
Absorbance at 280 nm
ABSTRACT
Purpose: A rapid, affinity- based HPLC quantitation method for monitoring the
concentration of a Fc fusion protein in a cell culture sample was demonstrated using
a POROS® CaptureSelect® IgG-Fc HPLC affinity column. In addition, the affinity
HPLC method was shown to function as a sample preparation method.
Glycosylation analysis was executed on the eluted affinity purified samples using
LC/MS/MS.
Methods: Varying quantities of Enbrel® (Etanercept), a TNF-Fc Fusion protein,
were injected onto a POROS® CaptureSelect® IgG-Fc HPLC column to generate a
standard curve for protein quantitation. The specific and complete capture of
Enbrel® spiked into CHO-S® parental conditioned Gibco® medium was shown using
SDS-PAGE and recovery analysis. The eluted protein was collected and determined
to be suitable for further protein characterization. The affinity purified protein was
treated with PGNase F to release the N-linked glycan structures, which were then
characterized by LC/MS/MS. In addition purified protein was subjected to mild
hydrolysis to release the sialyl moieties which were then derivatized and quantified
by HPLC with fluorescence detection.
Results: POROS® CaptureSelect® IgG-Fc analytical affinity column(s) allowed for
the quantitative recovery of a target protein added to conditioned cell culture media
in under 3 minutes. The standard curve exhibited excellent linearity (R2 0.9992) and
a dynamic range suitable for monitoring a wide range of expressed protein
concentrations. The affinity HPLC column also functioned as a sample preparation
step and yielded sufficiently pure protein from a complex sample for further
assessment of the protein glycosylation patterns and other protein attributes.
Conclusions: A POROS® CaptureSelect® IgG-Fc analytical affinity HPLC column
was used to develop a rapid method for monitoring the concentration of a Fc fusion
protein in cell culture samples and as a sample preparation method for the
purification of protein suitable for further analytical characterization such as glycan
analysis. Specific and complete capture of protein from complex samples was
demonstrated with SDS-PAGE and traditional recovery analysis. The method also
yielded enough pure protein for further assessment of the protein glycosylation
patterns using LC/MS/MS and sialyl content by HPLC with fluorescence detection.
Eluted peak (mole SA/
mole protein*)
Fetuin control (mole SA/
mole protein*)
Glycolyl
1.33
1.04
Acetyl
13.67
9.40
Total
15.01
10.44
Approximately 43µg of the eluted peak and 140µg of bovine fetuin control were subjected to mild acid hydrolysis in a
solution of acetic acid (2.5M) for 45 minutes at 800C. Note: fetuin is a well characterized glycoprotein and was added
as an internal control for the sample processing. The released sialic acids were derivatized with o-Phenyldiamine and
then chromatographed using a C18 column (Phenomenex15cm x 0.46 cm). Elution was performed using a linear gradient
at a flow rate 0.6 mL/min with mobile phase A: 0.5% phosphoric acid/0.3% butylamine/1% THF in water and B, 50%
acetonitrile in mobile phase A.
CONCLUSIONS
POROS® CaptureSelect® IgG Fc analytical columns can be used to rapidly quantitate the levels of target Fc
containing analytes in a 3 min assay.
• In the case of our model analyte, Enbrel®, we were able to construct a highly linear standard curve(s) in the
range 0.5-10.0 mg/mL at 280 nm and 0.02–1.0 mg/mL at 215 nm.
• In small scale preparative mode, the same column could be used to prepare sufficient sample (500-600µg) to
carry out a range of product characterization assays such as: N-Glycan profiling, intact protein mass by ESI-ToF
MS, sialic acid content and analytical ultracentrifugation (AUC) to look at molecular aggregation.
•
ACKNOWLEDGEMENTS
We would like to acknowledge the help of Michelle Sabourin and Graziella Piras of the GIBCO® cell culture group
at Life Technologies, Frederick, MD for supplying the CHO cell supernatant. Expert technical guidance and
editorial support was provided by Christine Gebski and Rick Garretson of the POROS® Application and R&D
groups in Bedford, MA.
For Research Use Only. Not intended for any human or animal therapeutic or diagnostic use, unless otherwise
stated. © 2012 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the
property of Life Technologies Corporation or their respective owners. All other trademarks are the sole property of
their respective owners. Enbrel is a registered trademark of Immunex Corporation. Epon is a registered trademark
of Momentive Specialty Chemicals, Inc. Microcon is a registered trademark EMD Millipore. Agilent is a registered
trademark of Agilent Technologies, Inc. Shodex is a trademark of Showa Denko K.K. Corporation, Japan.
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