Post on 10-Jul-2020
Detection: UV 280 nm (sampling rate 10 Hz, 0.2 s filter
time constant)
Sample Diluent: 25 mM sodium phosphate, pH 6.8,
0.15 M NaCl
Other conditions are specified in the figure captions.
1 2 3 4 5 6 min5 10 15 20 25 30 35 40 min
HPLC SEC7.8 x 300 mm, 5 µm
ACQUITY UPLC BEH200 SEC4.6 x 150 mm, 1.7 µm
Aggregates10.3%
Aggregates10.7%
Figure 1: Comparison of traditional HPLC and ACQUITY UPLC SEC for the separation of a human-
ized monoclonal antibody (IgG). Injection volumes: 20 µL for HPLC and 5 µL for ACQUITY UPLC.
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Batch “A”
Batch “B”
Batch “C”
90 1 2 3 4 5 6 7 8
Figure 2: Batch-to-batch reproducibility for a mixture of protein standards. Buffer: 100 mM
sodium phosphate, pH 6.8. Flow rate is 0.3 mL/min. Peaks: (1) thyroglobulin, (2) IgG,
(3) bovine serum albumin, (4) myoglobin, (5) uracil.
A nA lysis o f B iomo l ec u l e s By s iz e- e xc lusio n u lt r A P e r fo rmA n c e l iqu i d c h romAt og r A P h y
Kenneth J. Fountain, Paula Hong, Susan Serpa, Edouard S. P. Bouvier, Damian Morrison
Waters Corporation, Milford, MA, USA
INT RODUCT ION
In the production of biopharmaceuticals, there may be different
analytical requirements for groups performing clone section,
formulations and stability, and quality control (QC). Depending
on the goal of the separation, methods may be optimized for fast
analysis time, highest possible resolution, and/or reproducibility.
Size-exclusion (SEC) chromatography is often used throughout the
biopharmaceutical production process for the analysis of proteins
and their aggregates. While SEC has traditionally been used in
conjunction with low pressure HPLC instrumentation, the advent
of UPLC® Technology and new sub-2 µm packing materials allows
for substantial improvements in chromatographic resolution and
throughput. This application note will demonstrate the use of the
new ACQUITY UPLC® SEC solution for the improved detection and/
or faster analysis of protein aggregates in biopharmaceuticals.
EX PERIMENTAL
UPLC System: ACQUITY UPLC System with TUV
(with stainless steel flow cell)
HPLC System: Waters 2796 Separations Module with
2487 dual λ detector
UPLC Column: ACQUITY UPLC BEH200 SEC,
4.6 x 150 mm, 1.7 µm
HPLC Column: Traditional silica diol-coated SEC,
7.8 x 300 mm, 5 µm
Mobile Phase A: 25 mM sodium phosphate, pH 6.8, 0.15 M NaCl
Flow rate: 0.4 mL/min
Temperature: 30 ˚C
Analysis of Biomolecules by Size-Exclusion UltraPerformance Liquid Chromatography 1
Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com
©2010 Waters Corporation. Waters, The Science of What’s Possible, ACQUITY UPLC and UPLC are trademarks of Waters Corporation.
April 2010 WA64266 KK-PDF
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France 33 1 30 48 72 00, Germany 49 6196 400600, Hong Kong 852 2964 1800, Hungary 36 1 350 5086, India and India Subcontinent 91 80 2837 1900, Ireland 353 1 448 1500, Italy 39 02 265 0983, Japan 81 3 3471 7191, Korea 82 2 6300 4800, Mexico 52 55 5524 7636, The Netherlands 31 76 508 7200, Norway 47 6 384 60 50, Poland 48 22 6393000,
Puerto Rico 1 787 747 8445, Singapore 65 6593 7100, Spain 34 936 009 300, Sweden 46 8 555 11 500, Switzerland 41 56 676 70 00, Taiwan 886 2 2543 1898, United Kingdom 44 208 238 6100,All other countries: Waters Corporation U.S.A. 1 508 478 2000/1 800 252 4752
Analysis of Biomolecules by Size-Exclusion UltraPerformance Liquid Chromatography 2
Injection #16
Injection #615
2 3 4 5 6 7 8 9 10 min0 1
Figure 3: Lifetime study for a humanized IgG sample on the ACQUITY UPLC BEH200
SEC column.
RESULTS AND DISCUSSION
Figure 1 shows the chromatographic comparison of traditional
HPLC and ACQUITY UPLC for the separation of a humanized mono-
clonal antibody. Equivalent aggregate quantification in significantly
shorter run times is possible with the ACQUITY UPLC SEC solution
as compared to traditional SEC. This is especially important for
those scientists performing clone selection who may need increased
throughput for large numbers of samples.
In regulated environments, the use of SEC is often required for the
characterization of biopharmaceutical therapeutics. Given these
demands, columns are expected to be reproducible from batch-to-
batch and have long lifetimes. Figure 2 shows the batch-to-batch
performance for three different batches of 1.7 µm, BEH200 SEC
packing material. These data show the consistent performance of
the BEH200 SEC columns regardless of the batch of material being
used, which provides confidence for those performing aggregate
determination in biopharmaceutical drugs. Figure 3 demonstrates
the lifetime of the ACQUITY UPLC BEH200 SEC columns with the
same humanized IgG sample shown in Figure 1. No deterioration
in peak shape or retention was observed, which ensures accurate
identification and quantitation of the antibody monomer and dimer
over several hundred injections.
CONCLUSIONS
The Waters ACQUITY UPLC SEC solution provides the speed,
resolution, and sensitivity of UPLC for proteins (antibodies) in a
molecular weight range of 10,000 to 450,000 Daltons. It can be
adapted to varying requirements of run time and resolution that are
often needed in clone selection and/or quality control testing. This
work extends UPLC technology to a wider range of bioseparations.