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.

0.00

0.02

0.04

0.06

0.08

AU

0.00

0.02

0.04

0.06

0.08

0.00

0.02

0.04

0.06

0.08

10 min

1

23

4

5

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.