A New High-Resolution Size Exclusion Chromatography Column for MAb Aggregate Analysis

October 2, 2010

The Application Notebook

The Application Notebook, The Application Notebook-10-02-2010, Volume 0, Issue 0

Dionex Application Note

Srinivasa Rao, Xiaodong Liu and Chris Pohl, Dionex Corporation, Sunnyvale, California, USA.

The new MAbPac size exclusion (SEC) column is based on high-purity, spherical, porous (300 Å), 5 µm silica covalently modified with a proprietary diol hydrophilic layer. The column is packed into nonmetallic, biocompatible, PEEK column housing to eliminate compromised chromatography as a result of the presence of metal from the column hardware. This offering results in minimal nondesired interactions between the biomolecules and the stationary phase. Stable surface bonding leads to low column bleed and enhances compatibility with MS, ELSD and Corona CAD detection. The column is available as 4 × 300 mm analytical and 4 × 50 guard column formats.


A Dionex ICS 3000 HPLC system with a DP pump, VWD absorbance detector, autosampler and a TC thermostatted column compartment was used.

Chromatography was controlled by Chromeleon Chromatography Data System software (Dionex).

Monoclonal antibody was a gift from a local biotech company. Other analytical grade chemicals used in the buffers were obtained from Sigma. Super SW3000 SEC column was obtained from Tosoh Bioscience.

Results and Discussion

MAbPac SEC-1 is a size exclusion chromatography (SEC) column specifically developed for characterization and analysis of monoclonal antibody (MAb) aggregates, papain, or other enzyme digested fragments and for other size based separation applications.1 The diol stationary phase is designed to minimize undesired interactions to handle both high and low salt mobile phase eluents and mass spectrometry compatible volatile eluents.

Figure 1: MAb aggregate analysis using MAbPac SEC-1 column. Mobile phase: 300 mM NaCl in 50 mM phosphate buffer (pH 6.8); flow rate = 0.2 mL/min; sample = MAb 10 mg/mL; injection volume = 2 µL; detection; UV at 280 nm; temperature = 30 °C.

Size-exclusion chromatography (SEC) is a well-accepted technique for the detection and accurate quantification of protein aggregates in MAbs and other biological drug products. An example separation of MAb aggregation using MAbPac SEC-1 is shown in Figure 1. A comparison of MAb analysis on MAbPac SEC-1 and Super SW3000 using mass spectrometry compatible mobile phase (100 mM ammonium acetate) is shown in Figure 2. The MAbPac SEC-1 column provided superior efficiency, peak shape and asymmetry, sensitivity and better recovery when compared with Tosoh's Super SW3000 SEC column.

Figure 2: Comparison of MAbPac SEC-1 with Tosoh's Super SW 3000 using volatile eluents. Mobile phase: 100 mM NH4OAC (pH 5.0); sample = MAb 1 mg/ mL; flow rate = 0.25 mL/min (Dionex); 0.33 mL/min (Tosoh); injection volume = 2 µL (Dionex); 2.5 µL (Tosoh); detection: UV at 280 nm; temperature = 30 °C.


1. MAbPac SEC-1 manual and data sheets are available at www.dionex.com

PEEK is a trademark of Victrex PLC. CAD, Chromeleon and Corona are registered trademarks, and MAbPac is a trademark of Dionex Corporation.

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