Technical Information

High resolution analysis of monoclonal antibodies utilizing cation exchange column BioPro IEX SF

The Universities of Geneva and Lausanne performed a comparative examination of dissimilar ion exchange columns of different manufacturers within an independent study.
The capacity of ion exchange columns for therapeutic monoclonal antibodies (MAb) was chromatographic analyzed in the pH and salt gradient mode. BioPro IEX SF scoring highest selectivity and resolution.
BioPro IEX SF column is an outstanding stationary phase for the characterization of charge variants mAbs in cation exchange mode.


  Sample Speacies Isotype/Subclass pI
1 Natalizumab Humanized IgG4 7.3
2 Cetuximab Chimeric IgG1 7.9
3 Adalimumab Human IgG1 8.4
4 Denosumab Human IgG2 8.8

Comparison of monoclonal antibodies separation on pH and salt gradient mode

The chromatograms obtained on BioPro IEX SF, Protein-Pak Hi Res SP (waters), Antibodix WCX NP3 (Sepax) and on MabPac SCX-10RS (Thermo Scientific) columns in pH and salt gradient modes for natalizumab, cetuximab, adalizumab and denosumab is shown below.
BioPro IEX SF is able to provide good peak shapes and achieve higher resolution.

1. natalizumab
2. cetuximab
3. adalimumab
4. denosumab

pH gradient mode

Eluent A) CX-1 pH Gradient Buffer A* (pH 5.6)
B) CX-1 pH Gradient Buffer B* (pH 10.2)
0-100%B (0-20 min) for 100 X 4.6 mmI.D.
0-100%B (0-30 min) for 150 X 4.6 mmI.D.
Flow rate 0.6 mL/min

*Purchased Thermo Fisher Scientific Inc.

salt gradient mode

Eluent A) 10 mM MES-NaOH (pH 5.7)
B) 10 mM MES-NaOH (pH 5.7)
     containing 1 M NaCl
0-20%B (0-20 min) for 100 X 4.6 mmI.D.
0-20%B (0-30 min) for 150 X 4.6 mmI.D.
Flow rate 0.6 mL/min

High resolution by column coupling

A column coupling approach could help to improve antibody cation exchange analysis. This allows to compare the chromatographic profiles of different batches of bio-similar products.
Because BioPro IEX SF showed the highest resolution for the separation of natalizumab, cetuximab, adalimumab and denosumab, it was selected to perform high resolution separation by using a column coupling approach. The improved resolution of the different variants through the extension of column length and gradient time is illustrated below.

salt gradient mode
Column BioPro IEX SF 5 μm,
100 X 4.6 mmI.D. (L=10 cm)
100 X 4.6 mmI.D. X 2 (L=20 cm)
Eluent A) 10 mM MES-NaOH (pH 5.7)
B) 10 mM MES-NaOH (pH 5.7)
     containing 1 M NaCl
Flow rate 0.6 mL/min

S. Fekete, A. Beck, D. Guillarme, Characterisation of cation exchanger stationary phases applied for the separations of therapeutic monoclonal antibodies, J. Pharm. Biomed. Anal., 2015, 111, 169–176.