Comparison of Camelid Antibody Ligand to Protein A for Monoclonal Antibody Purification - A stable alternative to Protein A chromatography. - BioPharm International

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Comparison of Camelid Antibody Ligand to Protein A for Monoclonal Antibody Purification
A stable alternative to Protein A chromatography.


BioPharm International
Volume 22, Issue 9

Exploration of Binding Mechanism

The binding mechanism of IgGs on Protein A ligand has been studied in detail by x-ray crystallography and it has been shown that the interactions consist of hydrophobic interactions along with some hydrogen bonding and two salt bridges.15,38 To explore the differences in the binding mechanisms of Protein A ligand and the IgSelect ligand, linear gradient experiments were conducted with different modifiers in the mobile phase. Molecule A and D (one Fc-fusion protein and one antibody) were used as the test proteins. Only one of the representative Protein A resins (MabSelect) was used for comparison.


Table 2. Elution pH values with different mobile phase modifiers
As summarized in Table 2, the results for both molecules with no additive in mobile-phase buffer showed similar elution pH on both MabSelect and IgSelect resins, which proved the reproducibility of the data previously included in Figure 1. Change in the elution pH on addition of mobile-phase modifiers was used as an indicator of the change in the strength of binding. An increase in the elution pH would suggest a decrease in binding strength and vice versa. Ethylene glycol (20%) was used in the mobile phase to test the role of hydrophobic interactions in IgG binding on IgSelect resin because this mobile-phase modifier can reduce the effect of hydrophobic interactions. Compared to the control (i.e., with no additives), both molecules A and D had higher elution pHs on MabSelect and IgSelect resins. This indicated that 20% ethylene glycol weakened bindings on both the ligands to a similar extent.

A suppressant of surface charge interactions (300 mM NaCl) was added to the mobile phase to investigate the role of electrostatic interaction in IgG binding on the IgSelect resin. Compared to control, both proteins had lower elution pHs on IgSelect resin on adding salt to the mobile phase. In fact, molecule D did not elute from IgSelect even at pH 2.5 under this condition. This indicated that the binding between IgGs and the new ligand was significantly increased after the addition of a medium concentration of salt. In comparison, MabSelect actually showed slightly higher elution pH for both molecules under the same conditions. These results showed that a medium concentration of NaCl affected IgG binding on the two resins in different ways and it required further investigation to determine if the stronger binding on IgSelect was because of decreased electrostatic interactions or increased hydrophobic interactions or a combination of both.

NaCl used in the previous set of experiments could have contributed to increased hydrophobic interactions while decreasing electrostatic interactions. To explore this effect further, a stronger kosmotropic salt (100 mM sodium citrate) was tested as the third mobile-phase modifier to explore the effect of increased hydrophobic interactions with minimum surface charge shielding. Compared to the data with no additives, 100 mM sodium citrate caused a significant binding increase on IgSelect resin while the effect on MabSelect resin was very small. This proved that salt played a much bigger role in the binding on IgSelect resin than MabSelect resin. When the mobile phase contains medium concentration of Kosmotropic salt or even NaCl, the binding of IgG molecules with CaptureSelect ligand can be significantly increased. In comparison, the Protein A ligand did not seem to be significantly affected by the tested salt concentration.

Gaining a fundamental understanding of the interaction mechanism on this new ligand requires additional sophisticated analytical techniques (such as x-ray crystallography and docking calculations) and was beyond the scope of this study. The results shown here demonstrate a clear difference in the way Protein A and the new ligand interact with IgGs.


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