FAQs on Purification with Mixed-Mode Chromatography
Can I apply high salt concentrations (similar to HIC) for protein adsorption to MX-Trp-650M?
The ligand of MX-Trp-650M has a weak cation exchange and a hydrophobic moiety. Capacity of the resin for proteins is usually much higher when applying conditions dedicated to charge-based adsorption. Hydrophobic interactions support salt tolerance and selectivity during protein elution.
Applying pH or salt gradients; which one does provide higher selectivity with MX-Trp-650M?
In general, both is possible, and the preferred gradient mode may depend upon an individual application. Most often, robust sodium chloride gradients allow for successful separation. Occasionally, a combination of salt and pH gradients may increase selectivity of the resin.
How can I improve the reproducibility of my hydroxyapatite step?
Performance of Ca⁺⁺Pure-HA can be improved for non-mAb molecules, such as lysozyme, by conditioning the media prior to use with a phosphate wash. Using 5 mmol/L NaPO4, pH 6.8, for 10 CV delivers reproducibility from run-to-run.
Is Ca⁺⁺Pure-HA pH stable?
Uncontrolled pH variations that occur in conjunction with changes in conductivity take place with all charged media, such as HA, and are referred to as pH excursions. During elution with a salt in cation exchange chromatography, the positively charged ion of the salt will displace hydronium ions adsorbed to the media. As a result of the ion exchange, the pH of the solution will decrease due to the increase in hydronium ion concentration in the eluate. This transient pH drop, or excursion, during elution can be problematic for competitive hydroxyapatite chromatography media, as the hydroxyapatite is vulnerable to dissolution at low pH. Ca⁺⁺Pure-HA® experiences less pH excursions, which demonstrates the increased durability of this media.