HA Ultrogel® Hydroxyapatite Chromatography SorbentPall Life Sciences Products - Biopharmaceutical Division
Hydroxyapatite Chromatography Sorbent
• Effective purification mechanism in a variety of processes
• High porosity
• Easy cleaning
• Used in large scale step
Biosepra HA Ultrogel sorbent available from Pall is a hydroxyapatite agarose composite sorbent for the separation of biomolecules from research and development scale to manufacturing.
Hydroxyapatite chromatography is considered to be a “pseudo-affinity” chromatography, or “mixed-mode” ion exchange. It has proven to be an effective purification mechanism in a variety of processes, providing biomolecule selectivity complementary to more traditional ion exchange or hydrophobic interaction techniques. HA Ultrogel is easy scaleable and is currently used in research scale to multi-liter column applications.
HA Ultrogel hydroxyapatite sorbent is composed of cross-linked agarose beads with microcrystals of hydroxyapatite entrapped in the agarose mesh. The particle size ranges between 60 and 180 µm. The agarose moiety in HA Ultrogel is chemically stabilized with epichlorohydrin under strongly alkaline conditions. Thus HA Ultrogel can be regularly treated with 0.1 - 1.0 M NaOH for regeneration and sanitization. This creates glycerol bridges between the polysaccharide chains and gives the sorbent beads an excellent rigidity and stability to pH and ionic strength changes, as well as to high temperature.
HA Ultrogel porosity is comparable to an agarose gel, with an exclusion limit for globular proteins of 5,000,000 daltons. This macroporosity avoids any moleculear sieving effect during the separation.
The sorbent is shipped in 1 M NaCl containing 20% ethanol and is available in a range of package sizes. Special packaging to meet specific manufacturing requirements is available on request.
The recommended flow rates to be used with HA Ultrogel sorbent depend on the column geometry and on the separation phase (capture, elution or washing steps). At process scale, typical flow rates from 30 to 200 cm/h are currently applied with multi-liter column sizes.
Hydroxyapatite crystals are naturally resistant to most chemical agents, except solutions with a pH less than 4 and complexing agents. Hydroxyapatite is dissolved by acidic solutions, while EDTA, citrate and other complexing agents decrease the adsorption capacity of the resin. Complexing agents may be used in extreme cases, e.g. when the desorption of certain compounds irreversibly bound to the matrix is required.
HA Ultrogel sorbent is resistant to denaturing agents: it can be treated with 8 M urea, 6 M guanidine-HCl, 1% SDS and chaotropic agents such as 3 M KSCN. The agarose moiety of HA Ultrogel sorbent is chemically stabilized by cross-linking with epichlorohydrin in a strong alkaline medium. HA Ultrogel sorbent is stable in alkaline conditions, and can be regularly treated with 0.1 to 1M sodium hydroxide for regeneration and depyrogenation. The chromatographic behavior of the sorbent was not significantly modified of after 5 weeks of incubation in 1M NaOH, pH 13. HA Ultrogel sorbent should not be treated with solutions at pH < 4 due to the nature of the hydroxyapatite crystals.
HA Ultrogel sorbent is stable a high temperature (up to 121 °C). It can be sterilized by autoclaving without undergoing any changes to its chromatographic properties. However, the operation should be performed in buffered conditions at pH 7 to avoid the presence of phosphate which may precipitate.
HA Ultrogel sorbent should never be frozen.