HA Ultrogel® Hydroxyapatite Chromatography Sorbent

Introduction

HA Ultrogel®  sorbent 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 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. 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.    

Features

• Unique selectivity and separation mechanism
• Protein purification in neutral , non-denaturating conditions
• Laboratory scale to large-scale production columns
  
  

Hydroxyapatite chromatography provides a gentle, neutral pH separation mechanism, different from other conventional methods such as ion exchange or affinity. The most well-known application of hydroxyapatite is the separation of basic proteins (cytochrome c, lysozyme, etc.) and phosphoproteins.

HA Ultrogel sorbent can be used for the separation of human serum proteins and plant proteins such as  lectins, glycoproteins, glycosidases, phospholipidases, sulfohydrolases, sphingomyelinases, transferases, trehalases and kinases.

As a phosphate-containing sorbent, HA Ultrogel can be used for the separation of phosphate-dependent proteins and enzymes as well as DNA-dependent enzymes.

Other key applications include :

• Vaccine purification processes (e.g. Bordetella pertussis toxin)
• Removal of aggregates in antibody purification
• Separation of protein isoforms
• Impurity removal during recombinant protein purification
• Separation of phosphoproteins, enzymes, glycoproteins, EPO, receptors

Figure 1. Separation of Glycohydrolases from a Crude Enzyme Extract of Buckwheat

Column: 2 x 6 cm; Sample: 40 mg of lyophilized extract in 1 ml of 1 mM phosphate buffer, pH 6.8; Discontinuous elution gradient of phosphate buffer; Flow rate: 7.1 cm/h; Temperature: 4 °C. Peak a: proteins with no glycohydrolase activity; peak b: β-glucosidase. Courtesy of R. Rourbouze & F. Percheron, Biochemistry Lab., Faculty of Pharmacy, Paris.

 

Figure 2. Purification of Damage-specific DNA Binding Protein from Human Placenta

The sample was previously prepurified from human placenta by ammonium sulfate precipitation, ion exchange chromatography and gel filtration. Column: 2.5 x 6 cm; Initial buffer: 10 mM potassium phosphate, pH 8 containing 5% glycerol and 13 mM 2-mercapto-ethanol; 1st elution (arrow): 0.15 M potassium phophate buffer; 2nd (arrow): 0.5 M potassium phosphate buffer;
Fraction volume: 1.8 ml. ●—● A280 nm ■—■ DNA binding activity o—o conductivity. Courtesy of R.S. Feldberg, et al., J. Biol. Chem. 257 (1982) 6394-401.

 

Figure 3. Separation of Trypsin and Chymotrypsin from a Porcine Pancreatic Enzyme Extract

Column: 1.6 x 5 cm; Sample: 30 mg protein in 1 ml of 5 mM phosphate buffer, pH 6.8; Gradient: 5 to 200 mM sodium phosphate, pH 6.8; Flow rate: 10 cm/h; Temperature: 10 °C; Histogram with broken line: trypsin activity; Histogram with solid line: chymotrypsin activity. Spec. Ac.: specific activity in U/mg. tr: trypsin, ch: chymostrypsin. Trypsin activity was primarily found in the peak eluted by 50 mM phosphate where the chymotrypsin was eluted by 100 mM phosphate. The final yield was approximately 50%.

Main Properties of HA Ultrogel Sorbent

Particle Size	60 – 180 µm
Hydroxyapatite Content	40 %
Agarose (weight/volume)	4 %
Exclusion Limit	> 5,000,000 dt
Working pH	5 – 13
Cleaning pH	5 – 14
Thermal Stability	4 – 121 °C
Capacity for Cytochrome c*	> 7 mg/mL
Capacity for BSA**	< 7 mg/mL

* Determined using 5 mg/mL cytochrome c diluted 50/50 in 10mM sodium phosphate buffer pH 6.9 at 30 cm/h
** Determined using 1mg/mL BSA diluted 50/50 in 10mM sodium phosphate buffer pH 6.9 at 12.5 cm/h

Chemical Stability

HA Ultrogel sorbent is stable in alkaline conditions, and can be treated with 0.1 M to 1M sodium hydroxide for regeneration and cleaning in place. HA Ullrogel sorbent can be stored  long term in  0.1 M NaOH, pH 13 at room temperature.

HA Ultrogel must not be treated with pH< 4 acidic solutions that dissolve the hydroxyapatite crystals.

Mechanical Stability

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.

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.