Calcium-derivatized apatities

The inoic state of HA columns can be varied by changing the ionic nature of the buffers with which they are equilibrated. Thus, equilibration with PO4 buffer should make the surface of the column negative due to complexation with HA-C12+ stes. Equilibration with CaCl2 should have the opposit effect, while NaCl should lead to a neutral column surface (Gorbunoff, Analytical Biochemistry 136, 440-445 (1984). 

Native hydroxyapatite and fluorapatite can be converted to calium derivatized forms by exposure to soluble calcium in the absence of phosphate. This converts P-sites into secondary C-sites, abolishing phosphoryl cation-exchange interactions, increasing the number of C-sites, and fundamentally altering the selectivity of the apatite support. Small alkaline proteins typified by lysozyme (13.7-14.7 Kda, pI 10.7) fail to bind to calcium derivatized apatites, but most other proteins bind so stronly that even 3M calcium chloride is inadequate to achieve leution. Other chloride salts also fail to achieve elution. Calcium derivatized apatites are restored to their native forms by exposure to phosphate buffer, at which point they may be eluted by methods commonly applied for elution of native apatite supports. (Gagnon, US 2009/0186396). 

Ceramic hydroxyapatite (CHT or cHA): 

Ceramic hydroxyapatite refers to forms of the respective minerals in which nanocrysals are agglomerated into particles and fused at high termperature to create stable ceramic microspheres. New production procedures developed in the 1980s resulted in HPLC compatible HA beads. A major breakthrough in this context was the development of a sintering process for the production ofceramic HA variants. The ceramic material was mechanically stable and combined excellent flow properties with high particle porosity, while essentially maintaining the retention properties of the Tiselius HA. Sun (US 2005/0107594) teaches that ceramic hydroxyapatite was a known chromatography medium with the advantages of providing for durability and for a fast flow rate (¶6).

Ceramic hydroxyapatite is available in 2 types, Type I, with a medium porosity and relatively high binding capacity and Type II, with a larger porosity and a lower binding capacity. Either porosity can be used, and the optimal porosity for nay particular protein separation will vary with the proteins or the composition of the source mixture (Cummings, US 13/205354).

1. Type I (Ca5(PO4)3OH)2: Ceramic spherical HA particles have been commercialized since 1983 by Bio-Rad Laboratories Inc under the name CHT®(e.g., CHT Type I and CHT Type II). The binding mechanisms of resins such as type I is based on a mixed-mode interaction with Ca2+ as the positively charged functional group and PO43- as the negatively charged functional group. It has the separation properties of crystalline hydroxyapatite but can be used reproducibly for several hundred cycles at high flow rates and in large column. (US2009/0124790).

Type I has a high protein binding capacity and better capacity for acidic proteins.

2. Type II (Ca5(PO4)3OH)2: Type II has a lower protein binding capacity but better resolution of nucleic acids and certain proteins. The type II material also has a very low affinity for albumin and is especially suitable for the purification of many species and classes of immunoglobulins.

Other Modifications

Surface modification with Hexanoic and Decanoic Acids: 

Tanaka teaches calcium hydroxyapatite Ca10(PO4)6(OH)2 having the application as an adsorbent for protein (mixed mode chromatography matrix) (abstract; p. 31, line 4) modified with hexanoic acid CH3(CH2)4(COOH) (title). Tanaka further teaches that that hexanoic acid is hydrogen-bonded to the surface P-OH groups of the CaHAP (hexanoic acid is linked directly to a hydroxyl-functionalized solid support).

Fluoroapatite: 

Fluoroapatite refers to a mixed mode support comprising an insoluble fluoridated miniral of calcium phosphate with the structural formula Ca10(PO4)F2. Fluorapatite is prepared by fluoridating hydroxyapatite. Its dominant modes of interaction are phosphoryl cation exchange and calcium metal affinity. Fluorapatite is commercially available in various forms, including ceramic and crystalline composite forms. Fluoroapatite is markedly more stable under acidic pH conditions than hydroxylapatite (US 7,939,643).

I. Ceramic fluorapatite (CFT): Since 2006 ceramic FA has also been commercially available as a chromatographic stationary phase from Bio-Rad under the name ofCFT®(e.g., CFT Type I and CFT Type II).