Performance and characterization of a nanophased porous hydroxyapatite for protein chromatography

Abstract

Nanophased porous hydroxyapatite beads with particle diameters of 25 Am and 30 Am intended for use in protein and biomolecule separation are characterized with respect to chromatographic characteristics. These particles were produced from a hydroxyapatite gel by a controlled spray process yielding microspheres containing hydroxyapatite nanocrystals. By calcification of the microspheres, nanophased porous hydroxyapatite beads were obtained. As a reference material, ceramic hydroxyapatite Types I and II with a particle diameter of 40 Am was chosen. SEM pictures show that the surface of the nanophased hydroxyapatite is very rough compared to ceramic hydroxyapatite Types I and Type II. The calcium-to-phosphorous ratio of this nanophased hydroxyapatite is 1.6, which is slightly below the theoretical ratio of 1.67 of pure hydroxyapatite. The porosity is greater than 60%. An IgG binding capacity of 60.7 mg/ml for Bio-Rad Type I and 36.0 mg/ml for Type II, 42.0 mg/ml for the nanophased material with 25 Am and 19.7 mg/ml for the nanophased material with 30 Am were observed. The nanophased material with 30 Am had the lowest mass transfer resistancy as indicated by the dependency of the dynamic binding capacity on velocity. It is assumed that the mass transport properties are characterized by a low particle diffusion resistancy or by slight intraparticle convection. The material also showed high selectivity for IgG. When culture supernatant with 5% FCS containing 3 mg/ml was loaded, pure IgG could be eluted by linear gradient with increasing sodium phosphate concentration. This nanophased material comprises a novel stationary phase for IgG separation.

Development and characterization of porous silver‐incorporated hydroxyapatite ceramic for separation and elimination of microorganisms

“…The enhanced specific surface area and permeability makes the porous ceramic a promising material not only for microbial filtration but also for chromatography, because it has more adsorptive sites and interconnected passages that can fulfill both the requirements of high loadability and fast operation. According to Jungbauer et al, 2 this kind of HA ceramic is a perspective stationary phase for protein separation.…”

Monoclonal antibody purification with hydroxyapatite

“…Intermediate and polishing purification HA has been used for intermediate purification of many IgG [14,15,[19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36], IgA [37][38][39][40][41], and IgM antibodies [17,37,38,[42][43][44]. The majority of these applications employ simple phosphate gradients.…”

Isolation and purification of recombinant proteins, antibodies and plasmid DNA with hydroxyapatite chromatography

“…A major breakthrough in this context was the development of a sintering process for the production of ceramic HA variants [15]. 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 [17]. Ceramic spherical HA particles have been commercialised since 1983 by Bio-Rad Laboratories Inc. (Hercules, CA, USA) under the name of Macro-Prep® HA (today CHT®).…”