Hydroxyapatite high-performance liquid chromatography: column performance for proteins

Kawasaki, Tsutomu; Takahashi, Shoichi; Ideda, Koetsu

doi:10.1111/j.1432-1033.1985.tb09206.x

Cited by 207 publications

(119 citation statements)

References 16 publications

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“…C-sites may also be capable of anion exchange interactions with negatively charged solute residues [4][5][6][7][8]. Chelation and coordination bonds can be eluted with phosphate, which has a strong affinity for HA-calcium, but they are stronger than electrostatic interactions and usually survive exposure to 4 M sodium chloride in the absence of phosphate.…”

Section: Composition and Retention Mechanismsmentioning

confidence: 99%

“…Each C-site comprises a doublet of positively charged calcium residues [4]. Their dominant interactions are calcium chelation by protein carboxyl clusters and calcium coordination by phosphoryl groups on DNA and other phosphorylated solutes.…”

Section: Composition and Retention Mechanismsmentioning

confidence: 99%

“…Each P-site comprises a triplet of phosphate groups; each phosphate with a pair of negatively charged oxygen residues [4]. They act as cation exchangers, binding to protein amino groups under appropriate conditions [4][5][6][7][8].…”

Section: Composition and Retention Mechanismsmentioning

confidence: 99%

“…They act as cation exchangers, binding to protein amino groups under appropriate conditions [4][5][6][7][8]. Small strongly alkaline proteins such as lysozyme bind almost exclusively by cation exchange.…”

Section: Composition and Retention Mechanismsmentioning

confidence: 99%

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Monoclonal antibody purification with hydroxyapatite

Gagnon¹

2009

New Biotechnology

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Section: Composition and Retention Mechanismsmentioning

confidence: 99%

Section: Composition and Retention Mechanismsmentioning

confidence: 99%

Section: Composition and Retention Mechanismsmentioning

confidence: 99%

Section: Composition and Retention Mechanismsmentioning

confidence: 99%

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Monoclonal antibody purification with hydroxyapatite

Gagnon¹

2009

New Biotechnology

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“…A high level of chromatographic separation was obtained by selecting from very extensive ranges the optimum overall column length and the optimal slope of the molarity gradient for each particular sample based on chromatographic theory; except in some cases, the chromatographic resolution increases when the slope of the molarity gradient decreases and when the column length increases at the same time [4,5].…”

mentioning

confidence: 99%

High-performance liquid chromatography using spherical aggregates of hydroxyapatite micro-crystals as adsorbent

et al. 1986

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High-performance liquid chromatography (HPLC) using spherical aggregates of hydroxyapatite (HA) microcrystals as adsorbent has been developed ; preliminary performance tests were carried out by using several types of protein. In comparison with previously developed plate-like HA packed columns for HPLC, spherical HA packed columns show considerably high chromatographic resolutions in spite of extremely reduced column lengths of 0.5-3 cm. The pressure generated by the latter columns is much higher than that generated by the former, however.Hydroxyapatite (HA) chromatography, ideally used for separation of proteins, nucleic acids and viruses [l-31, is unique in that the geometrical arrangement of the adsorbing sites on the packed particles (i.e. the HA particles) is highly regular in any loci in the column [4, 51. Therefore, the adsorption configuration of a macromolecule on the HA surface can be deduced to be the same in any locus in the column; the elution position of the molecule is determined by how the geometrical arrangement of adsorption groups (i. e. carboxyl, phosphate, &-amino and guanidinyl groups) on a local molecular surface (that can keep in contact with the crystal surface) fits that of adsorbing crystal sites [4, 51.HPLC columns packed with plate-like HA micro-crystals (with thicknesses of about 1-3 pm and diameters of several times 10 pm) were recently developed in our laboratory (KB columns; Japan Biomedical Material Research Center, Koken Co. Ltd, Tokyo, Japan). A high level of chromatographic separation was obtained by selecting from very extensive ranges the optimum overall column length and the optimal slope of the molarity gradient for each particular sample based on chromatographic theory; except in some cases, the chromatographic resolution increases when the slope of the molarity gradient decreases and when the column length increases at the same time [4,5].The purpose of the present paper is to make another independent approach to high chromatographic resolutions by improving the shape of the packed particles in the column; this procedure would considerably reduce the flow heterogeneity occurring within the column. Thus, HPLC columns packed with spherical aggregates (with diameters of 1-10 pm) of extremely small HA micro-crystals (with diameters of the order of 0.1 pm) were prepared and preliminary performance tests were carried out by using several types of protein. Considerably high chromatographic resolutions were obtained even by using very compact columns with lengths of 0.5-3 cm. MATERIALS AND METHODSHA micro-crystals with diameters of the order of 0.1 pm as starting material were precipitated by mixing an aqueous Ca(OH)2 suspension and an aqueous NH4 a H2P04 solution. The aqueous HA suspension obtained above was sprayed; before being captured, the sprayed drips were dried into spherical crystal aggregates with diameters of 1 -10 pm (Fig. 1). These spherical aggregates, which will later be shown to be composed of stoichiometric HA crystals (see Discussion), were packed in SUS316 st...

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