CHT Ceramic Hydroxyapatite Type II Support

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Overview

Functional groups	Ca²⁺, PO₄^3-, OH
Particle sizes available	20, 40 and 80 µm (nominal)
Recommended linear flow rate	50–1,000 cm/hr
Operating pH range	6.5–14
Chemical compatibility (>24 hr)	1 M NaOH, 8 M urea, 8 M guanidine-HCl, ethanol, methanol, 100% acetonitrile
Regeneration	0.4–1.0 M phosphate buffer, 0.5 M sodium phosphate pH 7 1.0 M trisodium phosphate pH 11–12
Sanitization	1–2 N NaOH
Autoclavability (121µC, 20 min)	Yes
Packing density (g/ml packed bed)	0.63 g/ml
	Type I	Type II
Dynamic binding capacity	> 25 mg lysozyme/g	> 12.5 mg lysozyme/g
Typical IgG binding capacities at 500 cm/hr	25–60 mg/ml	15–25 mg/ml
Nominal pore diameter	600–800 Å	800–1,000 Å
Maximum operating pressure	100 bar (1,500 psi)	100 bar (1,500 psi)

CHT Binding Mechanisms

Hydroxyapatite contains two types of binding sites, positively charged calcium and negatively charged phosphate groups. These sites are distributed regularly throughout the crystal structure of the matrix. Solute species dominantly interact through cation exchange via the phosphate groups and/or metal affinity via the calcium atoms.

CHT Characteristics

Characteristics of Cermaic Hydroxyapatite

	Type I	Type II
Observed dynamic binding capacity IgG	25–60 mg IgG/ml CHT*	15–25 mg IgG/ml CHT**
Typical linear flow rate range	50–1,000 cm/hr
pH stability***	6.5–14 pH
Base stability	at least 21 months in 1 N NaOH
Regeneration	500 mM sodium phosphate, pH 7
	1,000 mM trisodium phosphate, pH 11–12
Autoclavability (bulk)	121°C, 20 min in phosphate buffer, pH 7
Sanitization	1–2 N NaOH
Recommended column storage	0.1 M NaOH
Shelf life (dry, unused material)	85 months stored dry, sealed and at room temperature

* 40 µm particles, 300 cm/hr, 5 mM sodium phosphate, pH 6.5
** 40 µm particles, 300 cm/hr, 5 mM sodium phosphate, pH 6.5
*** For pH 5.5–6.0, see Section 3.1.1 Buffers and Table 1

IgG Protocol Using CHT

Optimizing the bioprocess requires management of multiple variables ranging from matrix interaction, elution characteristics, scale-up from bench, regulation and drug safety requirements, and process robustness and economics. This IgG protocol has been developed as general starting guidelines for the purification of most proteins and nucleic acids and may help to reduce time spent in methodology development.

Purification of IgG

Purification of IgG using CHT

Elution of monoclonal antibody in a 40 column volume linear gradient to 1 M NaCl.

Globular Proteins Protocol

Globular Protein Protocol Using CHT

Optimizing the bioprocess requires management of multiple variables ranging from matrix interaction, elution characteristics, scale-up from bench, regulation and drug safety requirements, and process robustness and economics. This protocol has been developed as general starting guidelines for the purification of most proteins and nucleic acids and may help to reduce time spent in methodology development.

Plasmid Protocol

Plasmid Protocol Using CHT

Optimizing the bioprocess requires management of multiple variables ranging from matrix interaction, elution characteristics, scale-up from bench, regulation and drug safety requirements, and process robustness and economics. This protocol has been developed as general starting guidelines for the purification of most proteins and nucleic acids and may help to reduce time spent in methodology development.

Acidic Protein Protocol

Acidic Proteins Protocol Using CHT

Optimizing the bioprocess requires management of multiple variables ranging from matrix interaction, elution characteristics, scale-up from bench, regulation and drug safety requirements, and process robustness and economics. This protocol has been developed as general starting guidelines for the purification of most proteins and nucleic acids and may help to reduce time spent in methodology development.

CHT ceramic hydroxyapatite, (Ca₅(PO₄)3OH)₂, support is a leading purification medium for today's demanding downstream process industry. Its mixed-mode support offers unique selectivities and often separates biomolecules that appear homogeneous using other chromatographic methods. The diverse binding capabilities of CHT for host cell proteins, leached protein A, antibody dimmers and aggregates, nucleic acids, and viruses allow its use at any stage from initial capture to final polishing.

The robust properties of CHT ceramic hydroxyapatite improve efficiency, yield, and financial value through:

Excellent capture at elevated flow rates, enabling processing at all scales
Large capacity for high-titer upstream feedstocks
Exceptional selectivity, allowing a two-step chromatographic process

Hydroxyapatite (Ca₅(PO₄)3OH)₂ is a form of calcium phosphate used in the chromatographic separation of biomolecules. Sets of five calcium doublets (C-sites) and pairs of hydroxyl-containing phosphate triplets (P-sites) are arranged in a repeating geometric pattern. Repeating hexagonal structures can be seen in electron micrographs of the material; space-filling models and repeat structure from Raman spectroscopy have also been constructed.

Two types of CHT ceramic hydroxyapatite, Type I and Type II, are available in three particle sizes, 20, 40, and 80 µm. Both types have elution characteristics similar to crystalline hydroxyapatite, but also have some important differences:

Type I, sintered at 400°C, has a higher protein binding capacity and better capacity for acidic proteins
Type II, sintered at 700°C, has a lower protein binding capacity, but offers better resolution of nucleic acids and certain proteins; it also has a very low affinity for albumin and is particularly well-suited for the purification of many species and classes of immunoglobulins

Regulatory support files are available upon request. Bio-Rad Laboratories is an ISO9001 registered corporation.