CHT Ceramic Hydroxyapatite Type I Support

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Overview

Specifications

	Type I	Type II
Functional groups	Ca²⁺, PO₄^3-, OH	Ca²⁺, PO₄^3-, OH
Dynamic binding capacity, lysozyme (Lys)	≥25 mg Lys/g CHT	≥12.5 mg Lys/g CHT
Nominal pore diameter	600–800 Å	800–1,000 Å
Maximum backpressure	100 bar (1,500 psi)	100 bar (1,500 psi)
Nominal mean particle size	20 ± 2, 40 ± 4 and 80 ± 8 µm	20 ± 2, 40 ± 4 and 80 ± 8 µm
Bulk density	0.63 g/ml	0.63 g/ml

CHT Mechanism

CHT Hydroxyapatite

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

	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, pH7 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

Acidic Proteins Protocol

Purity of Materials

Purification of IgG

In the preparation of ceramic hydroxyapatite, use of high-purity raw materials results in low levels of contaminants as determined by ICP mass spectrometry for metal analysis and ion chromatography for anions.

Impurity	Levels
Chloride	0.005%
Sulfate	0.01%
Carbonate	0.01%
Lead	0.001%
Cadmium	0.0001%
Barium	0.001%
Arsenic	0.001%

Globular Proteins Protocol

Globular Protein Protocal

Protocol II: Globular Proteins

Flow rate: 300 cm/hr
Buffer A: 5 mM NaPO4, 150 mM NaCl, pH 6.8
Buffer B: 500 mM NaPO4, 150 mM NaCl, pH 6.8
Buffer C: 500 mM NaPO4, pH 6.8

Equilibrate the column with approximately 10 column volumes of buffer A. Prepare the sample, adjusting the pH and conductivity to those of buffer A. Load the sample in 5 mM NaPO4, pH 6.8; wash the column with approximately 5 column volumes of buffer A; and elute with approximately 20 column volumes of buffer B linear gradient. Slope and amplitude can be adjusted based on initial results. After elution clean the column with approximately 5 column volumes of buffer C followed by a sanitation step using approximately 5 column volumes of 1 M NaOH.

Acidic Proteins Protocol

Acidic proteins protocol

Protocol IV: Acidic Proteins

Flow rate: 300 cm/hr

Buffer A: 5 mM NaPO4, pH 6.8
Buffer B: 500 mM NaPO4, pH 6.8

Equilibrate the column with approximately 10 column volumes of buffer A. Prepare the sample, adjusting the pH and conductivity to those of buffer A. Load the sample in 5 mM NaPO4, pH 6.8; wash the column with approximately 5 column volumes of buffer A; and elute with approximately 20 column volumes of buffer B linear gradient. After elution clean the column with approximately 5 column volumes of buffer B followed by a sanitation step using approximately 5 column volumes of 1 M NaOH.

Plasmid Protocol

Plasmid protocol

Protocol III: Plasmids

Flow rate: 300 cm/hr

Buffer A: 10 mM NaPO4, 1 mM EDTA, pH 7.0
Buffer B: 400 mM NaPO4, 1 mM EDTA, pH 7.0

Equilibrate the column with approximately 10 column volumes of buffer A. Prepare the sample, adjusting the pH and conductivity to those of buffer A. Load the sample in 0.5 M NaCl pH 7.0; wash the column with approximately 5 column volumes of buffer A; and elute with approximately 20 column volumes of buffer B linear gradient. Slope and amplitude can be adjusted based on initial results. After elution, clean the column with approximately 5 column volumes of buffer A followed by a sanitation step using approximately 5 column volumes of 1 M NaOH.

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.

CHT ceramic hydroxyapatite 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

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.