CHT™ Ceramic Hydroxyapatite Type I Media

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Overview

	Type I	Type II
Functional groups	Ca²⁺, PO₄, OH	Ca²⁺, PO₄, OH
Observed dynamic binding capacity lysozyme (Lys)	≥25 mg Lys/g CHT	≥12.5 mg Lys/g CHT
Nominal pore diameter	600–800 A	800–1,000 A
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

Tutorial: CHT™ for Commercial Purification

Ceramic Hydroxyapatite (CHT™) for Commercial Purification

Ceramic hydroxyapatite is one of the leading media for biomolecule purification. It's a spherical ceramic form of crystalline hydroxyapatite and this form allows it to be used in production-scale columns at high flow rates while maintaining its unique separation properties.

CHT Binding Mechanism

CHT Ceramic Hydroxyapatite Mechanism

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 Ceramic Hydroxyapatite

	Type I	Type II
Functional groups	Ca²⁺, (PO₄)^3-	Ca²⁺, (PO₄)^3-
Dynamic binding capacity	>25 mg lysozyme per gram CHT	>12.5 mg lysozyme per gram CHT
Typical IgG binding capacities at 500 cm/hr	25–60 mg/ml	15–25 mg/ml
Particle sizes available	20, 40, and 80 µm (nominal)	20, 40, and 80 µm (nominal)
Nominal pore diameter	600–800 Å	800–1,000 Å
Maximum operating pressure	100 bar (1500 psi)	100 bar (1500 psi)
Recommended operating flow rates	10–1,500 cm/hr	10–1,500 cm/hr
pH stability	6.5–14	6.5–14
Regeneration	0.4-1.0 M phosphate buffer 0.5 M sodium phosphate pH 7 1.0 M trisodium phosphate pH 11–12	0.4-1.0 M phosphate buffer 0.5 M sodium phosphate pH 7 1.0 M trisodium phosphate pH 11–12
Sanitization	in 1–2 N NaOH	in 1–2 N NaOH
Autoclavability	20 min at 121°C	20 min at 121°C

IgG Protocol Using CHT

Purification of IgG on UNOsphere Rapid S 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 Ceramic Hydroxyapatite. Read more.

Globular Protein Protocol

Globular Protein Protocol Using CHT

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.

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 Using CHT

3.2.3 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.

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 Proteins Protocol

Acidic Proteins Protocol Using CHT

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. This 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 dimers 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

For more information, request bulletin 5709.

Regulatory support files are available upon request. Bio-Rad Laboratories is an ISO 9001-registered corporation.