CHT Ceramic Hydroxyapatite Type I Support

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Overview

Specifications
  Type I Type II
Functional groups Ca2+, PO43-, OH Ca2+, PO43-, 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 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

Acidic Proteins Protocol
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 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

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

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.

CHT Ceramic Hydroxyapatite #158-2000
CHT Ceramic Hydroxyapatite

158-2000
10 g, CHT ceramic hydroxyapatite multimodal chromatography media, Type I, 20 μm particle size

List Price:   $155.00
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CHT Ceramic Hydroxyapatite #157-0020
CHT Ceramic Hydroxyapatite

157-0020
100 g, CHT ceramic hydroxyapatite multimodal chromatography media, Type 1, 20 μm particle size

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CHT Ceramic Hydroxyapatite #157-0021
CHT Ceramic Hydroxyapatite

157-0021
1 kg, CHT ceramic hydroxyapatite multimodal chromatography media, Type I, 20 μm particle size

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CHT Ceramic Hydroxyapatite #157-0025
CHT Ceramic Hydroxyapatite

157-0025
5 kg, CHT ceramic hydroxyapatite multimodal chromatography media, Type I, 20 μm particle size

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CHT Ceramic Hydroxyapatite #158-4000
CHT Ceramic Hydroxyapatite

158-4000
10 g, CHT ceramic hydroxyapatite multimodal chromatography media, Type I, 40 μm particle size

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CHT Ceramic Hydroxyapatite #157-0040
CHT Ceramic Hydroxyapatite

157-0040
100 g, CHT ceramic hydroxyapatite multimodal chromatography media, Type I, 40 μm particle size

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CHT Ceramic Hydroxyapatite #157-0041
CHT Ceramic Hydroxyapatite

157-0041
1 kg, CHT ceramic hydroxyapatite multimodal chromatography media, Type I, 40 μm particle size

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CHT Ceramic Hydroxyapatite #157-0045
CHT Ceramic Hydroxyapatite

157-0045
5 kg, CHT ceramic hydroxyapatite multimodal chromatography media, Type I, 40 μm particle size

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CHT Ceramic Hydroxyapatite #158-8000
CHT Ceramic Hydroxyapatite

158-8000
10 g, CHT ceramic hydroxyapatite multimodal chromatography media, Type I, 80 μm particle size

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CHT Ceramic Hydroxyapatite #157-0080
CHT Ceramic Hydroxyapatite

157-0080
100 g, CHT ceramic hydroxyapatite multimodal chromatography media, Type 1, 80 μm particle size

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CHT Ceramic Hydroxyapatite #157-0081
CHT Ceramic Hydroxyapatite

157-0081
1 kg, CHT ceramic hydroxyapatite multimodal chromatography media, Type I, 80 μm particle size

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CHT Ceramic Hydroxyapatite #157-0085
CHT Ceramic Hydroxyapatite

157-0085
5 kg, CHT ceramic hydroxyapatite multimodal chromatography media, Type I, 80 μm particle size

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Number Description Options
5739 Packing and Slurry-in-Place Procedures for CHT Ceramic Hydroxyapatite Using a Bio-Rad InPlace Process-Scale Chromatography Column, Rev A Click to download [ Add to Cart (Free) ]
6278 High-Throughput Calcium Analysis of Hydroxyapatite Column Effluent, Rev A Click to download [ Add to Cart (Free) ]
6086 Ceramic Hydroxyapatite Application Guide for Process Development and Scale-Up, Rev C Click to download [ Add to Cart (Free) ]
6067 Hydroxyapatite Column Effluent Analysis Tech Note, Rev A Click to download
2524 Purification of Horse IgGT Using Macro-Prep DEAE and CHT Ceramic Hydroxyapatite Type I Supports, Rev C Click to download
RP0033 A Ceramic Hydroxyapatite–Based Purification Platform — Simultaneous Removal of Leached Protein A, Aggregates, DNA, and Endotoxins from MAbs Click to download [ Add to Cart (Free) ]
2774 Purification of Transgenic Antibody From Corn Seed Using UNOsphere S and CHT Ceramic Hydroxyapatite Supports, Rev A Click to download
2026 Evaluation of Different Approaches for the Chromatographic Purification of Monoclonal Antibodies Click to download
5853 Chimeric IgG Monoclonal Antibody Purification: A Comparative Study Using CHT Ceramic Hydroxyapatite and CFT Ceramic Fluoroapatite Chromatographic Media, Rev A Click to download [ Add to Cart (Free) ]
5667 CHT Ceramic Hydroxyapatite Product Information Sheet, Rev B Click to download [ Add to Cart (Free) ]