Pleiad CHT Type I 80 µm

Hydroxyapatite [Ca₁₀(PO₄)₆(OH)₂] is an inorganic pure phase chromatography resin, which is calcined into spheres at high temperature. Its crystal structure is the same as that of bone hydroxyapatite (HAP); the spherical structure is stable and the particle size is uniform. There are multiple modes of interaction between HAP and biomolecules, which can achieve two separation mechanisms of cation exchange and calcium metal affinity simultaneously. The cation exchange mainly depends on the negatively charged phosphate groups on hydroxyapatite. At the same time, carboxyl clusters and phosphorus clusters on biomolecules can form with calcium on hydroxyapatite through a metal-affinity, stronger bond. Due to these unique separation mechanisms, hydroxyapatite is an indispensable chromatographic resin in today’s extremely demanding downstream processes in antibodies, vaccines (such as bacterial polysaccharides, pneumococcal polysaccharides and viruses), nucleic acids, enzymes and recombinant proteins, playing an important role in the field of biopharmaceuticals.

Method of use

Chromatographic conditions

Buffer selection. Select buffer salts whose buffer groups do not interact with the chromatography resin. If the binding and elution mode is used, the equilibration buffer should be a low pH — usually 1 pH unit lower than the isoelectric point of the target molecule, but not lower than 6.5 — to facilitate binding of the target molecule. Consider the stability of the sample in the buffer. The elution buffer is usually the equilibration buffer with a high concentration of salt added, such as 1–2M NaCl.

Flow rate. Generally choose a linear flow rate of 90–500 cm/h according to the bed height of the column.

Sample pretreatment. Filter the sample through a 0.45 µm microporous membrane before loading to prevent the sample from clogging the column.

Chromatography steps

1. Pre-equilibration. Use pre-equilibration buffer (for example 0.4M Na₃PO₄, pH 6.8) to quickly and fully equilibrate the column to the corresponding pH. This step usually requires 3–4 CV.
2. Equilibration. Use equilibration buffer (for example 0.005M Na₃PO₄, 0.1M NaCl, pH 6.8) to fully equilibrate the column until the pH and conductivity are stable and basically consistent with the equilibration buffer. This step usually requires 5–10 CV.
3. Loading*. Determine the loading volume and loading amount of the sample on the Pleiad CHT according to the binding capacity measured in the small test experiment.
4. Washing*. Use equilibration buffer or another suitable buffer to wash the column until the UV stabilises and returns to the baseline.
5. Elution*. Elute by increasing the concentration of salt ions or phosphate ions, applied as a linear or step gradient (for example linear gradient 0–100%, 0.005M Na₃PO₄, 1–2M NaCl or 0.1M NaPB, pH 6.8), to elute molecules with different binding strengths. Collect fractions from the eluted samples.
6. Regeneration. Flush the column with a high-concentration phosphate solution (such as 0.4M Na₃PO₄, pH 8.0). This step typically requires 5 CV.
7. Cleaning. Rinse the column with 0.005M NaPB buffer, pH 7.0. Reduce the concentration of phosphates to prevent the formation of bottoms.

* If the flow-through mode is used, set the sample loading step to collect; at the washing step, ensure that all the target molecules have flowed through before collection is stopped; at the elution step, use high-salt buffer directly to wash impurities away.

Cleaning and sterilisation

Contaminants (such as lipids, endotoxins and proteins) accumulate on the column as the number of uses of the chromatography resin increases. Regular cleaning-in-place (CIP) is essential to keep the column in a stable working condition. Determine the frequency of CIP based on the degree of contamination of the chromatography resin. Where contamination is considerable, CIP after each use is recommended to ensure reproducible results and to prolong the working life of the chromatography resin.

For different types of impurities and contaminants, the recommended cleaning conditions are as follows:

• Stronger binding proteins: wash with 5 CV of 2M NaCl solution.
• Strongly hydrophobic proteins and precipitated proteins: first wash with 5 CV of 1M NaOH solution, then use 5–10 CV of 0.1M NaOH to decrease the lye concentration.
• Lipoproteins and lipids: first wash with 5 CV of 70% ethanol or 30% isopropanol, then rinse with 5–10 CV of 0.005M NaPB buffer, pH 7.0.

Degas 70% ethanol or 30% isopropanol before use. Use a flow rate of 30–60 cm/h during CIP. Use reverse cleaning where the blockage is severe.

To reduce the microbial load, treat the chromatography resin with 1M NaOH solution for 15–30 minutes. Dry or wet sterilisation can also be used, as long as the solution is still alkaline at high temperature.

Storage

Keep the unopened chromatography resin in the original container and store at 4–30°C in a well-ventilated, dry and clean place. Do not freeze. Wash the used column with 2–3 CV of 1M NaOH solution and store at 2–8°C.

Destruction and recycling

Chromatography resin is difficult to degrade in nature, but it is homologous to bone and has no effect on the natural environment. For chromatography resin that has been in contact with biologically active samples such as viruses and blood, follow the local biosafety requirements before destroying or disposing of it.

Ordering information

• 804-00010 — 10 g
• 804-00025 — 25 g
• 804-00100 — 100 g
• 804-01000 — 1 kg
• 804-05000 — 5 kg