Bio-Rad’s CHT Ceramic Hydroxyapatite XT (CHT XT) calcium-affinity cation-exchange chromatography medium is used for purifying numerous types of biomolecules, with single-step clearance of impurities and aggregates in monoclonal antibody (MAb) purification. This is an easy-to-use mixed-mode medium, which is both ligand and support matrix in one. In an “Ask the Expert” webinar on 18 July 2018, Mark Snyder (manager of the process chromatography R&D applications group at Bio-Rad Laboratories) discussed its application and use at process scale, providing guidelines on where to begin in selecting hydroxyapatite media.

Snyder’s Presentation 
Ceramic Hydroxyapatite (CHT) is a crystalline mineral of calcium and phosphate, Ca₁₀(PO₄)₆(OH)₂. In CHT chromatography media, ligand and matrix are the same. This mixed-mode medium works as a universal polishing matrix through both calcium affinity and cation exchange. It is unequaled in clearing a full range of impurities: aggregates, protein A, host-cell proteins, DNA, endotoxins, and minute virus of mice (MVM). Snyder demonstrated the mixed-mode properties by showing how CHT works on amino acid residues, carboxyl clusters, and phosphoryl residues.

By crystallizing a mixture of calcium hydroxide and phosphoric acid, spray drying and high-temperature sintering produce the familiar CHT Type I and II from Bio-Rad Laboratories. CHT XT formation adds a jet-milling step in which a homogenizer breaks larger crystals down into smaller crystals before the spray-drying step. This addition conveys increased particle robustness.

Snyder provided case studies of monoclonal antibody and Dengue virus purification showing how different protein species and virus particles had been separated. He advised that users selecting among the CHT types consider the size of molecule to be purified. For very large biomolecules (e.g., adenovirus), use CHT Type II. For large to medium-sized biomolecules (e.g., small viruses), use CHT Type II first and then consider CHT XT. For smaller biomolecules (e.g., antibodies), use CHT Type I in acidic solutions and CHT XT in basic or neutral solutions.

For CHT purification, users should not include anhydrous sodium phosphate, dodecahydrates (12-hydrate), or chelators. Never try to compress CHT when packing or running columns in upflow mode, Snyder cautioned. He said that CHT products and samples come as bulk media (10-g samples to 5-kg containers), in prepacked columns, or in 96-well plates for screening applications. Application notes and a packing tutorial can be found online at www.bio-rad.com/proteinpurification. For technical support, send general questions to [email protected], or contact a process specialist for specific assistance at [email protected].

Questions and Answers
What is the maximum pressure that you can use? For larger columns the pressure range is generally 3–5 bar. For acrylic or glass columns, 3 bar tends to be the maximum, whereas stainless steel columns can go up to 5 bar. CHT handles those pressures with no issues.

I’ve heard that CHT is difficult to pack. Any suggestions? I’ve packed a 1.8-m column with CHT in half an hour. CHT, by far, is the easiest media to pack — compressible or not — once you know how. It’s not a compressible resin, so it’s much like packing silica or controlledpore glass. Contact a Bio-Rad process specialist with questions about packing.

Will disposable column formats become available? They will be available in the last quarter of 2018.

What is the CHT XT particle size? It is a 40-µm particle. CHT Type I and II are available in both 40-µm and 80-µm sizes.

Does load temperature affect binding? There have been no detailed studies on this, but CHT binding mechanisms are well known and have been well studied. Not a lot of temperature effects come with either ion exchange or metal affinity — by contrast with hydrophobic-interaction chromatography (HIC) resins, for which temperature can play a large role.

How about cleaning columns? Typically all CHT types are cleaned with high-phosphate (≥0.4 M) buffer, then sanitized in normal hydroxide and stored in 0.1 M. Do not add NaCl to the cleaning solution as this will reduce effectiveness of the step. All three types of CHT use the same cleaning regimen. You should validate your own cleaning in developing your process. Email us with any questions.

Does flow rate affect binding? Yes and no. Flow rate certainly affects binding because CHT processes are diffusion controlled. If you want the maximum binding capacity for any resin (CHT or otherwise), then you want to use the slowest flow rate that is practical for your process.

Because of the more specific binding of CHT, is it suitable for primary capture? It depends on what is in the feed stream. Email our process specialists if you want to know more.

More Online
Watch the full presentation of this webcast now. Bio-Rad and CHT are trademarks of Bio-Rad Laboratories, Inc. in certain jurisdictions.