Hydroxyapatite is used for the fractionation and purification of a wide variety of biological molecules. At commercial scale, the release of protons during elution can detract from ceramic hydroxyapatite performance. The authors developed a simple methodology using a single additional buffer step called the surface neutralization system (SNS) that removes these protons at near-neutral pH. The insertion of the SNS step does not alter the desirable basic properties of ceramic hydroxyapatite.
Hydroxyapatite is a unique form of calcium phosphate used for the fractionation and purification of a wide variety of biological molecules, such as subclasses of IgG, enzymes, antibody fragments, and nucleic acids (1). Hydroxyapatite chromatography can be used as a polishing step to separate biomolecules that otherwise closely copurify (2–5, 6) or for the initial purification of crude samples (6). Ceramic hdroxyapatite is manufactured through sintering and heat treating hydroxyapatite crystals, thus forming a robust, easy-to-pack beaded form (7).
CHT is a mixed-mode resin. The calcium ions on the surface bind to either carboxyl clusters or phosphoryl groups on target molecules in a metal affinity-type mechanism. The phosphate groups on the surface, on the other hand, interact with amines or other positively-charged groups on the surface of proteins (or other molecules) through a classical cation-exchange mechanism. CHT is used to purify many manufactured biotherapeutics and for general proteins, owing to its superior removal of all process-stream impurities (e.g., aggregates, host-cell protein, viruses, endotoxin, protein A, and DNA).
At commercial scale, the release of protons during elution can hinder CHT performance. The surface of CHT under typical processing conditions has a high degree of protonation on the surface phosphate groups (see Figure 1) (8). These protons can change places with other cations in the mobile phase. When the concentration of cations increases (e.g., step gradients range from low ionic strength to high ionic strength), protons are released into the mobile phase, temporarily reducing the pH of the solution. This release has been mathematically modeled (9). Such uncontrolled pH excursions can, in the long run, detract from CHT performance (10, 11).
Figure 1: Surface chemistry of ceramic hydroxyapatite with reversible binding of protons to the surface under the influence of increasing or decreasing amounts of cation, in this case, sodium.
The pH excursion could be eliminated if the protons were replaced by other cations, thus converting all phosphate hydroxyls into their salt (e.g., sodium) form and forming water from the protons (see Figure 2). Taking this concept, a simple methodology was developed using a single additional buffer step immediately before elution, which removes protons at a near-neutral pH. This patent-pending technology is called the surface neutralization system (SNS) and is mechanistically similar to the action shown in Figure 2, except that water provides the neutralizing hydroxyls. This article presents data demonstrating that the SNS technique significantly extends the useful life of CHT (i.e., improves robustness) and prevents the pH drop during elution without desorbing the target protein when the target protein is a monoclonal antibody. Data also show that this technology does not affect the common quality outputs typically monitored during antibody purification.
Figure 2: Action of proton removal.