Bind and Elute Mode of Operation

A. Equilibrium buffer:

Sun (US8058407) discloses a method of purifying at least one acidic protein by applying an equilibration buffer of CaCl2 to HA, containing the HA with a load buffer, washing with CaCL2 and eluting with a buffer with 2-50 mM phosphate.

B. Binding/Loading

There are two different types of adsorbing sites on the surface of HA, calcium and phosphate sites. The former appear to be responsible for the binding of acidic groups, carboxyls and phosphates whereas the latter ones for the binindg of basic groups (Bernadi, Biochimica et Biophysica acta, 278 (1972) 409-420). 

Prior to protein loading, the resin is commonly equilibrated with a buffer of the same strenght as the loading buffer and at the same pH (Cummings US 13/205354). 

–Polyethylene glycol and glycine have been used to improve the performance of hydroxyapatite chromatography (Arakawa “Solvent Modulation of Column Chromatography” Protein & Peptide Letters, 2008, 15, 544-555).

Sheldon (WO/2010/148143) discloses methods of isolating recombinant adeno-associated virus (rAAV) particles by capturing the rAAV particles on an apatite chromatography meidum in the presence of polyethylene glycol (PEG). 

–Ca or Mg ion: Phosphate within the HA can be blocked (or bridged) by Ca ion or Mg ion which provides strengthened interaction between positive charge within a protein (Gorbunoff (Protein Chromatography on Hydroxyapatite Column, Method in Enzymology, Academic Press Inc., 1985, pp370-380) (US Patent Application 12/355,686).

–Phosphate buffer: 

In HA, the column is normally quilibrated, and the sample applied, in a low concentration of phosphate buffer and the adsorbed proteins are then eluted in a concentration gradient of phosphate buffer (Sun US2005/0107594).

Protein loading of a HA column is commonly conducted at pH 6.5 with phosphate buffer at 2mM ot 5mM, conditions that promote the adsorption of protein to the hydroxyapatite surface (Cummings US 13/205354).

–Salts:

In some cases, adsorption is further promoted by the inclusion of minor amounts of NaCl or KCl (Cummings US 13/205354).

Sun, US 2005/0107594 disclose using NaCl with HA for the purification of immunoglobulins and removal of high molecular weight aggregates. In one embodiment, the invention discloses using an elution or load buffer that contains from 1-20 mM sodium phosphate and 0.2-2.5 M NaCl, wherein the elution buffer has a pH from 6.4-7.6. Conditions vary depending on whether a binding or flow-through mode are used. 

Mitsui (EP256836) discloses that the degree of tPA adsorption to HA is governed by pH and/or salt concentration and also varies depending on the MW of tPA. Namely, the binding strenght of tPA to HA becomes greater as the pH decreases or the salt concentration drops. At the same pH and salt concentration, tPA having higher MWs is adsorbed more tightly.

C. Washing 

 E. Elution:

Elution on HA is caused by anions (usually phosphate) which compete for the calcium sites of HA with the carboxyl or phosphate groups of the macromolecules; or by cations (Na+, K+ or, more effectively Ca2+ or Mg2+) which compete for the phosphate groups of HA with the basic groups of proteins ((Bernadi, Biochimica et Biophysica acta, 278 (1972) 409-420). 

Gorbunoff, (Analytical Biochemistry, 136, 425-432 (1984)) discloses elution behavior as a function of their isoelectric point. Basic proteins having isoelectric points above 8 elute with PO4, F-,, Cl-, SCN- and ClO4- at moderate molarities, Proteins with isoelectric points between 7 and 8 elute with PO4, F- and CL- ions but not SCN- or Ca2+. Acidi proteins do not elute with Ca2+ ion. 

–Salts:

(Gorbunoff (Protein Chromatography on Hydroxyapatite Column, Method in Enzymology, Academic Press Inc., 1985, pp370-380) (US Patent Application 12/355,686) as well as other salts such as KCl, NaCL, KAc and NaAc can be used as eluting agent for HA. (Gagnon (232nd National Meeting of the American Chemical Society, Sep 10-14, 2006, Sanfransico, California, USA)  (US Patent Applicaiton 12/355,686).

The effects of different salts on the selectivity of a given apatite are unpreditable. For example, in the absence of phosphate, sodium chloride is unable to elute most IgG monoclonal antibodies from native HA, even at concentrations in excess of 4 moles per liter. When eluted with a combination of lower concentration of salts, such as 0.25M sodium chloride and 50 mM phosphate, IgG is one of the earliest eluting proteins (compare to when just phosphate is used above) (Gagnon, US2009/0186396). 

(a) Phosphate:

Phosphate refers to salts based on phosphorus (V) oxoacids such as, but not limited to, sodium phosphate and potassium phosphate (Gagnon, US2009/0186396). 

Once bound, the most common elution mechaism has been a gradient of increasing phosphate concentration. This would seem to be the most convenient choice of elution bufers since it serves as a displacement agent, disrupting both the COO:Ca+ interaction as well as the NH3+PO4 interaction. The strong calcium affinity of phosphate suspends calcium chelation and coordination iteractions, while its ionic character suspends phosphoryl cation exchange interactions. In exclusively phosphate gradients, IgG is typically one of the latest eluting proteins, using requiring 100-150 mM phosphate (Gagnon, US2009/0186396). 

In the increasing phosphate concentration method, all proteins bound to the column can be eluted and resolved based on the strenght of the interaction with the phosphate group on the hydroxyapatite matrix. Therefore, by using a gradient of increasing phosphate concentration, the most weakly bound proteins bound by the NH3+:P (V interaction (more acidic proteins) will elute earlier than those bound by the COO:Ca+ interaction (more basic proteins). In addition, the elution time of various bound proteins and resolution between them can be altered significantly through changes in the pH of the elution buffer (Mazzola, WO/2009/017491). Elution is accomplished by displacing the non-specific protein-hydroxyapatite pairing with ions such as Ca2+ or Mg2+. Negatively charged protein groups are displaced by negatively charged compounds, such as phosphates, thereby eluting a net-negatively charged protein.

Mitsui (EP256836) discloses a method of separating a mixture of tissue plasminogen activator (tPA) species having different MW by contacting the mixture with hydroxyapatite to adsorb the tPA species and then treating the HA with eluents to elute the fractions sucessively. The eluents are different in pH, salt concentrations or both pH and salt concentrations. In one embodiment the eluent is phosphate. In a second, the eluent is sodium chloride (see salts below).

In the rare cases where alternatives to phosphate as a primary eluting salt have been discussed in the literature, suggestions have included calcium chloride, citrate and fluoride salts, but without mention of sulfates (Gagnon, US2009/0186396). 

—-Phosphate + chloride salts: Some applications elute HA with combinations of phosphate and chloride salts. Chlorides preferentially elute the phosphoryl cation exchange interaction while having relatively little effect on calcium affinity interactions Gagnon (US 2009/0186396). 

———–Phosphate + calcium cloride (CaCL) or calcium sulfate (CaSO4):  

Jensen (US2009/0047723) discloses elution of FVII protein using either an appropriate combination of pH and phosphate or calcium in the form of calcium chloride or calcium sulfate).

Cummings (US 13/205354) discloses that the deteriorated of HA during protein elution can be mitigated by eluting adsorbed proteins by the use of an elution buffer that contains a combination of calcium and phosphate ions. Calcium ion can be supplied by an calcium salt such as calcium chloride that is soluble in the elution buffer. Phosphate ion can also be supplied from any phosphate salt such as sodium phosphate that is soluble in the elution buffer. In certain cases, sodium chloride is also included in the elution buffer for enhanced desorption of the protein.

———Phosphate + sodium chloride (NaCL):  Sun (US2005/0107594)  discloses HA for the purification of immunoglobulins using an elution buffer or load buffer that contains from 1-20 mM soium phosphate and from 0.2 to 2.5 M NaCL, wherein the elution buffer or load buffer has a pH from 6.4 to 7.6.

Cummings (US201101782760 discloses an elution buffer (0.55 M NaCl, 5 mM sodium phosphate at pH 6.5).

(b) sodum chloride (NaCL): Gagnon (US2009/0186396) discloses purification of a biomolecule by binding in the presence of calcium, convertion to native apatite and elution in a soidum chlorida gradient.

(c) calcium chloride (CaCL2):  Elution with Ca2+ is characterized by a drastic difference between the elution behavior of proteins as a funciton of their isoelectric point. All acidic proteins, as well as those with isoelectric points between 7 and 8, do not elute with Ca2+ ion even at 3 M sale. Basic proteins, to the contrary, elute at a Ca2+ molarity of 0.001-0.003 (Gorbunoff, Analytical Biochemistry, 136, 425-432 (1984).

—-CaCl2 + sodium sulfate: Gagnon (US2009/1086396) discloses binding DMA to HA and elution with 3 mM CaCL2, 1.0 M sodium sulfate, pH 6.7. 

 (d) sulfate: refers to salts based on sulfur (VI) oxoacides such as sodium sulfate and ammonium sulfate (Gagnon, US2009/0186396).

–Amino Acids:

——-Monocarboxylic zwitterion: refers to a molecule containing a single carboxyl moiety and at least one moeity with a positive charge. Examples include amino acids glycine, proline, lysine and histidine.  Gagnon (US 2009/0186396) discloses elution in the presence of a monocarboxylic awitterion.

Flow-through Mode of Operation

Vedantham (US 2003/0166869) teaches a method for separating a protein from another using HA in which the protein does not bind to HA but the other protein(s) does. In one embodiment, the protein is an antibody and the other protein is Protein A. 

a. Loading buffer: Sun (US2005/0107594A1) discloses antibody purification using a flow-through mode for HA with a load buffer containing 0.2 to 2.5 M NaCl at slightly acidic to slightly basic pH. The antibody preparation is then allowed to flow through a HA, while impuritied such as HMWA bind to the column. The column is optionally washed to allow additional purified antibodies to flow through.

Overload and Elute Mode

Nadarajah ((Nadarajah, US14/355818 (US2014/0301977)) teaches a method for purifying a polypeptide such as an antibody by a) loading the composition onto a mixed mode chromatography material such as CaptoAdhere in an amount in excess of the dynamic binding capacity of the material for the polypeptide, b) eluting the polypeptide under condtiions where contaminants remain bound and c) pooling fractions comprising the polypeptide in the chromatography effluent from steps a0 and b). In some embodiments, the partition coefficient of the chromatography material for the polypeptide is greater than 30 or greater than 100. 

Column Considerations

1. Column Dimensions: Sun, US2005/0107594 discloses many types of column diameters which can be used with cHA.