Introduction

Hydroxyapatite is commonly used for purification of antibodies Gagnon (US2008/0177048). 

Hydroxyapatite is chemically similar to the mineral component of bones and hard tissues in mammals. it is seldom pure in nature but rather often occurs mixed with fluorapatite. 

Along withe crystalline form of hydroxylapatite it is also possible to sue a creamic form which can be obtained by sintering. HA can be bought for example from Bio-Rad. Its ceramic HA is provided in two forms (type 1 and type 2). On account of larger surface areas, the type 1 material has a greater binding capacity for relatively small molecuels whereas the type 2 has larger pores which enalbe the penetration and thus better bidning of large molecules such as DNA or large proteins. (Kretschmar, US 7.939,643)

Other names: hydroxyapatite, hydroxylapatite, calcium hydroxide phosphate

Complexity: Due to the complexity of the mechanisms involved, antibody separations on HA remain difficult to predict and are not very widespread. However, HA can be efficiently used when applied after Protein A chromatography for the recovery of therapetuic antibodies form cell culture supernatants. (Roque, J. Chromatography A, 1160 (2007) 44-55. 

Modes of Operation

Bind and Elute mode:

Gagnon (US2009/0270596) disclsoes methods of pruifying antibodies by containing a sample containing the antibodies to a mixed mode chromatography matrix such as ceramic hydroxyapatite (CHT) or ceramic fluoroxyapatite (CFT), spearating the matrix with the bound antibodies from the solution and eluting the antibodies by chaing the pH or salt concentration.

–single-hydroxyapatite chromatographic step:

Ahn (1989, 17(3), 269-72) discloses development of purification of monoclonal antibody from ascites fluid using a single hydroxyapatite step.

Engelhorn (US 4745183) discloses purification of monoclonal antibodies of the same isotype but different light chain composition by hydroxylapatite and concentration gardient elution phosphate buffer, pH 6.0-8.0.

–Ceramic hydroxyapatite: IgG1 can be resolved form an IgG1-Protein A complex in unfractionated media on CHT ceramic hydroxyapatite (Chromatography, tech Bio-Rad note 2849; S.G. Franklin, Bio-Rad laboratories, Inc., 2000 Alfred Nobel Drive, hercules CA 94547 USA).

Flow through mode:

Vedantham (US 7,476,722) disclsoes a method of separating a protein such as an antibody from a second protein such as Prtoein A that is affixed to an affinity chromatography support which has been used to previously purify the antibody where the second protein/Protein A binds to the hydroxyapatite but the antibody/protein does not. The protein of interest which comprises an Fc region may also be TNFR:Fc. 

Weak Partitioning Mode:

Kelly (US 8,067,182) teachings using weak partitioning mode with hydroxyapatite chromatography for product purification.

Euilibration:

The column is usually equilibrated and the sample applied in a buffer that contains a low concentrate of phosphate. Adsorbed antibodies are often eluted in an increasing gradient of phosphate salts. Gradients of phosphate combined with non-phosphate salts such as sodium choloride have also been used for protein purification, including antibody purification. One such approach involves the application of a gradient of sodium chloride or potassium chloride while a low level of phosphate is held constant. Both hydroxyapatite and fluroapatite have been shown to be effective for removal of aggregates from many antibody preparations. Antibody aggregates usually elute after antibodies. (Gagnon (US2008/0177048)

Native hydroxyapatite and fluorapatite can be converted to calcium-derivatized forms by exposure to soluble calcium in the absence of phosphate. This converts P-sites into secondary C sites, agolishing phosphoryl cation exchange interactions, increasing the number of C sites and fundamentally altering the selectivity of the apatite support. Calcium derivatized apatites are restored to their native forms by exposure to phosphate buffer, at which poiht they may be eluted by methods commonly applied for elution of antive apatite supports. (Gagnon, US 8,093,364). 

Binding

Organic polymers: 

Gagnon discloses that the use of an aqueous-soluble (i.e., hydrophilic) nonionic organic polymer such as polyethylene glycol (PEG), polypropylene glycol, polyvinylpyrrolidone, dextran, cellulose and starch) enhances retention of antibody on mixed mode chromatography supports in comparison to most contaminating proteins. In one embodiment, a protein preparation may be applied to the mixed mode chromatography support under conditions that prevent the binding of non-aggregated protein while permitting the binding of aggregated protein and other large molecules (i.e., “flow-through” mode). In other embodiment, the preparation is applied under conditions that permit the binding of virus or non-aggregated protein and contaminants with fractionation of the virus or non-aggregated protein being achieved by changing the conditions such that the non-aggregated protein (.e.g, antibody) is eluted while contaminant remain bound to the support (e.g., “bind-elute” mode). (US13/178970). 

Gagnon (US2008/0177048) also discloses methods of purifyig intact non-aggregated antibody from an antibody preparation by contacting the preparation with a mixed mode chromatography support in the presence of an aqueous-soluble (i.e., hydrophilic) nonionic organic polymer such as PEG. The presence of a nonionic organic polymer enhances binding capacity of antibody on the support, thereby enabling higher levels of productivity to be acheived. The presence of nonionic organic polymer also preferentially enhances the retention of antibody on the support.

–Salt (NaCL):

Sun (WO/2005/044856 and US2005/0107594) discloses method for removal of HWMA from antibody preparations by contacting a mixture with a hydroxyapatite resin and selectively eluting the antibody. Alternatively, the antibody preparation may be buffer-exchanged into an equilibration buffer and then allowed to flow through a hydroxyapatite resin. A combination of these binding/flow through hydroxyapatite chromatography methods may also be used. In one procedure an elution buffer or load buffer that contains from 1-20 mM sodium phosphate and from 0.2-2.5M NaCl, wherein the elution bufer or load buffer has a pH from 6.4-7.6 was used.

Washing:

Morii (EP0333474) discloses removal of endotoxin contaminants from an aqueous solution of a valuable protein such as an antibody using hydroxyapatite which includes a wash with pH in the range of 6-8 and an amino compound such as arginine at a concentration 1-100 mM.

Elution:

Most non-antibody protein contaminants elute before antibodies on HA but different clones elute in different areas of pI of the elution profile and may therefore overlop to varying degrees with containating proteins. Antiibody aggregates usually elute after antibodies but may coelute to varying degrees Gagnon (US2008/0177048).

The effects of different salts on the selectively of a given apatite are unpredictable. For example, in the absence of phosphate, sodium chloride is unable to elute most IgG monoclonal antibodeis from native hydroxyapatite, even at concentrations in excess of 4 moles per liter. This iplies extremely strong binding. In exclusively phsophate gradients, IgG is typcially one of the latest eluting proteins, usually requiring 100-150 mM phosphate. When eluted with a combination of lower concentrations of both salts, such as 0.25 M sodium chloride and 50 mM phospahte however, IgG is one of the earliest eluting proteins. Other paradoxxes reinforce the point: increasing the sodium chloride concentration in the presence of phosphate, which casues IgG to bind less strongly, has the opposite effect on DNA. Additionaly, lysozyme elutes at a higher phosphate concentration than BSA in the absence of sodium chloride but fails to bind in the presence of 1 M sodium chloride. (Gagnon, US 14/819,334)

Sulfate, borate, and certain monocarboxylic acids or zwitterions are able to elute antibodies form apatite supports in the absence of phsophate and such elution produces unique selectivities that permit effective fractionation of, cinluding removal of aggregates, that may not be adequately served by elution with phosphate or by combinations of phsophate and chloride. (Gagnon, US 14/819,334)

Isocratic Elution:

Mazzola (WO 2009/017491) discloes a emthod of purifying antibody using ceramic HA wehre the ab is eluted by isocratic elution that simultaneously removes at least one impurity. 

Phosphate:

—-Phosphate gradients:

Hydroxyapatite is most commonly eluted with phosphate gradients. The strong calcium affinity of phosphate suspends calcium chelation and coordination interactions, while its ionic character suspends phosphoryl cation exchange interactions  (Gagnon, US 14/819,334, now US 9,938,317). Hydroxyapatite has been used in the chromatographic separation of proteins, nucleic acids as well as antibodies. The column is normally equilibrated adn the same applied in a low concentraiton of phosphate buffer and the absorbed proteins are then eluted in a concentraiton gradient of phosphate buffer ((Sun, US 2013/0107198) 

(Gagnon, US 14/819,334, now US 9,938,317) discloses mAb antibody cpature on calcium derivatized HA, conversion to native HA, and elution in a phosphate gradient. Specifically, HA was eluilibrated with CaCl2, cell culture supernatant continaing a mAb was equilibrated to 5 mM calcium by addition of 1 M calcium chloride and the sample applied to the column. The column was washed with 5 mM sodum phosphate and eluted with a linear gradient to 300 mM phsophate, pH 6.7. The antibody eluted in a single peak at about q165 mM dosium phosphate.  

—-Sodium Phosphate – Salt (NaCL): 

Some applications elute hydroxyapatite 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 14/819,334, now US 9938317). Elution of hydroxyapatite and fluorapatite with chloride gradients at low fixed concentraiotns of phosphate has been shown to be more effective than simple phosphate gradients for antibody preparations. (Gagnon US 7,691,980). 

Mazzola (US2010/0234577) discloses that a limitation of the repeated re-use of hydroxyapatite is the instability of hydroxyapatite in calcium chelating buffers, such as citrate. The elimiation of citric acid or alternatively filtration though an anion exchange filter so that a Protein A eluate can be directly loaded onto a cHA column can be ahevied by eluation from Protein A using sodium phosphate at pH 2.1-3.5. 

Nadkarni (US 15/778,290, published as US2019/0367556) disclsoes methods for purifying antibody drug conjugate monters form aggregates using HA using a low concentration of sodium phosphate buffer for the wash and eluting the ADC with a dosium phosphate buffer increasing concentration. 

Sun (WO/2005/044856 and US2005/0107594) discloses method for removal of HWMA from antibody preparations by contacting a mixture with a hydroxyapatite resin and selectively eluting the antibody. Alternatively, the antibody preparation may be buffer-exchanged into an equilibration buffer and then allowed to flow through a hydroxyapatite resin. A combination of these binding/flow through hydroxyapatite chromatography methods may also be used. In one procedure an elution buffer or load buffer that contains from 1-20 mM sodium phosphate and from 0.2-2.5M NaCl, wherein the elution bufer or load buffer has a pH from 6.4-7.6 was used.

NaCl gradients:

(Gagnon, US 14/819,334, now US 9,938,317) discloses intermediate purification of a mAb by binding in the presence of calcium, converstion to native apatite and elution in a sodium chloride gradient. Specifically, HA was equilibrated with CaCl2, about 50 mg of protein A purified mAb was equilibrated with CaCl2 and the sample was applied to the column. The column was washed with buffer having sodium phosphate and then eluted with a linear gradeint to 20 mM Hepes, `0 MM phosphate, 2 M soidum chloride, pH 6.7. The antibody eluted in a single peak at 0.6 M soidum chloride, follwed by a well separate aggregate peak. 

Schubert (J. Chromatography A, 1142 (2007) 106-113) discloses binding antibody at pH 6, 7, 7,8 and 8.2 and to HA and elution in a NaCL gradient.

Ionic Species

–Borate

(Gagnon, US 14/819,334, now US 9938317) discloses purificaitona of an IgG mAb from cell culture supernatant on native HA, eluted with a broate gradeint. The column was eluted with a linear gradient fo 1M sodium borate, 5 mM sodium phosphate, pH 7.0. The majority of contaminating proteins eluted before the antibody. Non-aggregated antibody eluted at an average conductivity of about 5 mS/cm. Aggregates eluted later. 

–Monocarboxylic acid (lactate)

(Gagnon, US 14/819,334, now US 9938317) discloses purificaiton of an IgG mAb from cell culture supernatant which includes elution onf HA using a linear gradient of sodium lactate. The majority of contaminating proteins eluted before the antibody. 

–Monocarboxylate (e.g., acetate) or Monocarboxylate zwitterion (e.g., glycine) 

Gagnon (US 14/819,334, now US 9938317; US 15/910,702, published as US 2018/0186831) discloses elution from apatite using a monocarboxylic which refers to organic acid salts having a single carboxylic acid moeity such as sodium or potassium salts of formic, acetic, propionic, lactic, pyruvic, gluconic or glucuronic acid OR with a monocarboxylic zwitterion which refers to a molecule containing a site carboxyl moiety and at least one moiety with a positive charge such as glycine, proline, lysine and histidine. In one embodiment, the elution is conducted in the presence of a nonionic organix polymer such as dextrans, starches, celluloses, polyvinylpyrrolidones, polypropylene glycols and polyetheylene glycol (PEG). 

–Sulfate

Gagnon, (US 14/819,334, now US 9938317) discloses purification of an IgG mAb form a cell culture sueprnatant on calcium derivatized HA, eluted with a borate gradient. The majority of contaminating proteins eluted before the antibody. Antibody aggregate eluted after non-aggregated antibody. The column was eluted with a linear gradient to 1M sodium borate, 2.5 mM calcium chloride, 10% PEG 600 pH 7.0. 

Gagnon (US 8,093,364) discloses methods of fractionating a desired antibody or fragemtn using a native or calciu-derivatized apatite chromatography support which incuddes eluting the support in the presence of an ionic species which is a sulfate, borate, monocarboxylic organic acid salt or monocarboxylic zwitterion. In certian embodiments the ionic species is the primary eluting ion in the eluent. In certain embodiments the eluent is substantially free of phosphate as an eluting ion. 

Non-ionic Polymers:

–Polyethylene glycol (PEG):

PEG modulates elution of antibodies in hydroxyapatite purificaiton processes (Protein & Peptide Letters, 2008, 15, 544-555). PEG imposes a size discriminating selectivity on HA which dramatically enhances aggregate removal efficiency. (Gagon “Nonionic polymer enhancement of aggregate removal in ion exchange and hydroxyapatite chromatography” 12th Annual Waterside condference, San Juan, Puerto Rico, April 23-25, 2007). 

Gagnon (US 7,691,980) disclsoes a bind-elute mode enhanced revmoal of non-antibody protein contaimants and aggregates from a preparation of unpurified mAb which includes HA, CHT Type II that includes elution with a gradient to 5 mM sodium phosphate, 2.0 M sodium chloride, pH 6.7. The run is repeated but eluted with linear gradient to about 5 mM sodium phosphate, 2.0 M sodium chloride and about 5% PEG-6000. 

Hydroxyapatite in Combination with other Chromatography

Affinity chromatography – hydroyapatite: 

Vedantham (US 2003/0166869 and WO/2003/059935) discloses a method for separating a protein such as an antibody by affinity chromatography and then subjecting the protein to HA in flow through mode.

–Affinity (Protein A) – Ion Exchange – Hydroxyapatite: Mazzolo teaches a method of capturing IgG using protein A followed by at least one ion exchange prior to adsorbing the IgG to HA and selectively eluting.

–HCPLC – hdroxyapatite: Yamakawa (J. Liquid Chromatography, 1988, 11(3) 665-81) teach HPLC on spherical beads of hydroxyapatite to purify moncolconal antiobdies secreted into mouse ascitic fluid.