Removal of high molecular weight aggregates using hydroxyapatite chromatography

Abstract

"REMOVAL OF HIGH MOLECULAR WEIGHT AGGREGATES USING HYDROXIAPATITE CHROMATOGRAPHY". This invention relates to the application of hydroxyapatite chromatography to the purification of at least one antibody from a preparation containing high molecular weight aggregates. In addition, this invention relates to an integration of ceramic hydroxyapatite chromatography in a combination chromatographic protocol for the removal of high molecular weight aggregates from an antibody preparation.

Description

Patent Descriptive Report for "REMOVAL OF HIGH MOLECULAR WEIGHT AGGREGATES USING HYDROXYAPATHITE CHROMATOGRAPHY". This application claims the priority benefit of US provisional patent application no. 60 / 523,335, filed November 20, 2003, and US provisional patent application no. 60 / 514018, filed October 27, 2003, the descriptions of which are hereby incorporated by reference in their entirety. Field of Invention The present invention relates to methods of removing high molecular weight aggregates from an antibody preparation using hydroxyapatite chromatography. In some embodiments of the invention, the amount of high molecular weight aggregates present in the final preparation can be significantly reduced, such as from 40% to less than 1%. Background of the Invention It is desirable to identify usable methods of purifying proteins that do not destroy, or significantly reduce, the biological activity of the protein. Contaminants must be removed from antibody preparations before they are used in diagnostic applications, therapeutic applications, applied cell biology and functional studies. Antibody preparations collected from hybridoma cell lines, for example, often contain undesirable components, such as high molecular weight a-clusters (HMWA) from the antibody produced by the cell line. This formation of aggregates can adversely affect the safety of the product by causing complementary activation or anaphylaxis upon administration. Furthermore, the formation of aggregates can impede manufacturing processes by causing decreased product yield, peak broadening and loss of activity. The most common protein purification methods are attributed to differences in size, charge, and solubility between the protein to be purified and the contaminants. Protocols based on these parameters include affinity chromatography, ion exchange chromatography, size exclusion chromatography, and hydrophobic interaction chromatography. These chromatographic methods, however, sometimes present technical difficulties in separating aggregated or multimeric antibody species. Techniques such as ion exchange or hydrophobic interaction chromatography, for example, can induce the formation of aggregates due to increased protein concentration or required changes in buffer concentration and / or pH during elution. Furthermore, in several cases, the antibodies show differences in isoelectric points that are too small to allow their separation by ion exchange chromatography. Tarditi, J. Immunol. Methods. 599: 13-20 (1992). Size exclusion chromatography is bulky and results in significant dilution of the product, which is an impediment in efficiency-based and large-scale manufacturing processes. Binder leakage from affinity chromatography columns can also occur, which results in undesirable contamination of the eluted product. Steindl, J. Immunol. Methods, 235: 61-69 (2000). Applicants have attempted to remove HMWA from an anti-GDF-8 antibody preparation using anion exchange chromatography, cation exchange chromatography, as well as hydrophobic interaction chromatography. However, all of these methods were unable to substantially remove HMWA from the anti-GDF-8 antibody preparation. Hydroxyapatite chromatography is a protein purification method that uses an insoluble hydroxylated calcium phosphate [Caium(P0 4 ) 6 (OH) 2 ], which forms both the matrix and the linker. The functional groups consist of pairs of positively charged calcium ions (C sites) and clusters of negatively charged phosphate groups (P sites). Interactions between hydroxyapatite and proteins are complex and multimodal. In one interaction method, however, positively charged amino groups on proteins associate with negatively charged P sites, and protein carboxyl groups interact by coordination complexation to C sites. Shepard. J. of Chromatography 891: 93-98 (2000). Crystalline hydroxyapatite was the first type of hydroxyapatite used output in chromatography, but was limited by structural difficulties. Ceramic hydroxyapatite (cHA) chromatography was developed to overcome some of the difficulties associated with crystalline hydroxyapatite, such as limited flow rates. Ceramic hydroxyapatite has high durability, good protein binding capacity, and can be used at higher flow rates and pressures than ceramic hydroxyapatite. Vola et al, BioTechniques, 14: 650-655 (1993). Hydroxyapatite has been used in the chromatographic separation of proteins, nucleic acids, as well as antibodies. In hydroxyapatite chromatography, the column is normally equilibrated, 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. Giovan-nini, Biotechnology and Bioengineering, 73: 522-529 (2000). Sometimes, shallow sodium phosphate gradients are used successfully to elute proteins, while in other cases, concentration gradients up to 400 mM sodium phosphate have been used successfully. See, for example, Stan-ker, J. Immunolgical Methods 76: 157-169 (1985) (10 mM to 30 mM sodium phosphate elution gradient); Shepard J. Chromatography 891: 93-98 (2000) (10 mM to 74 mM sodium phosphate elution gradient). Tarditi, J. Chromatography 599: 13-20 (1992) (10 mM to 350 mM sodium phosphate elution gradient). Although salts such as NaCl have been incorporated into the binding buffer to purify an antibody using hydroxyapatite chromatography, Giovannini, R. Biotechnology and Bioengineering 73: 522-529 (2000), salts such as NaCl and (NH 4 ) 2 SO4 are not known to affect protein elution on hydroxyapatite chromatography. Karlsson et al, Ion Exchange Chromatography, in Protein Purification, VCH Publishers, Inc. (Janson and Ryden ed. 1989). In several cases, investigators are unable to selectively elute antibodies from hydroxyapatite or have found that hydroxyapatite chromatography does not result in a sufficiently pure product. Junbauer J. Chromatography, 476:257-268 (1989), Giovannini Biotechnology and Bioengineering 73: 522-529 (2000). The applicants tried unsuccessfully to process to separate high molecular weight aggregates from an antibody preparation using hydroxyapatite chromatography and a sodium phosphate elution based on prior art teachings (Figure 1). Furthermore, severe elution conditions, when used in an attempt to break the tight binding of a protein in a matrix, are known to destroy the biological activity of a protein. Thus, there is a need for effective methods of removing impurities, such as high molecular weight aggregates, from antibody preparations that do not destroy the biological activity of the antibodies. Summary of the Invention Applicants have surprisingly found that NaCl can be used in a new hydroxyapatite chromatography method for the purification of immunoglobulins and removal of HMWA from different starting materials (figure 2). Thus, the present invention relates to methods of removing high molecular weight aggregates from antibody preparations by contacting said preparation with hydroxyapatite resin and selectively eluting the antibody from the resin. Alternatively, the antibody preparation can be buffer exchanged in an equilibration buffer and then allowed to flow through the hydroxyapatite resin. A combination of these cross-flow hydroxyapatite chromatography / binding methods can also be used to purify antibody preparations. The invention relates to an elution buffer or loading buffer containing from 1 to 20 mM sodium phosphate and from 0.2 to 2.5 M NaCl, wherein the elution buffer or loading buffer has a pH from 6.4 to 7.6. In a combination binding / flow through mode, the invention relates to an equilibration buffer and wash buffer containing 1 to 20 mM sodium phosphate, 0.01 to 2.0 M NaCl, 0 200 mM arginine, and 0 to 200 mM HEPES, wherein the equilibration buffer and wash buffer have a pH of 6.2 to 8.0. In one embodiment, the purified antibody contains less than 5% high molecular weight aggregates. In another embodiment, the purified antibody contains less than 1% high molecular weight aggregates. In a further embodiment, the antibody preparation contains at least one IgG antibody. More specifically, the antibody preparation contains at least one antibody selected from anti-IL-21, anti-GDF-8, anti-Abeta, anti-CD22, anti-Lewis Y, anti-IL-13, or anti-IL receptor -22. At least one purification method can be used in combination with the hydroxyapatite chromatography of the invention. A variety of purification methods can be used, including, but not limited to, Protein A chromatography, affinity chromatography, hydrophobic interaction chromatography, immobilized metal affinity chromatography, size exclusion chromatography, diafiltration, ultrafiltration, stripping filtration. viral, anion exchange chromatography, and / or cation exchange chromatography. In one embodiment, anion exchange and Protein A chromatography are used in combination with ceramic hydroxyapatite chromatography. Anion exchange and Protein A chromatography may be used in combination, for example, contacting the antibody preparation with a Protein A support, allowing the antibody to adsorb onto the support, washing the support and the adsorbed antibody with at least one wash buffer of Protein A, eluting the adsorbed antibody, with at least one Protein A elution buffer, contacting the preparation with an ion exchange support, allowing the antibody to flow through the support, washing the support with at least one exchange wash buffer ionic flow by contacting the ion exchange throughflow with a hydroxyapatite resin, allowing the throughflow to adsorb onto the resin, washing the resin with at least one hydroxyapatite wash buffer, and eluting the purified antibody from the resin with at least one elution buffer of hydroxyapatite. In another embodiment, anion exchange and Protein A chromatography may be used in combination, for example, contacting the pre- Protein A support, allowing the antibody to adsorb onto the Protein A support, washing the Protein A support and adsorbed antibody with at least one Protein A wash buffer, eluting the adsorbed antibody with at least one buffer of Protein A elution, contacting the Protein A eluent with an ion exchange support, allowing the antibody to flow through the ion exchange support, washing the ion exchange support with at least one ion exchange wash buffer, changing the flow ion exchange bypass in a loading buffer comprising 1 to 20 mM sodium phosphate and 0.2 to 2.5 M NaCl, contacting the ion exchange bypass with a hydroxyapatite resin, allowing the antibody to flow through of the hydroxyapatite resin, and washing the hydroxyapatite resin with at least one hydroxyapatite wash buffer. In yet another embodiment, anion exchange and Protein A chromatography are used in combination with ceramic hydroxyapatite chromatography. Anion exchange and Protein A chromatography can be used in combination, for example, by contacting the antibody preparation with a Protein A support, allowing the antibody to adsorb onto the support, washing the support and adsorbed antibody with at least one wash buffer of Protein A, eluting the adsorbed antibody with at least one Protein A elution buffer, contacting the preparation with an ion exchange support, allowing the antibody to flow through the support, washing the support with at least one ion exchange wash buffer , contacting the ion exchange throughflow with a hydroxyapatite resin, letting the throughflow adsorb onto the resin, letting the HMWA bind more tightly than the antibody monomer, and, as the charge continues, displacement of the bound monomer by the HMWA, washing the hydroxyapatite resin with at least one hydroxyapatite wash buffer and collecting the displaced antibody monomer. Additional objects and advantages of the invention will be specified in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention will be realized and achieved through the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the above general description and the following detailed description are exemplary and explanatory only and not limiting of the invention as claimed. The attached drawings, which are incorporated and constitute part of this report, and together with the description, serve to explain the principles of the invention. Brief Description of the Drawings Figure 1 demonstrates the inability of prior art phosphate gradient elution to separate HMWA from an anti-GDF-8 antibody preparation. Figure 2 demonstrates that the use of NaCl gradient elution results in the separation of a large portion of the HMWA from an anti-GDF-8 antibody preparation. Figure 3 shows the separation of HMWA from an anti-CD22 antibody preparation using cHA chromatography. Figure 4 shows the separation of HMWA from an anti-Abeta antibody preparation using cHA chromatography. Detailed Description of the Invention A. Definitions In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are specified throughout the detailed description. The term "antibody" refers to any immunoglobulin or fragment thereof, and encompasses any polypeptide comprising an antigen-binding site. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, polyspecific, nonspecific, humanized, human, single chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitro generated antibodies. The term "antibody" also includes antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, dAb, and other antibody fragments that retain antigen-binding function. Typically, these fragments would comprise a binding domain the antigen. Antibodies that may also be purified by the invention include chemically modified forms such as by PEG treatment, and fusion proteins comprising an immunoglobulin moiety. The antibody or fragment thereof may be selected from any of the known antibody isotypes and their conformations, for example IgG, IgA, IgD, IgE, IgM monomers, IgA dimers, IgA trimers, or IgM pentamers. The term "antibody preparation" refers to any composition containing an antibody and / or unwanted components such as high molecular weight aggregates of these antibodies. "Ceramic hydroxyapatite" or "cHA" refers to an insoluble hydroxylated calcium phosphate of the formula [Caium(P04) 6 (OH) 2 ], which was sintered at elevated temperatures into a macroporous, spherical ceramic form. The term "cHA" encompasses, but is not limited to, type I and type II ceramic hydroxyapatite. Unless specified, "cHA" refers to any particle size including, but not limited to, 20, 40 and 80 pm. The term "high molecular weight aggregates" or "HMWA" refers to an association of at least two antibodies. Association can arise by any method including, but not limited to, covalent, non-covalent, disulfide, or non-reducible crosslinking. At least two antibodies can bind to the same or different antigens. The at least two antibodies can be in the form of an antibody, antibody fragment, or other forms described in the definition of "antibody" above. The term "flow-through mode" refers to an antibody preparation separation technique in which at least one antibody contained in the preparation is intended to flow through a chromatographic resin or support, while at least one contaminant or impurity potentially binds to the resin or chromatographic support. The flow-through mode can be used, for example, in hydroxyapatite chromatography and ion exchange chromatography. "Binding mode" refers to an antibody preparation separation technique in which at least one antibody contained in the preparation tion binds to a resin or chromatographic support while at least one contaminant or impurity flows through it. The binding mode can be used, for example, in hydroxyapatite chromatography or ion exchange chromatography. B. Description of the method The present invention provides methods for removing high molecular weight aggregates (HMWA) from antibody preparations using hydroxyapatite chromatography in binding mode, flow through mode, or a combination thereof. The present invention finds application in the large scale purification of antibody preparations. In binding mode, the method uses a phosphate-loaded hydroxyapatite support at neutral pH and low ionic strength to bind both the antibody and HMWA. The column is then washed with a phosphate buffer to remove loosely bound impurities. Next, the antibody is selectively eluted using a high ionic strength phosphate buffer containing 0.2 to 2.5 M NaCl at a slightly acidic to slightly basic pH. HMWA is optionally subsequently washed from the resin using an even higher ionic strength and a higher phosphate concentration buffer at neutral pH. Lastly, the resin is optionally regenerated using sodium hydroxide and potassium phosphate solution. In flow-through mode, an antibody preparation is buffer exchanged in a loading buffer containing 0.2 to 2.5 M NaCl at a slightly acidic to slightly basic pH. The antibody preparation is then allowed to flow through a hydroxyapatite column while impurities such as HMWA bind to the column. The column is optionally subsequently washed to let additional purified antibodies flow through the column. Lastly, the column can optionally be stripped and then regenerated using a sodium hydroxide and potassium phosphate solution. In a combination binding / through-flow mode, the antibody preparation is allowed to flow through a hydroxyapatite column, with both antibody monomer and HMWA binding initial- mind. However, as the charge continues, the incoming HMWA is able to bind more tightly than the antibody monomer and thus displaces the bound monomer. Consequently, the displaced monomer flows through the column. The column is optionally subsequently washed to allow additional displaced antibodies to flow through the column. Lastly, the column can optionally be extracted with a high salt, high phosphate solution, and then regenerated using a sodium hydroxide and potassium phosphate solution. In one embodiment of the invention, the purified antibody contains less than 5% HMWA, in one embodiment less than 3% HMWA, and in another embodiment less than 1% HMWA. 1. Antibodies Antibody preparations of the invention can be isolated from a variety of sources including, but not limited to, serum from immunized animals, ascites fluid, hybridoma or myeloma supernatants, conditioned medium derived from cultivation of a recombinant cell line that expresses the antibody molecule, and of all cell extracts from cells producing antibodies. In one embodiment of the invention, antibodies from conditioned cell culture media of recombinant cell lines producing antibodies are purified. Although some variation from cell line to cell line and among the various antibody products is to be expected based on the description herein, those skilled in the art are well aware of an adaptation of the invention herein to a particular combination of antibody protein and antibody production. cell line. For purposes of illustration only, this invention has been applied to the purification of various IgG and basic pl isotype antibodies. More specifically, this invention has been applied to monoclonal antibodies against GDF-8 described by US patent application 60 / 419 964 (hereinafter "Myo-29"), monoclonal antibodies specifically reactive with the CD22 antigen described in the patent application US 10 / 428 894 (hereinafter "anti-CD22"), and monoclonal antibodies against Abeta antigen described in international application no. PCT / US01 / 46587 (hereinafter "anti-Abeta"). The construction of systems but recombinants for the production of Myo-29 and the CD22 and A-beta antibodies is detailed in the aforementioned applications. 2. The hydroxyapatite resin Various hydroxyapatite chromatographic resins are commercially available, and any available form of the material can be used in the practice of this invention. In one embodiment of the invention, the hydroxyapatite is in crystalline form. Hydroxyapatites for use in this invention may be those which are agglomerated to form particles and sintered at elevated temperatures into a stable porous ceramic mass. The particle size of hydroxyapatite can vary widely, but a typical particle size is in the range of 1 gm to 1000 pm in diameter, and can be from 10 pm to 100 pm. In one embodiment of the invention, the particle size is 20 µm. In another embodiment of the invention, the particle size is 40 µm. In yet another embodiment of the invention, the particle size is 80 µm. Several chromatographic supports can be employed in the preparation of cHA columns, the most extensively used are type I and type II hydroxyapatite. Type I has a high protein binding capacity and better capacity for acidic proteins. Type II, however, has a lower protein binding capacity but has better resolution of nucleic acids and some proteins. Type II material also has a very low affinity for albumin and is especially suited for the purification of many species and classes of immunoglobulins. The choice of a particular hydroxyapatite type can be determined by one skilled in the art. This invention can be used with hydroxyapatite resin that is loose, packed in a column, or in an annual continuous chromatograph. In one embodiment of the invention, ceramic hydroxyapatite resin is packed in a column. The choice of column dimensions can be determined by one skilled in the art. In one embodiment of the invention, a column diameter of at least 0.5 cm with a bed height of about 20 cm can be used for small scale purification. In a In a further embodiment of the invention, a column diameter of about 35 cm to about 60 cm can be used. In yet another embodiment of the invention, a column diameter of 60 cm to 85 cm can be used. In some embodiments of the invention, a suspension of ceramic hydroxyapatite resin in Na 2 HPO 4 of 200 mM at pH 9.0 can be used to pack the column at a constant flow rate of about 4 cm / min or with gravity. 2. Buffer compositions and loading conditions Before contacting the hydroxyapatite resin with the antibody preparation, it may be necessary to adjust parameters such as pH, ionic strength, and temperature and, in some cases, the addition of substances of different types. Thus, it is an optional step to carry out an equilibration of the hydroxyapatite matrix by washing it with a solution (for example a buffer to adjust the pH, ionic strength, etc., or for the introduction of a detergent), with the necessary characteristics for purification. of the antibody preparation. In hydroxyapatite chromatography in combination binding / through-flow mode, the hydroxyapatite matrix is ​​equilibrated and washed with a solution, thus bringing about the necessary characteristics for purification of the antibody preparation. In one embodiment of the invention, the matrix can be equilibrated using a solution containing 0.01 to 2.0 M NaCl at a slightly basic to slightly acidic pH. For example, the equilibration buffer can contain 1 to 20 mM sodium phosphate, in another embodiment it can contain 1 to 10 mM sodium phosphate, in another embodiment it can contain 2 to 5 mM sodium phosphate, in another embodiment it can contain 2 mM sodium phosphate, and in another embodiment may contain 5 mM sodium phosphate. The equilibration buffer may contain 0.01 to 2.0 M NaCl, in one embodiment, 0.025 to 0.5 M NaCl, in another embodiment, 0.05 M NaCl, and in another embodiment 0.1 M NaCl. NaCI. The pH of the loading buffer can be in the range of 6.2 to 8.0. In one embodiment, the pH can be from 6.6 to 7.7, and in another embodiment, the pH can be 7.3. The equilibration buffer can contain 0 to 200 mM arginine, in another embodiment it can contain 120 mM arginine, and in another embodiment may contain 100 mM arginine. The equilibration buffer can contain 0 to 200 mM HEPES, in another embodiment it can contain 20 mM HEPES, and in another embodiment it can contain 100 mM HEPES. The antibody preparation can be buffer exchanged into an appropriate buffer or loading buffer in the preparation for flow-through mode hydroxyapatite chromatography. In another embodiment of the invention, the antibody preparation can be buffer exchanged into a loading buffer containing 0.2 to 2.5 M NaCl at slightly acidic to slightly basic pH. For example, the loading buffer can contain 1 to 20 mM sodium phosphate, in another embodiment it can contain 2 to 8 mM sodium phosphate, in another embodiment it can contain 3 to 7 mM sodium phosphate, and in another embodiment may contain 5 mM sodium phosphate. The loading buffer may contain 0.2 to 2.5 M NaCl, in one embodiment, 0.2 to 1.5 M NaCl, in another embodiment, 0.3 to 1.09 M NaCl, and in another modality, 350 mM NaCl. The pH of the loading buffer can be in the range of 6.4 to 7.6. In one embodiment, the pH can be from 6.5 to 7.0, and in another embodiment, the pH can be 6.8. Contacting an antibody preparation to the hydroxyapatite resin in binding mode, flow-through mode, or combinations thereof can be performed in a packed bed column, a fluidized / expanded bed column, containing the solid phase matrix , and / or a simple batch operation, where the solid phase matrix is ​​mixed with the solution for a period of time. After contacting the hydroxyapatite resin with the antibody preparation, a washing procedure is optionally performed. However, in some cases, where very high purity of the immunoglobulin is not critical or additional through-flow of antibody is not required, the washing procedure can be omitted, saving a process step as well as washing solution. The wash buffers employed will depend on the nature of the hydroxyapatite resin, the mode of hydroxyapatite chromatography being employed, and so on. can be determined by one skilled in the art. In the through flow mode, and binding / through flow in combination mode, the purified antibody through flow obtained after an optional column wash can be pooled with other purified antibody fractions. In binding mode, antibody can be eluted from the column after an optional wash procedure. For elution of antibody from the column, the invention uses a high ionic strength phosphate buffer containing about 0.2 to 2.5 M NaCl at slightly acidic to slightly basic pH. For example, the elution buffer can contain 1 to 20 mM sodium phosphate, in another embodiment it can contain 2 to 8 mM sodium phosphate, in another embodiment it can contain 2 to 6 mM sodium phosphate, in another embodiment it can contain 3 mM sodium phosphate, and in another embodiment may contain 5 mM sodium phosphate. The elution buffer may contain 0.2 to 2.5 M NaCl, in one embodiment 0.2 to 1.5 M NaCl, in another embodiment 0.3 to 1.1 M NaCl, in another embodiment 1 .0 M NaCl, and in another embodiment, 0.35 M NaCl. The pH of the elution buffer can be in the range of 6.4 to 7.6. In one embodiment the pH can be from 6.5 to 7.3, in another embodiment the pH can be 7.2, and in another embodiment the pH can be 6.8. The elution buffer can be changed to elute the antibody from the column in a step or continuous gradient. In both binding and through-flow mode, and combinations thereof, a solid-phase matrix can optionally be cleaned, i.e., extracted and regenerated, after elution or through-flow of the antibody. This procedure is typically performed regularly to minimize the accumulation of impurities on the surface of the solid phase and / or to sterilize the matrix to prevent contamination of the product with microorganisms. Buffer components can be adjusted according to the knowledge of one skilled in the art. Sample buffer composition ranges and examples for binding mode, flow-through mode, and binding / flow-through mode in combination are given in table 1, table 2 and table 3, respectively. Not all plugs or steps are required, but are provided for illustration only. For example, it may not be necessary to have two distinct equilibration steps, and it may not be necessary to extract, regenerate, or store the hydroxyapatite resin. A high throughput screen as described in the example 11, can be used to effectively optimize buffer conditions for cHA column chromatography. Table 1 - Example buffer composition ranges for binding mode Buffer Composition Range Composition(s) Example Equilibrium 1 10 mM to 500 mM Sodium Phosphate 1.0 M NaCl pH 6.4 to 7.4 0.3 M Sodium Phosphate 1.0 M NaCl pH 6.8 Equilibrium 2 1 to 20 mM sodium phosphate 0 to 200 mM NaCl pH 6.4 to 7.4 5.0 mM sodium phosphate 50 mM NaCl pH 7.2 Wash 1 to 20 mM sodium phosphate 0 to 200 mM NaCl pH 6.4 to 7.4 5.0 mM sodium phosphate 50 mM NaCl pH 7.2 Elution 1 to 20 mM sodium phosphate 0.2 to 2.5 M NaCl pH 6 .4 to 7.6 5.0 mM sodium phosphate 350 mM NaCl pH 6.8; or 3.0 mM sodium phosphate 1.0 M NaCl pH 7.2 Extraction 10 mM to 500 mM sodium phosphate 1.0 M NaCl pH 6.4 to 7.4 0.3 M sodium phosphate 1.0 M NaCl pH 6.8 Regeneration 0.5 to 1.0 M Potassium Phosphate 1.0 M NaOH 0.5 M Potassium Phosphate 1.0 M NaOH Storage 10 to 50 mM NaOH 20 mM NaOH Table 2. Example buffer composition ranges for through flow mode Buffer Composition Range Example Composition Equilibrium 1 10 mM to 500 mM Sodium Phosphate 1.0 M NaCl pH 6.4 to 7.4 0.3 M Sodium Phosphate 1.0 M NaCl pH 6.8 Equilibrium 2 1 to 20 mM Sodium Phosphate 0.2 to 2.5 M NaCl pH 6.4 to 7.6 5.0 mM Sodium Phosphate 350 mM NaCl pH 6.8 Loading Buffer 1 to 20 mM NaCl Sodium Phosphate 0.2 to 2.5 M NaCl pH 6.4 to 7.6 5.0 mM Sodium Phosphate 350 mM NaCl pH 6.8 Wash 1 to 20 mM Sodium Phosphate 0.2 to 2.5 M NaCl pH 6.4 to 7.6 5.0 mM Sodium Phosphate 350 mM NaCl pH 6.8 Extraction 10 mM to 500 mM Sodium Phosphate 1.0 M NaCl pH 6.4 to 7.4 0.3 M sodium phosphate 1.0 M NaCl pH 6.8 Regeneration 0.5 to 1.0 M potassium phosphate 1.0 M NaOH 0.5 M potassium phosphate 1.0 M NaOH Storage 10 to 50 mM NaOH 20 mM NaOH Table 3. Example buffer composition ranges for binding mode / throughflow in combination Buffer Composition Range Composition(s) Example Equilibrium 1 10 mM to 500 mM Sodium Phosphate 1.0 M NaCl pH 6.4 to 7.4 0.3 M Sodium Phosphate 1.0 M NaCl pH 6.8 Equilibrium 2 1 to 20 mM Sodium Phosphate 0.01 to 2.0 M NaCl 0 to 200 mM Arginine 0 to 200 mM HEPES 2.0 mM Sodium Phosphate 50 mM NaCl, 100 mM Arginine 100 mM HEPES, pH 7.3; Buffer Composition Range Composition(s) Example pH 6.2 to 8.0 or 5.0 mM Sodium Phosphate 100 mM NaCl, 120 mM Arginine, 20 mM HEPES, pH 7.3 Wash 1 to 20 mM Phosphate Sodium 0.01 to 2.0 M NaCl 0 to 200 mM Arginine 0 to 200 mM HEPES pH 6.2 to 8.0 2.0 mM Sodium Phosphate 50 mM NaCl, 100 mM Arginine 100 mM HEPES, pH 7.3; or 5.0 mM Sodium Phosphate 100 mM NaCl, 120 mM Arginine, 20 mM HEPES, pH 7.3 Extraction 10 mM to 500 mM Sodium Phosphate 1.0 M NaCl pH 6.4 to 7.4 0.3 M sodium phosphate 1.0 M NaCl pH 6.8 Regeneration 0.5 to 1.0 M potassium phosphate 1.0 M NaOH 0.5 M potassium phosphate 1.0 M NaOH Storage 10 to 50 mM NaOH 20 mM NaOH In one embodiment of the invention, the loading on the cHA resin can be, for example, at a loading challenge of < 20 mg / ml and a starting aggregate level in the loading of < 40% HMWA. In some embodiments of the invention, a loading challenge of 1.8 to 10.4 mg / ml can be used with a starting aggregate level in the loading of about 15%. In a further embodiment of the invention, the cHA resin is loaded at a load challenge of at least 20 mg / ml and a starting aggregate level in the load of < 40% HMWA. In some embodiments of the invention, a 30 to 40 mg / ml loading challenge can be used with a starting aggregate level in the loading of about 27%. 3. Additional Optional Steps Although it has been found that hydroxyapatite chromatography can be used alone to separate monomeric IgG from aggregates, as mentioned above, the purification method of the invention can be used in combination with other protein purification techniques. In one embodiment of the invention, one or more steps preceding the hydroxyapatite step may be desirable to reduce the loading challenge of contaminants or impurities. In another embodiment of the invention, one or more purification steps following the hydroxyapatite step may be desirable to remove additional contaminants or impurities. The described cHA purification procedure can optionally be combined with other purification steps including, but not limited to, Protein A chromatography, affinity chromatography, hydrophobic interaction chromatography, immobilized metal affinity chromatography, size exclusion chromatography, diafiltration , ultrafiltration, viral removal filtration and / or ion exchange chromatography. In one embodiment, prior to the cHA purification step, the collection media may optionally be purified initially by a Protein A chromatography step. For example, PROSEP-A® (Millipore, U.K.), which consists of covalently coupled Protein A in controlled pore glass, can be generally employed. Other usable Protein A formulations include FAST FLOW® Protein A Sepharose (Amersham Biosciences, Piscataway, NJ), TOYOPEARL® 650M Protein A Sepharose (TosoHaas Co., Philadelphia, PA), and MABSELECT® (Amersham Biosciences, Piscataway, NJ) columns. NJ). As an optional step prior to cHA purification, ion exchange chromatography can be employed. In this regard, various anionic or cationic substituents can be attached to the matrices in order to form anionic or cationic supports for chromatography. Anion exchange substituents include diethylaminoethyl (DEAE), trimethylaminoethyl acrylamide (TMAE), quaternary aminoethyl (QAE) and quaternary amine (Q) groups. Cationic exchange substituents include carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P) and sulfonate (S). Cellulose ion exchange resins Materials such as DE23, DE32, DE52, CM-23, CM-32 and CM-52 are available from Whatman Ltd. Maidstone, Kent, U.K. Cross-linked and Sephadex-based ion exchangers are also known. For example, DEAE-, QAE-, CM-, and SP-Sephadex, and DEAE-, Q-, CM- and S-Sepharose, and Sepharose are all available from Amersham Biosciences, Piscataway, NJ. Furthermore, ethylene glycol methacrylate copolymer derivatized from both DEAE and CM, such as TOYOPEARL® DEAE-650S or M and TOTYPEARL® CM-650S or M are available from Toso Haas Co., Philadelphia, PA. In one embodiment of the invention, ion exchange chromatography can be used in binding mode or flow-through mode. In some embodiments, the Protein A chromatography step is conducted first, the anion exchange step is conducted second, and the cHA step is conducted third. 4. Removal of Additional Impurities In addition to HMWA removal, cHA chromatography has been shown to be usable for removing other impurities from antibody preparations. Other impurities that can be removed by the cHA chromatography methods of the invention include, but are not limited to, DNA, host cell protein, adventitious virus, and Protein A contaminants from previous purification steps. In one embodiment of the invention, the invention is capable of removing Protein A from the antibody preparation. In some embodiments of this invention, the amount of Protein A present in the final preparation can be significantly reduced, such as from 300 ppm to less than 1 ppm. C. Examples The following examples are offered for illustration purposes only. Example 1: Purification of an Anti-GDF-8 Antibody The purification process described below was developed for an anti-GDF-8 monoclonal antibody (referred to herein as "Myo-29"). The Myo-29 antibody is an IgG1 subtype antibody and has a pl of approximately 8.1. The purification process consisted of three chromatographic steps (Protein A affinity, anion exchange, and hydroxyapatite), a viral inactivation step, and an ultrafiltration / diafiltration step to concentrate and exchange the product into a final buffer. All steps were performed at 18 to 25°C, except the Protein A chromatography step, which was cycled at 2 to 8°C. The purification process can be normalized to any scale. Linear flow rates listed are independent of column diameter and charge ratios are mass per unit volume. The Protein A chromatography step can be cycled multiple times per batch to accommodate varying amounts of cell culture titers in the collector bioreactor. Each cycle is considered a separate unit operation and the elution pool is retained for the next step. The Protein A stage has a capacity of approximately 35 grams of Myo-29 per liter of MabSelect. Downstream process steps (e.g. anion exchange chromatography and ceramic hydroxyapatite chromatography) are scaled to accommodate approximately 15 grams of Myo-29 per liter of anion exchange resin and around 10 grams of Myo-29 per liter of ceramic hydroxyapatite resin. 1. Removal of Culture Cells The Myo-29 antibody was expressed in Chinese Hamster Ovary (CHO) cells and was cultured in a 2500 L stirred tank bioreactor. To collect culture fluid, cells were removed using a Prostak microfiltration device (Millipore, Billerica, MA). Purged conditioned medium (CCM) was collected for the first chromatographic step, Protein A chromatography. 2. Protein A affinity chromatography purification step A 17.7 L MabSelect column (30 cm diameter x 25 cm height) of recombinant Protein A resin (Amersham Biosciences, Piscataway, NJ) was equilibrated with 5 column volumes of an equilibration buffer (10 mM of Tris, 100 mM NaCl, pH 7.5). CCM was applied to the column at a flow rate of 2.5 cm / min and a load challenge of 35 g of Myo-29 per liter of resin. After loading the column, it was washed with 5 column volumes of a high salt wash buffer (20 mM Tris, 1.0 M NaCl, pH 7.5) and then with 10 column volumes in a low salt wash buffer (10 mM Tris, 100 mM NaCl, pH 7.5). Myo-29 was eluted by applying 6 column volumes of an elution buffer (100 mM arginine, 50 mM NaCl, pH 3.0). The elution pool was then maintained at a pH of 3.6 ± 0.5 for 1.5 ± 0.5 hours as a prophylactic measure to facilitate inactivation of potential adventitious viral contaminants. The elution pool was then neutralized to pH 7.3 with 2 M HEPES buffer pH 8.0 to prevent degradation of acid labile portions of Myo-29. Column effluents were monitored for several parameters, including visual inspection of chromatographic conductivity and UV absorbance profiles, and product recovery using HPLC Protein A for loading titer and absorbance at 280 nm for elution pool concentration . The column was extracted with 6M guanidine HCl and then washed with extraction wash buffer (10 mM Tris, 100 mM NaCl, pH 7.5). The column was stored in 16% ethanol. 3. Anion exchange chromatography purification step The Protein A column eluent was further purified by anion exchange chromatography on a 75 L column (80 cm diameter X 15 cm length) of Q SEPHAROSE FF resin (Amersham Biosciences, Piscataway, NJ). The column was equilibrated with 5 column volumes of a first equilibration buffer (20 mM HEPES, 1000 mM NaCl, pH 7.3) and then with 5 column volumes of a second equilibration buffer (100 mM arginine, 50 mM NaCl, 100 mM HEPES, pH 7.3). Protein A column eluent was applied to the equilibrated column at a flow rate of 2.5 cm / min and a loading ratio of 15 grams of Myo-29 per liter of resin. After loading, the column was washed with 5 column volumes of the second equilibration buffer. The flow through the anion exchange column was collected. The collected column throughflow was monitored by several parameters, including visual inspection of chromatographic conductivity and UV absorbance profiles, and product recovery using absorbance at 280 nm. The anion exchange column was extracted with extraction buffer (20 mM HEPES, 1 M NaCl, pH 7.3) and regenerated with regeneration buffer (500 mM NaOH, 1 M NaCl, pH 13.3). The column was stored in 0.01 M NaOH. 4. Filtering to retain viruses The purpose of this optional step was to remove retroviral-like particles that may be present in cultured CHO cells and to provide additional safety by removing potential adventitious viral contaminants. Anion exchange column flow through was collected and passed through a 35 nm Pla-nova single-use filter (Asahi-Kasei Corp., New York, NY). The remaining product in the module was recovered by passing anion exchange column wash buffer (100 mM Arginine, 50 mM NaCl, 100 mM HEPES, pH 7.3) through the device. Product recovery after virus-retaining filtration was evaluated by absorbance at 280 nm and Plano-va pool SDS-PAGE analysis compared to historical performance data. 5. cHA chromatography purification step The viral filtered solution was further purified with a hydroxyapatite column (60 cm x 20 cm) packed with cHA type II resin, 40 pM particle size (BioRad, Hercules, CA). The column was equilibrated with 3 column volumes of equilibration buffer 1 (0.3 M sodium phosphate, 1.0 M NaCl, pH 6.8). A second equilibration step was conducted with 4 column volumes of equilibration buffer 2 (5 mM sodium phosphate, 50 mM NaCl, pH 7.2). The partially purified medium was loaded onto the resin in a 1:1 (v / v) loading buffer (10 mM sodium phosphate, pH 7.2) and at a flow rate of 2.5 cm / min. The cHA column was washed with 3 column volumes of a wash buffer (5 mM sodium phosphate sodium, 50 mM NaCl, pH 7.2). The Myo-29 antibody was eluted from the cHA resin using 6 column volumes of an elution buffer (5 mM sodium phosphate, 350 mM NaCl, pH 6.8). The cHA purification step was monitored by visual inspection of chromatographic conductivity and UV absorbance profiles, product recovery by absorbance at 280 nm, removal by HMWA as determined by size exclusion chromatography (SEC) analysis, and removal of of Protein A, as determined by a competitive enzyme-linked immunosorbent assay (ELISA). The cHA column was extracted with extraction buffer (0.3 M sodium phosphate, 1.0 M NaCl, pH 6.8) and regenerated with regeneration buffer (0.5 M potassium phosphate, 1, 0 M NaOH, pH 13.3). The column was stored in 0.02M NaOH. Table 4 demonstrates that cHa chromatography effectively removes HMWA impurities from antibody preparations. Furthermore, cHA chromatography is capable of removing other impurities such as Protein A. Table 4. Antibody monomer yields and clearance from HMWA Sample HMWA Load % HMWA Peak Load ProA (ppm) Peak ProA 1 14.6 1.0 17 BLOCK* 2 16.4 0.7 20 BLOCK 3 15.1 1.6 23 BLOCK 4 16.0 0, 9 13 BLOCK *BLOCK = below the quantitation limit of 1 ng / ml. 6. Ultrafiltration / diafiltration and final filtration The cHA elution pool was passed through a tangential flow ultrafiltration system using Pellicon 2 PLCTK cassettes (Milli-pore, Billerica, MA) composed of a composite regenerated cellulose membrane, with a nominal molecular weight cutoff of 30,000 . The objectives of this step were to concentrate and exchange, in buffer, the puddle of the cHA product in the formulation buffer. The pHA product pool was sprinkled with a 50% sucrose (w / v) solution to bring the sucrose concentration to 2%. The Myo-29 antibody was concentrated to approximately 20 gl - then diafiltered with approximately > 9 wash volumes of formulation buffer (0.01 M L-histidine, 2% sucrose, pH 6.0). Upon completion of the diafiltration, the product was further concentrated to approximately 60 gL' 1 and retrieved from the apparatus by gravity drain and a downward air puff, followed by flushing the retention channels with the formulation buffer. The target concentration of Myo-29 in the drug substance is > 35 g / L. Myo-29 drug substance was finally filtered through a 0.22 micron filter, equilibrated with formulation buffer (0.01 M L-histidine, 2% sucrose, pH 6.0), aliquoted into vials, and then stored at -80 °C. Example 2: Purification of cHA from an anti-GDF-8 antibody Preparation using type I resin The Myo-29 antibody was also successfully purified using cHA type I resin, 40 pM particle size, packed into a 3.1 L column. The column was equilibrated with 3 column volumes of 1 equilibration buffer ( 0.3 M sodium phosphate, 1.0 M NaCl, pH 6.8). A second equilibration step was conducted with 4 column volumes of equilibration buffer 2 (5 mM sodium phosphate, 50 mM NaCl, pH 7.2). Partially purified medium from an anion exchange purification step was loaded onto the resin at a loading challenge of 35 mg / ml and at a flow rate of 1.5 cm / min. The cHA column was washed with 3 column volumes of a wash buffer (5 mM sodium phosphate, 50 mM NaCl, pH 7.2). The Myo-29 antibody was eluted from the cHA resin using 6 column volumes of an elution buffer (3 mM sodium phosphate, 1.0 M NaCl, pH 7.2). The cHA column was extracted with extraction buffer (0.3 M sodium phosphate, 1.0 M NaCl, pH 6.8) and regenerated with regeneration buffer (0.5 M potassium phosphate, 1, 0 M NaOH, pH 13.3). The column was stored in 0.02M NaOH. The cHA purification step was monitored by visual inspection of chromatographic conductivity and UV absorbance profiles, product recovery by absorbance at 280 nm, removal by HMWA as determined by size exclusion chromatography (SEC) analysis, and removal of of Protein A, as determined by a competitive enzyme-linked immunosorbent assay (ELISA). As shown in Table 5, purification of cHA using type I resin was able to decrease percent HMWA from 27% to 0.9% in the first cycle, and further decrease percent HMWA to 0.6% in the second cycle. Furthermore, the type I resin accommodated a greater loading challenge than the type II resin, while still producing sufficient monomer yield. Finally, purification of cHA using type I resin is able to decrease the amount of Protein A impurity. Table 5, HMWA clearance and antibody monomer yields using type I resin Sample % HMWA % monomer yield Protein A (ppm) cHA load 27.0 N / A 169 cHA peak (Run #1) 0.9 86.6 LOCK* cHA peak (Run #2) 0.6 86, 6 BLOCK *BLOCK = below the quantification limit of 1 ng / ml Example 3: Purification of cHA from an antibacterial preparation anti-CD22 body and an anti-Abeta antibody preparation using type I resin The cHA purification process described in example 2 was also able to sufficiently remove HMWA from both an anti-CD22 and an anti-Abeta example 2, with the exception that the purified antibody monomer was gradient eluted in 15 volumes of column elution buffer (3 mM sodium phosphate, 1.5 M NaCl, pH 7.2). As shown in Table 6, purification of cHA using type I resin was able to decrease HMWA in the 0.5% anti-CD22 antibody preparation and in the anti-Abeta antibody preparation to below the limit of detection. Furthermore, the cHA purification step was able to remove Protein A contaminants from the antibody preparation. Table 6. HMWA clearance and monomer yields from antibodies using type I resin in the purification of anti-CD22 and anti-Abeta antibody preparations Sample % HMWA % monomer yield Protein A (ppm) anti-CD22 load 3.7 N / A 30 anti-CD22 peak 0.5 87 BLOCK* anti-Abeta load 1.87 N / A 40 anti-Abeta peak 0.0 89 BLOCK *BLOCK = below the quantification limit of 1 ng / ml Example 4: Purification of cHA in flow-through mode from an anti-GDF-8 antibody The Myo-29 antibody was also successfully purified using a flow-through cHA purification protocol. A 1.6 x 20 cm Vantage column (Millipore, Billerica, MA) was packed in 200 mM dibasic sodium phosphate, pH 9.0 using macro-prep type II ceramic hydroxyapatite, 40 pM of the resin the size of particle (BioRad, Hercules, CA). The column was equilibrated with 3 column volumes of equilibration buffer 1 (300 mM sodium phosphate, 1.0 M NaCl, pH 6.8) and 3 column volumes of equilibration buffer 2 (350 mM NaCl, 5 mM sodium phosphate, pH 6.8). The Myo-29 antibody preparation was buffer exchanged in a loading buffer containing 350 mM NaCl, 5 mM sodium phosphate, pH 6.8 and then loaded onto the cHA column. The column was washed with wash buffer (350 mM NaCI, 5 mM sodium phosphate, pH 6.8), extracted with extraction buffer (300 mM sodium phosphate, 1.0 M NaCI, pH 6, 8), and then regenerated with regeneration buffer (500 mM potassium phosphate, 1.0 M NaOH, pH 13.3). All flow rates were maintained at 2.5 to 3 cm / min. Column effluents were analyzed by SEC-HPLC using an HPLC system. A summary of Myo-29 recovery and HMWA removal from The antibody preparation is shown in Table 7. The preparation initially contained 14.4% HMWA (load), which was reduced to 0.2% HMWA (flow through) using the cHA purification method of the invention. Table 7. Myo-29 recovery and HMWA removal Sample % recovery of Myo-29 % HMWA in sample load N / A 14.4 flow through 79.6 0.2 wash 12.2 1.9 post wash 1.5 5.6 extraction 10.3 84 ,3 Example 5: Purification of cHA in flow through mode of an anti-GDF-8 antibody using type I resin A 1.1 x 21 cm Vantage column (Millipore, Billerica, MA) was packed in 200 mM dibasic sodium phosphate, pH 9.0 using type I macro-prep ceramic hydroxyapatite, 40 µM resin from the particle size (BioRad, Hercules, CA). The column was equilibrated with 3 column volumes of equilibration buffer 1 (300 mM sodium phosphate, 1.0 M NaCl, pH 6.8) and 3 column volumes of equilibration buffer 2 (1.0 M NaCl, 3 mM Sodium Phosphate, pH 7.2). The Myo-29 antibody preparation was buffer exchanged in a loading buffer containing 1.0 M NaCl, 3 mM sodium phosphate, pH 7.2, and then loaded onto the cHA column in a loading challenge of 26mg / ml. The column was washed with wash buffer (1.0 M NaCl, 3 mM NaCl, pH 7.2), extracted with extraction buffer (300 mM NaCl, 1.0 M NaCl, pH 6.8), and then regenerated with 5 column volumes of regeneration buffer (500 mM potassium phosphate, 1.0 M NaOH, pH 13.3). Flow rates were kept less than 90 cm / h for loading and washing and less than 240 cm / h for the remainder of the purification process. Column effluents were analyzed by SEC-HPLC using an HPLC system. The antibody preparation initially contained 27.2% HMWA (load), which was reduced to 6.1% HMWA (flow through). Beyond Furthermore, the antibody monomer recovery was 72%. Example 6: Purification of cHA from an anti-CD22 antibody preparation HA ceramic chromatography purification has also been shown to be usable in purifying an anti-CD22 antibody preparation. A 1.6 x 20 cm Vantage column (Millipore, Billerica, MA) was packed in 200 mM dibasic sodium phosphate at pH 9.0 using type II macro-prep ceramic hydroxyapatite, 40 pM resin size particle board (BioRad, Hercules, CA). The column was equilibrated with 3 column volumes of equilibration buffer 1 (300 mM sodium phosphate, 1.0 M NaCl, pH 6.8) and 3 column volumes of equilibration buffer 2 (50 mM Na-Cl, 5 mM sodium phosphate, pH 6.8). An anti-CD22 antibody preparation was buffer exchanged in 50 mM NaCI, 5 mM sodium phosphate, pH 6.8 and then loaded onto the cHA column. The column was washed with 3 column volumes of wash buffer (50 mM NaCl, 5 mM sodium phosphate, pH 6.8) and then gradient eluted with 5 mM sodium phosphate, 1.0 M NaCl , pH 6.8 in 15 column volumes. The column was extracted with extraction buffer (300 mM sodium phosphate, 1.0 M NaCl, pH 6.8) and then regenerated with regeneration buffer (500 mM potassium phosphate, 1.0 M NaOH) . All flow rates were maintained at 2.5 to 3 cm / min. Column effluents were analyzed by SEC-HPLC using an HPLC system. As shown in Figure 3, the cHA purification was successful in removing HMWA from the anti-CD22 antibody preparation. The percent HMWA in the batch was 1.7%, while the percent HMWA in the cHA eluent was 0.0%. Example 7: Purification of cHA from an anti-Abeta antibody HA ceramic chromatography purification has also been shown to be usable in purifying an anti-Abeta antibody preparation. A 1.6 x 20 cm Vantage column (Millipore) was packed in 200 mM dibasic sodium phosphate at pH 9.0 using type II macro-prep ceramic hydroxyapatite, 40 µM particle size resin (BioRad, Hercules , CA). The column was equilibrated with 3 column volumes of equilibration buffer 1 (300 mM sodium phosphate, 1.0 M NaCl, pH 6.8) and 5 column volumes of equilibration buffer 2 (50 mM NaCl, 5 mM sodium phosphate, pH 6, 8). An anti-Abeta antibody preparation was buffer exchanged in 50 mM NaCl, 5 mM sodium phosphate, pH 6.8 and then loaded onto the column. The column was washed with 5 column volumes of wash buffer (50 mM NaCl, 5 mM sodium phosphate, pH 6.8) and then gradient eluted with 5 mM sodium phosphate, 1.0 M NaCl , pH 6.8 in 15 column volumes. The column was extracted with extraction buffer (300 mM sodium phosphate, 1.0 M NaCl, pH 6.8) and then regenerated with regeneration buffer (500 mM potassium phosphate, 1.0 M NaOH) . All flow rates are maintained at 2.5 to 3 cm / min. Column effluents were analyzed by SEC-HPLC using an HPLC system. As shown in Figure 4, the cHA purification was successful in removing HMWA from the anti-Abeta antibody preparation. The percent HMWA in the batch was 2.6%, while the percent HMWA in the cHA eluent was 0.0%. Example 8: Evaluation of Purified Myo-29 Antibody Activity Anti-GDF-8 antibody purified according to the method described in Example 1, Myo-29, was tested for binding activity using a competitive enzyme-linked immunosorbent assay (ELISA). Competitive ELISA can be adapted to test the binding activity of other purified antibodies. See, for example, Antibodies: A Laboratory Manual, Harlow and Land (eds.), 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. A GDF-8 receptor, ActRlip.Fc (2 pg / ml), was adsorbed onto a 96-well microtiter plate in a volume of 100 µl / well. The plate was then incubated at 2 to 8°C overnight. The plate was washed twice with wash buffer (50 mM Tris-HCl, pH 8.0, 0.05% Tween 20) and blocked with 4% bovine serum albumin (BSA) solution to minimize the non-specific binding. The plate was incubated at room temperature for 1.5 to 3.0 hours and washed twice with wash buffer (50 mM Tris-HCl, pH 8.0, 0.05% Tween 20). The Myo-29 antibody reference standard was serially diluted 4 times in test diluent (0.5% BSA, 137 mM NaCl, 2.7 mM KCl) resulting in a total of 8 standard points. The test samples contained Myo-29 antibody fractions purified by a Protein A chromatography step as well as fractions purified after an additional cHA purification step. These test samples were also serially diluted 2 times in test diluent to yield 8 points that fall within the standard curve range. Standards and test samples were added to the appropriate test wells at 50 µl / well. The biotinylated competitor, biotin-labeled GDF-8 (50 ng / ml) was then added to each well at 50 µl / well. The plate was incubated overnight on a plate shaker at room temperature. The plate was washed four times with wash buffer (50 mM Tris-HCl, pH 8.0, 0.05% Tween 20) and biotin-bound labeled GDF-8 was detected with the 100 µl / well derivative of streptavidin-conjugated horseradish peroxidase (1:5,000; Southern Biotech, Birmingham, Alabama). The plate was incubated 50 to 70 minutes on the plate shaker at room temperature, and then developed by adding 100 µl / well of 3,3',5,5'-tetramethylbenzidine (BioFX, Owings Mills, MD). The absorbance for each well is determined at 450 nm with an ELISA plate reader (Molecular Devices, Sunnyvale, CA). The amount of active Myo-29 in the test sample is indirectly proportional to the signal generated in the test. The reaction was stopped by adding 100 pl / well 0.18 M H 2 ONLY 4 . A concentration of Myo-29 capable of binding biotinylated GDF-8 in the test samples was interpolated from the standard curve which was configured using a 4-parameter logistic equation. The bioactivity value (% active protein) of a test sample was then computed by dividing the active protein concentration (determined by ELISA) by the total protein concentration (determined by A 2 so) and multiplying this ratio by 100. If the same sample was purified in separate batches, averages of the bioactivity value were calculated. Bioactivity values ​​are recorded in Table 8. Table 8. Anti-GDF-8 antibody binding activity (% active protein) Sample Post-Protein A Purification Post-chHA Purification 1 88 108 2 86 104 3 94 108 As shown in Table 8, Myo-29 retains its ability ability to bind GDF-8 after being purified by the procedure described in example 1. The binding activity of purified Myo-29 is somewhat lower than that in the peak eluent fractions from the Protein A purification. However, the binding activity exceeds the reference antibody Myo-29 after an additional cHA purification step as described in example 1. Example 9 - Purification of cHA in binding / flow-through mode in combination with an anti-GDF-8 antibody Purification by ceramic HA chromatography in a combination binding / flow-through mode has also been shown to be usable in the purification of an anti-GDF-8 antibody preparation. The experiment detailed below was performed on an ÀKTA FPLC system (General Electric). A 1.1 x 21 cm Vantage column (Millipore) was packed in 200 mM dibasic sodium phosphate at pH 9.0 using hydroxyapatite ceramic macro-prep type II, 40 pM particle size resin (BioRad , Hercules, CA). The column was equilibrated with 3 column volumes of equilibration buffer 1 (300 mM sodium phosphate, 1.0 M NaCl, pH 6.8) and 5 column volumes of equilibration buffer 2 (50 mM NaCl, 2.0 mM sodium phosphate, 100 mM arginine, 100 mM HEPES, pH 7.3). An anti-GDF-8 antibody preparation was loaded onto the column with a loading challenge of 20 mg / ml. The column was washed with 2 equilibration buffer, extracted with extraction buffer (300 mM sodium phosphate, 1.0 M NaCl, pH 6.8), and then regenerated with regeneration buffer (500 mM potassium phosphate , 1.0 M NaOH, pH 13.3). The flow rate for loading and washing was 1.5 cm / min. Flow rates for the remainder of the purification process were maintained at 2.5 to 3.0 cm / min. Column effluents were analyzed by SEC-HPLC using an HPLC system. The results demonstrated that the operation of the cHA resin type II in the binding / flow-through mode in combination is effective in removing HMWA from antibody preparations. The preparation initially contained 27% HMWA (load), which was reduced to 1.1% HMWA (flow through) using the cHA purification method of the invention. Example 10 - Purification of cHA in binding / flow-through mode in combination with an anti-GDF-8 antibody using type I resin The procedure described in example 9 was repeated using cHA type I resin in place of cHA type II resin. Buffer conditions were identical to those used in Example 9, with the exception of Equilibration Buffer 2, which was composed of 5.0 mM sodium phosphate, 100 mM NaCl, 120 mM arginine, 20 mM HEPES, in a pH of 7.3. As shown in Table 9, operating the type I cHA resin in binding / flow through mode in combination is effective for removing HMWA from antibody preparations while maintaining antibody monomer yields. Furthermore, the binding / flow-through mode in combination is effective to remove Protein A impurities. Finally, the type I cHA resin allowed for an increased loading challenge of 55 mg / ml. Table 9 - HMWA clearance and antibody monomer yields from type I cHA resin operated in binding / flow-through mode in combination Sample % HMWA % monomer yield Protein A (ppm) Cycle load 27.0 NA 236 1 peak 0.9 75 3.3 Cycle load 27.2 NA — 2 peak 0.7 78 — Example 11 - High throughput screening of cHA buffer conditions A high throughput screening was performed to optimize the buffer conditions used to purify a Myo-029 antibody preparation using a type I cHA resin. fact of sodium, sodium chloride, arginine and Myo-029 on the type I cHA resin and examined the extent of binding of Myo-029 and high molecular weight aggregate (HMWA) to the resin. Type I cHA resin (50 µl) was added to each well of a 96-well filter plate. Each well, labeled A1, A2...H11, H12 in tables 10 to 12, was equilibrated in equilibration buffer made from 20 mM HEPES, pH 7.2 and a unique combination of phosphate (table 10), sodium chloride (table 11), and arginine (table 12). Table 10. Phosphate levels in each cavity 1 2 3 4 5 6 7 8 9 10 11 12 to 1mm 1mm 1mm 6mm 5mm 4mm 1mm 1mm 1mm 3mm 1mm 1mm 1mm 1mm 1mm 6mm 5mm 1mm 1mm 1mm 3mm 2mm 1mm 1mm 1mm 6mm 5mm 4mm 1mm 1mm 1mm 3mm 2mm 1mm 1mm 1mm 1mm 6mm 5mm 4mm 1mm 1mm 1mm 1mm 3mm 2mm 1mm and 2mm 4mm 8mm 16mm 2mm 3mm 4mm 6mm 8mm f 2mm 4mm 8mm 10mm 4mm 4mm 4mm 5mm 6mm 8mm 8mm 3mm 3mm 3mm 3mm 2mm 2mm 2mm 5mM 6mM 8mM H 2mM 3mM 4mM 8mM 10mM 16mM 2mM 3mM 4mM 5mM 6mM 8mM Table 11. NaCI levels in each well 1 2 3 4 5 6 7 8 9 10 11 12 A 25m M 25m M 25m M 25m M 25m M 25m M 750m M 625m M 500m M 500m M 400m M 300m M B 25m M 25m M 25m M 25m M 25m M 25m M M 625m m 500m m 500m m 400m m 300m m c 50m m 100m m 200m m 50m m 100m m 1,250 mm 1,750 mm 2,500 mm 600m m 800m m 1,000 mm d 50m m 100m m 50m m 100m m 200m m 1,250 mm 1,750 mM 2,500 mM 600m M 800m M 1,000 mM E 25m M 25m M 25m M 25m M 25m M 25m M 750m M 625m M 500m M 500m M 400m M 300m M F 25m M 25m M 25m M 25m2 M 5m5 M2 M 2 M 500m m 500m m 400m m 300m m g 50m m 100m m 200m m 100m m 200m m 1,250 mm 1,750 mm 2,500 mm 600m m 800m m 1 1,000 mm h 50m m 100m m 200m m 100m m 200m m 1,250 mm 1,750 mm 2,500 mM 600 mM 800 mM 1,000 mM Table 12. Arginine levels in each well 1 2 3 4 5 6 7 8 9 10 11 12 A 350 mM 300 mM 200 mM 12m M 12m M 12m M 12m M 12m M 12m M 75m M 50m M 25m M B 450 mM 500 mM 600 mM 40m M 120 mM 200 mM M 120 mM 200 mM 125 mM 150 mM 300 mM C 350 mM 300 mM 200 mM 12m M 12m M 12m M 12m M 12m M 12m M 75m M 50m M 25m M D 450 mM 500 mM 600 mM 40m M 120 mM 40m M 20mM 120 mM 200 mM 125 mM 150 mM 300 mM E 350 mM 300 mM 200 mM 12m M 12m M 12m M 12m M 12m M 12m M 75m M 50m M 25m M F 450 mM 500 mM 600 mM 40m M 120 mM 20 mM 120 mM 200 mM 125 mM 150 mM 300 mM G 350 mM 300 mM 200 mM 12m M 12m M 12m M 12m M 12m M 12m M 75m M 50m M 25m M H 450 mM 500 mM 600 mM 40m M 120 m1M 20mM 20mM mM 200 mM 125 mM 150 mM 300 mM After the single equilibration buffer was added to each well, a mixture of Myo-029 and aggregate was added to each well. The aggregate level in the load challenge was 25%. Buffer constituents were maintained at the same level as during equilibration. The material was stirred for 20 minutes, allowing it to reach equilibrium. The supernatant was removed from each well of the filter plate. Another stage of addition of the antibody preparation was done, and the plate shaken, and the supernatant removed. Up to seven stages were performed. Protein that had not bound at each stage was analyzed to determine overall protein concentration (by absorbance at A280 nM). The amount of monomer and aggregate was measured by size exclusion HPLC. Any decrease in aggregate indicates a condition conducive to purification. Tables 13 and 14 show the percentage of aggregate and monomer, per cavity, in a pool of the first four stages. Table 13. Aggregate level (%) 1 2 3 4 5 6 7 8 9 10 11 12 A 8% 6% 3% 4% 7% 16% 3% 3% 3% 5% 3% 3% B 12% 15% 17% 3% 3% 5% 3% 4% 4% 6% 5% 5% 1 2 3 4 5 6 7 8 9 10 11 12 c 7% 6% 3% 4% 4% 3% 5% 4% 4% 6% 6% 4% D 13% 14% 14% 6% 4% 7% 5% 6% 4% 8% 8% 7% E 11% 10% 6% 4% 19% 11% 5% 6% 7% 9% 8% 6% F 15% 19% 20% 4% 3% 10% 6% 8% 10% 10% 10% 17% G 10% 9% 6% 6% 3% 7% 8% 14% 17% 10% 16% 22% H 16% 18% 20% 4% 5% 17% 9% 15% 17% 12% 17% 18% Table 14. Monomer recovery (%) 1 2 3 4 5 6 7 8 9 10 11 12 A 51% 44% 24% 3% 3% 2% 43% 34% 25% 53% 32% 11% B 64% 70% 78% 4% 24% 43% 42% 40% 38% 57% 42% 44% C 49% 43% 27% 4% 6% 23% 60% 62% 62% 58% 60% 48% D 65% 68% 75% 5% 35% 54% 61% 64% 64% 63% 63% 62% E 61% 58% 44% 3% 3% 5% 58% 62% 64% 66% 65% 63% F 70% 76% 88% 5% 33% 63% 59% 63% 68% 67% 68% 74% G 57% 57% 50% 4% 7% 65% 72% 82% 85% 70% 80% 83% H 71% 75% 85% 5% 48% 75% 69% 82% 88% 71% 80% 87% High throughput screening was able to qualitatively predict monomer recovery and HMWA removal in a column purification scheme. For example, C8 well conditions (20 mM HEPES, 1 mM phosphate, 1750 mM NaCl, and 12 mM arginine, pH 7.2) were tested on a column packed with type I cHA resin Aggregate levels were reduced to 2.5% and monomer yield was 72% after column purification. D5 well conditions (20 mM HEPES, 5 mM phosphate, 100 mM NaCl, and 120 mM arginine, pH 7.2) were also tested on a column packed with cHA-1 resin. Aggregate levels were reduced to 0.7% and monomer yield was 73% after column purification. Finally, conditions equivalent to the C3 cavity (100 mM HEPES, 1 mM phosphate, 120 mM NaCl, and 200 mM arginine, pH 7.2, where the high level of HEPES contributes to ionic strength similarly to NaCl) , were tested on a column packed with cHA-1 resin. The levels of aggregates were reduced to 4%, with a monomer recovery of 69%, after column purification. These results demonstrate that high throughput screening underestimates column performance, as measured by both monomer recovery and HMWA removal. However, a high throughput screen is capable of qualitatively predicting both yield and purity. All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. To the extent that publications and patents or patent applications incorporated by reference contradict the description contained in this report, the report is intended to super-grant and / or take precedence over this contradictory material. All numbers expressing amounts of ingredients, reaction conditions and so on, used in the report and claims, are to be understood to be modified in all cases by the term "about". Consequently, unless otherwise stated, the numerical parameters set forth in the report and appended claims are approximations that may vary depending on the desired properties sought to be obtained by the present invention. Lastly, and not in an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter must be constructed in light of the number of significant digits and common rounding approaches. Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only and are not meant to limit the same in any way. It is intended that the report and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

Claims

CLAIMS 1. A method for purifying at least one antibody from an antibody preparation containing high molecular weight aggregates, comprising: (a) contact the antibody preparation with a hydroxyapatite resin and elute the purified antibody from the resin with at least one elution buffer comprising 1 to 20 mM sodium phosphate and 0.2 to 2.5 M NaCl; or (b) Contact a hydroxyapatite resin with an antibody preparation in loading buffer comprising 1 to 20 mM sodium phosphate and 0.2 to 2.5 M NaCl and allow the purified antibody to flow through the column.

2. Method according to claim 1, wherein the purified antibody contains less than 5% of high molecular weight aggregates.

3. Method according to claim 1, wherein the purified antibody contains less than 1% of high molecular weight aggregates.

4. Method according to claim 1, wherein the elution buffer or loading buffer has a pH of 6.4 to 7.

6.

5. Method according to claim 4, wherein the elution buffer or loading buffer contains 3 mM or 5 mM sodium phosphate.

6. Method according to claim 4, wherein the elution buffer or loading buffer contains 1 M or 0.35 M of NaCl.

7. Method according to claim 4, wherein the elution buffer or loading buffer has a pH of 6.8 or 7.

2.

8. Method according to claim 1, wherein the antibody is IgG, IgA, IgD, IgE, or IgM antibody.

9. Method according to claim 1, wherein the antibody is monoclonal, polyclonal, chimeric, humanized, or a fragment thereof.

10. Method according to claim 1, wherein the antibody is an anti-IL-21, anti-GDF-8, anti-Abeta, anti-CD22, anti-Lewis Y, anti-IL-13, or anti-IL-22 receptor antibody.

11. Method according to claim 1, wherein the antibody has a basic pI.

12. Method according to claim 1, wherein the resin is type I or type II ceramic hydroxyapatite.

13. Method according to claim 12, wherein the resin is type I ceramic hydroxyapatite.

14. Method according to claim 1, wherein the purified antibody contains less than 300 ppm of Protein A.

15. Method for purifying at least one antibody from an antibody preparation containing high molecular weight aggregates comprising subjecting the antibody preparation to (a) Protein A affinity chromatography, (b) ion exchange chromatography, and (c) hydroxyapatite chromatography, (i) wherein the purified antibody is eluted from the hydroxyapatite resin using an elution buffer comprising 1 to 20 mM sodium phosphate and 0.2 to 2.5 M NaCl; or (ii) contact a hydroxyapatite resin with an antibody preparation in loading buffer comprising 1 to 20 mM sodium phosphate and 0.2 to 2.5 M NaCl and allow the purified antibody to flow through the column.

16. Method according to claim 15, wherein Protein A affinity chromatography is performed first and hydroxyapatite chromatography is performed last.

17. Method according to claim 15, wherein the purified antibody contains less than 5% of high molecular weight aggregates.

18. Method according to claim 15, wherein the purified antibody contains less than 1% of high molecular weight aggregates.

19. Method according to claim 15, wherein the elution buffer or loading buffer has a pH of 6.4 to 7.

6.

20. Method according to claim 19, wherein the buffer The elution or loading buffer contains 3 mM or 5 mM sodium phosphate.

21. Method according to claim 19, wherein the elution buffer or loading buffer contains 1 M or 0.35 M of NaCl.

22. Method according to claim 19, wherein the elution buffer or loading buffer has a pH of 6.8 or 7.

2.

23. Method according to claim 15, wherein the antibody is an IgG, IgA, IgD, IgE, or IgM antibody.

24. Method according to claim 15, wherein the antibody is monoclonal, polyclonal, chimeric, humanized, or a fragment thereof.

25. Method according to claim 15, wherein the antibody is an anti-IL-21, anti-GDF-8, anti-Abeta, anti-CD22, anti-Lewis Y, anti-IL-13, or anti-IL-22 receptor antibody.

26. Method according to claim 15, wherein the antibody has a basic pI.

27. Method according to claim 15, wherein the resin is type I or type II ceramic hydroxyapatite.

28. Method according to claim 27, wherein the resin is type I ceramic hydroxyapatite.

29. Method according to claim 15, wherein ion-exchange chromatography is anion-exchange chromatography.

30. Method according to claim 15, wherein the purified antibody contains less than 300 ppm of Protein A.

31. Method for purifying at least one antibody from an antibody preparation containing high molecular weight aggregates, comprising: (a) contact the preparation with a Protein A support; (b) allow the antibody to adsorb onto the Protein A support; (c) wash the Protein A support and adsorbed antibody with at least one Protein A washing buffer; (d) elute the adsorbed antibody with at least one Protein A elution buffer; (e) contact the Protein A eluent with an exchange support ionic; (f) allow the antibody to flow through the ion exchange support; (g) wash the ion exchange support with at least one ion exchange washing buffer, (h) contact the ion exchange flow with a hydroxyapatite resin; (i) allow the antibody to adsorb onto the resin; (j) wash the resin with at least one hydroxyapatite washing buffer; and (k) elute the purified antibody from the resin with at least one hydroxyapatite elution buffer comprising 1 to 20 mM sodium phosphate and 0.2 to 2.5 M NaCl.

32. Method according to claim 31, wherein the purified antibody contains less than 5% of high molecular weight aggregates.

33. Method according to claim 31, wherein the purified antibody contains less than 1% of high molecular weight aggregates.

34. Method according to claim 31, wherein at least one hydroxyapatite elution buffer has a pH of 6.4 to 7.

6.

35. Method according to claim 34, wherein at least one hydroxyapatite elution buffer contains 3 mM or 5 mM sodium phosphate.

36. Method according to claim 34, wherein at least one hydroxyapatite elution buffer contains 0.35 M or 1.0 M NaCl.

37. Method according to claim 34, wherein at least one hydroxyapatite elution buffer has a pH of 6.8 or 7.

2.

38. Method according to claim 31, wherein the antibody is an IgG, IgA, IgD, IgE, or IgM antibody.

39. Method according to claim 31, wherein the anticor po is monoclonal, polyclonal, chimeric, humanized, or a fragment thereof.

40. Method according to claim 31, wherein the antibody is an anti-IL-21, anti-GDF-8, anti-Abeta, anti-CD22, anti-Lewis Y, anti-IL-13, or anti-IL-22 receptor antibody.

41. Method according to claim 31, wherein the antibody has a basic pI.

42. Method according to claim 31, wherein the resin is type I or type II ceramic hydroxyapatite.

43. Method according to claim 42, wherein the resin is type I ceramic hydroxyapatite.

44. Method according to claim 31, wherein ion-exchange chromatography is anion-exchange chromatography.

45. Method according to claim 31, further comprising filtering the ion exchange throughflow prior to application to the hydroxyapatite resin, thereby reducing viral contaminants.

46. ​​Method according to claim 31, further comprising subjecting the hydroxyapatite eluent to at least one of ultrafiltration or diafiltration.

47. Method according to claim 31, wherein the purified antibody contains less than 300 ppm of Protein A.

48. Method for purifying at least one antibody from an antibody preparation containing high molecular weight aggregates, comprising: (a) contact the preparation with a Protein A support; (b) allow the antibody to adsorb onto the Protein A support; (c) wash the Protein A support and adsorbed antibody with at least one Protein A washing buffer; (d) elute the adsorbed antibody with at least one Protein A elution buffer; (e) contact the Protein A eluent with an exchange support ionic; (f) allow the antibody to flow through the ion exchange support; (g) wash the ion exchange support with at least one ion exchange washing buffer; (h) replace the ion exchange throughflow in a loading buffer comprising 1 to 20 mM sodium phosphate and 0.2 to 2.5 M NaCl; (i) contact the ion exchange flow with a hydroxyapatite resin; (j) allow the antibody to flow through the hydroxyapatite resin; (k) wash the hydroxyapatite resin with at least one hydroxyapatite washing buffer.

49. Method according to claim 48, wherein the purified antibody contains less than 5% of high molecular weight aggregates.

50. Method according to claim 48, wherein the purified antibody contains less than 1% of high molecular weight aggregates.

51. Method according to claim 48, wherein the loading buffer has a pH of 6.4 to 7.

6.

52. Method according to claim 51, wherein the loading buffer contains 5 mM sodium phosphate.

53. Method according to claim 51, wherein the loading buffer contains 350 mM of NaCl.

54. Method according to claim 51, wherein the loading buffer has a pH of 6.

8.

55. Method according to claim 48, wherein the antibody is an IgG, IgA, IgD, IgE, or IgM antibody.

56. Method according to claim 48, wherein the antibody is monoclonal, polyclonal, chimeric, humanized, or a fragment thereof.

57. Method according to claim 48, wherein the antibody is an anti-IL-21, anti-GDF-8, anti-Abeta, anti-CD22 receptor antibody, anti-Lewis Y, anti-IL-13, or anti-IL-22.

58. Method according to claim 48, wherein the antibody has a basic urn pl.

59. Method according to claim 48, wherein the resin is type I or type II ceramic hydroxyapatite.

60. Method according to claim 59, wherein the resin is type I ceramic hydroxyapatite.

61. Method according to claim 48, wherein ion-exchange chromatography is anion-exchange chromatography.

62. Method according to claim 48, further comprising filtering the ion exchange throughflow prior to application to the hydroxyapatite resin, thereby reducing viral contaminants.

63. Method according to claim 48, further comprising subjecting the hydroxyapatite flow to at least one of ultrafiltration or diafiltration.

64. Method according to claim 48, wherein the purified antibody contains less than 300 ppm of Protein A.

65. Method for purifying at least one antibody from an antibody preparation containing high molecular weight aggregates, comprising: (a) equilibrate the hydroxyapatite resin with at least one equilibration buffer comprising 1 to 20 mM sodium phosphate and 0.01 to 2.0 M NaCl; (b) contact the antibody preparation with said hydroxyapatite resin under conditions that allow the binding of both monomer and high molecular weight aggregates; (c) allow high molecular weight aggregates to bind more firmly than antibody monomers and, as the charge continues, allow high molecular weight aggregates to displace the bound monomer; and (d) collect the displaced monomer mentioned.

66. Method according to claim 65, wherein the anticor Purified powder contains less than 5% high molecular weight aggregates.

67. Method according to claim 65, wherein the purified antibody contains less than 1% of high molecular weight aggregates.

68. Method according to claim 65, wherein the equilibrium buffer has a pH of 6.2 to 8.

0.

69. Method according to claim 68, wherein the equilibrium buffer contains 2 mM or 5 mM sodium phosphate.

70. Method according to claim 68, wherein the equilibrium buffer contains 50 mM or 100 mM of NaCl.

71. Method according to claim 68, wherein the equilibrium buffer has a pH of 7.

3.

72. Method according to claim 68, wherein the equilibrium buffer further comprises up to 200 mM of arginine or HEPES.

73. Method according to claim 68, wherein the equilibrium buffer further comprises 100 mM or 120 mM arginine and 100 mM or 20 mM HEPES.

74. Method according to claim 65, wherein the antibody is an IgG, IgA, IgD, IgE, or IgM.

75. Method according to claim 65, wherein the antibody is monoclonal, polyclonal, chimeric, humanized, or a fragment thereof.

76. Method according to claim 65, wherein the antibody is an anti-IL-21, anti-GDF-8, anti-Abeta, anti-CD22, anti-Lewis Y, anti-IL-13, or anti-IL-22 receptor antibody.

77. Method according to claim 65, wherein the antibody has a basic pI.

78. Method according to claim 65, wherein the resin is type I or type II ceramic hydroxyapatite.

79. Method according to claim 78, wherein the resin is type I ceramic hydroxyapatite.

80. Method according to claim 65, wherein the purified antibody contains less than 300 ppm of Protein A.

81. Method for the purification of at least one antibody from an antibody preparation containing high molecular weight aggregates comprising subjecting the antibody preparation to (a) Protein A affinity chromatography, (b) ion exchange chromatography, and (c) hydroxyapatite chromatography, wherein (i) the hydroxyapatite resin is equilibrated with at least one equilibration buffer comprising 1 to 20 mM sodium phosphate and 0.01 to 2.0 M NaCl; (ii) the antibody preparation is contacted with said hydroxyapatite resin under conditions that allow the binding of both the monomer and high molecular weight aggregates; (iii) the high molecular weight aggregates are allowed to bind more tightly than the antibody monomers and, as loading continues, the high molecular weight aggregates are allowed to displace the bound monomer; and (iv) the displaced monomers mentioned are collected.

82. Method according to claim 81, wherein Protein A affinity chromatography is performed first and hydroxyapatite chromatography is performed last.

83. Method according to claim 81, wherein the purified antibody contains less than 5% of high molecular weight aggregates.

84. Method according to claim 81, wherein the purified antibody contains less than 1% of high molecular weight aggregates.

85. Method according to claim 81, wherein the equilibrium buffer has a pH of 6.2 to 8.

0.

86. Method according to claim 85, wherein the equilibrium buffer contains 2 mM or 5 mM sodium phosphate.

87. Method according to claim 85, wherein the buffer The equilibrium solution contains 100 mM or 50 M of NaCl.

88. Method according to claim 85, wherein the elution buffer or loading buffer has a pH of 7.

3.

89. Method according to claim 85, wherein the equilibrium buffer further comprises up to 200 mM of arginine or HEPES.

90. Method according to claim 85, wherein the equilibrium buffer further comprises 100 mM or 120 mM arginine and 100 mM or 20 mM HEPES.

91. Method according to claim 81, wherein the antibody is IgG, IgA, IgD, IgE, or IgM.

92. Method according to claim 81, wherein the antibody is monoclonal, polyclonal, chimeric, humanized, or a fragment thereof.

93. Method according to claim 81, wherein the antibody is an anti-IL-21, anti-GDF-8, anti-Abeta, anti-CD22, anti-Lewis Y, anti-IL-13, or anti-IL-22 receptor antibody.

94. Method according to claim 81, wherein the antibody has a basic pI.

95. Method according to claim 81, wherein the resin is type I or type II ceramic hydroxyapatite.

96. Method according to claim 95, wherein the resin is type I ceramic hydroxyapatite.

97. Method according to claim 81, wherein ion-exchange chromatography is anion-exchange chromatography.

98. Method according to claim 81, wherein the purified antibody contains less than 300 ppm of Protein A.

99. Method for purifying at least one antibody from an antibody preparation containing high molecular weight aggregates comprising (a) contact the preparation with a Protein A support; (b) allow the antibody to adsorb onto the Protein A support; (c) wash the Protein A support and adsorbed antibody with minus one Protein A wash buffer; (d) elute the adsorbed antibody with at least one Protein A elution buffer; (e) contact the Protein A eluent with an ion exchange support; (f) allow the antibody to flow through the ion exchange support; (g) wash the ion exchange support with at least one ion exchange washing buffer; (h) equilibrate the hydroxyapatite resin with at least one equilibration buffer comprising 1 to 20 mM sodium phosphate and 0.01 to 2.0 M NaCl; (i) contacting the ion exchange flow with said hydroxyapatite resin under conditions that allow the binding of both the monomer and high molecular weight aggregates; (j) allow high molecular weight aggregates to bind more firmly than antibody monomers, and as loading continues, allow high molecular weight aggregates to displace the bound monomer; (k) wash the hydroxyapatite resin with at least one hydroxyapatite washing buffer; and (l) collect the displaced monomer mentioned.

100. Method according to claim 99, wherein the purified antibody contains less than 5% of high molecular weight aggregates.

101. Method according to claim 99, wherein the purified antibody contains less than 1% of high molecular weight aggregates.

102. Method according to claim 99, wherein the equilibrium buffer has a pH of 6.2 to 8.

0.

103. Method according to claim 102, wherein the equilibrium buffer contains 2 mM or 5 mM sodium phosphate.

104. Method according to claim 102, wherein the tam- Equilibrium bread contains 100 mM or 50 M of NaCl.

105. Method according to claim 102, wherein the equilibrium buffer has a pH of 7.

3.

106. Method according to claim 102, wherein the equilibrium buffer further comprises up to 200 mM of arginine or HEPES.

107. Method according to claim 102, wherein the equilibrium buffer further comprises 100 mM or 120 mM arginine and 100 mM or 20 mM HEPES.

108. Method according to claim 99, wherein the antibody is an IgG, IgA, IgD, IgE, or IgM.

109. Method according to claim 99, wherein the antibody is monoclonal, polyclonal, chimeric, humanized, or a fragment thereof.

110. Method according to claim 99, wherein the antibody is an anti-IL-21, anti-GDF-8, anti-Abeta, anti-CD22, anti-Lewis Y, anti-IL-13, or anti-IL-22 receptor antibody.

111. Method according to claim 99, wherein the antibody has a basic pI.

112. Method according to claim 99, wherein the resin is type I or type II ceramic hydroxyapatite.

113. Method according to claim 112, wherein the resin is type I ceramic hydroxyapatite.

114. Method according to claim 99, wherein ion-exchange chromatography is anion-exchange chromatography.

115. Method according to claim 99, further comprising filtering the ion exchange throughflow prior to application to the hydroxyapatite resin, thereby reducing viral contaminants.

116. Method according to claim 99, further comprising subjecting the hydroxyapatite eluent to at least one of ultrafiltration or diafiltration.

117. Method according to claim 99, wherein the purified antibody contains less than 300 ppm of Protein A.