Purification of acidic proteins

Medicilon's protein scientists have been working on protein expression and purification for many years. We can start your project even you have nothing in hand but the name of your protein. In Medicilon's laboratories, protein purification is performed in scales from micrograms and milligrams. All Protein Purification Services start with the analysis of physico-chemical and biological properties of a target protein resulting in the development of tailored procedures for its extraction, purification and characterization.Email:marketing@medicilon.com.cn Web:www.medicilon.com

It is desirable to identify useful methods of purifying proteins that do not destroy, or significantly reduce, the biological activity of the protein. Contaminants must be removed from protein preparations, such as acidic protein preparations (e.g., immunoglobulin (Ig)-fusion protein preparations), before they can be used in diagnostic applications, therapeutic applications, applied cell biology, and functional studies. For instance, protein preparations, e.g., acidic protein preparations, often contain unwanted components (impurities), such as inactive and/or partially active species and high molecular weight aggregates (HMWA). Presence of inactive and/or partially active species is undesirable because these species have significantly lower binding capacity to the target compared to the active protein; thus, the presence of inactive and/or partially active species can reduce product efficacy. The formation of aggregates, e.g., HMWA, can adversely affect product safety by causing complement activation or anaphylaxis upon administration. Further, aggregate formation may hinder manufacturing processes by causing decreased product yield, peak broadening, and loss of activity.

The most common protein purification methods are predicated on differences in the size, charge, and solubility between the protein to be purified and 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 the separation of aggregated or multimeric species of proteins, e.g., IgG-containing proteins. Techniques such as ion exchange and hydrophobic interaction chromatography, for instance, may induce the formation of aggregates due to an increased protein concentration or the required changes in buffer concentration and/or pH during elution. Further, in several instances proteins show differences in isoelectric points that are too small to allow for their separation by ion-exchange chromatography. Tarditi, (1992) J. Immunol. Methods 599: 13-20. Size exclusion chromatography is cumbersome and results in the significant dilution of the product, which is a hindrance in large-scale, efficiency-based manufacturing processes. Leakage of ligands from affinity chromatography columns can also occur, which results in undesirable contamination of the eluted product. Steindl (2000) J. Immunol, Methods 235:61-69. Of interest, Applicants were unable to remove the inactive or partially active species using either ion exchange, e.g., anion exchange, or hydrophobic interaction chromatography.

Hydroxyapatite chromatography is a method of purifying proteins that utilizes an insoluble hydroxylated calcium phosphate [CaIo(PO4)O(OH)2], which forms both the matrix and ligand. Functional groups consist of pairs of positively- charged calcium ions (C-sites) and clusters of negatively charged phosphate groups (P-sites). The interactions between hydroxyapatite and proteins are complex and multi-mode. In one method of interaction, positively charged amino groups on proteins associate with the negatively charged P-sites, and protein carboxyl groups interact by coordination complexation to C-sites. Shepard (2000) J. of Chromatography 891 :93-98. Thus, acidic and basic proteins usually interact with cHA resin through different mechanisms: an acidic protein usually binds to C-sites via a coordination bond to carboxyl group, while a basic protein binds to P-sites through charge interaction with the amine group. Crystalline hydroxyapatite was the first type of hydroxyapatite used in chromatography, but it 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 crystalline hydroxyapatite. VoIa et al. (1993) BioTechniques 14:650-655. Chromatographic separation using cHA can be performed in several distinct modes, such as binding mode, flow- through mode, or a combination binding/flow-through mode. Hydroxyapatite chromatography has been used in the chromatographic separation of proteins, nucleic acids, as well as antibodies. However, in several instances, researchers have been unable to selectively elute antibodies from hydroxyapatite or found that hydroxyapatite chromatography did not result in a sufficiently pure product. Junbauer, (1989) J. Chromatography Al '6:257 '-268; Giovannini, (2000) Biotechnology and Bioengineering 73:522-529. A successful separation of antibodies and other basic proteins from impurities, such as HMWA, using cHA chromatography either in binding, flow-through, or combination binding/flow-through mode has been demonstrated in U.S. Publication No. 2005-0107594, incorporated herein in its entirety by reference. The present invention provides a novel method for removing product-related partially active and/or inactive species, as well as other impurities, such as HMWA, from acidic proteins, e.g.. lg-fusion proteins, using cHA chromatography techniques. The present invention provides methods of removing impurities, such as high molecular weight aggregates, inactive and/or partially active species, as well as other impurities from acidic protein preparations using hydroxyapatite chromatography. Thus, in one embodiment of the invention, the invention provides a method for purifying at least one acidic protein of interest from a protein preparation containing impurities, wherein the method comprises applying an equilibration buffer comprising a divalent metal cation to hydroxyapatite resin, contacting the hydroxyapatite resin with the protein preparation in a load buffer, washing the hydroxyapatite resin with a wash buffer comprising the divalent metal cation, and eluting at least one acidic protein from the hydroxyapatite resin with an elution buffer comprising phosphate. In some embodiments of the invention, the impurities are inactive and/or partially active species of the at least one acidic protein. Thus, another embodiment of the invention provides a method of purifying at least one acidic protein of interest from a protein preparation containing inactive and/or partially active species of the at least one acidic protein, comprising contacting a hydroxyapatite resin with the protein preparation; and eluting the at least one acidic protein of interest separately from the inactive and/or partially active species. Another embodiment of the invention provides a method of purifying at least one acidic protein of interest from a protein preparation containing inactive and/or partially active species of the protein of interest, comprising applying an equilibration buffer comprising a divalent metal cation to hydroxyapatite resin, contacting the hydroxyapatite resin with a protein preparation in a load buffer comprising the divalent metal cation, washing the hydroxyapatite resin with a wash buffer comprising the divalent metal cation, and eluting at least one acidic protein from the hydroxyapatite resin with an elution buffer comprising phosphate.

In at least some embodiments of the invention, the impurities are high molecular weight aggregates, and in at least one embodiment, the method of the invention results in at least about 60% reduction in high molecular weight aggregates. In other embodiments of the invention, the method results in at least about 90% reduction in high molecular weight aggregates. In additional embodiments of the invention, the impurities are protein A and/or host cell proteins.

In at least some embodiments, the equilibration buffer comprises from about 1 to about 20 niM of the divalent metal cation, the load buffer comprises about 1 to about 20 mM of the divalent metal cation, the wash buffer comprises about 1 to about 20 mM of the divalent metal cation, and the elution buffer comprises about 2 to about 50 mM phosphate. In another embodiment, the elution buffer comprises about 1 to about 100 mM phosphate, or about 5 to about 50 mM phosphate or about 5 to about 20 mM phosphate. In one embodiment, the equilibration buffer, the load buffer, and the wash buffer comprise about 5 mM of the divalent metal cation, and the elution buffer comprises about 6 mM phosphate. In one embodiment, the load buffer comprises a monovalent cation, such as NaCl or KCl. In some embodiments, the divalent metal cation is either CaCl2 or MgCl2, and in one embodiment, the divalent metal cation is CaCl2. In at least some embodiments, the phosphate is either sodium phosphate or potassium phosphate, and in one embodiment, the phosphate is sodium phosphate. In some embodiments of the invention, the equilibration buffer, the wash buffer and/or the elution buffer further comprise about 10 mM to about 200 mM HEPES, e.g., about 10 mM HEPES. In at least some other embodiments, the equilibration buffer, the wash buffer and/or the elution buffer have a pH of about 6.1 to about 8.1, e.g., a pH of about 7.2.

In some embodiments of the invention, the acidic protein purified is an immunoglobulin-fusion protein, e.g., receptor fusion protein. In one embodiment of the invention, the receptor fusion protein is ActRIIB-Fc. In another embodiment, the fusion protein is slL2 Ir-Fc.

In some embodiments of the invention, the hydroxyapatite resin is ceramic hydroxyapatite Type 1 or Type II. In at least some embodiments, the method further comprises, prior to the step of applying the equilibration buffer, the step of subjecting the protein preparation to a purification method selected from the group consisting of Protein A chromatography, affinity chromatography, hydrophobic interaction chromatography, immobilized metal affinity chrornatography, size exclusion chromatography, diafiltration, ultrafiltration, viral removal filtration, ion exchange chromatography, and combinations thereof.