MXPA00005708A - Novel tgf-beta protein purification methods - Google Patents

Abstract

Method of purifying TGF-b superfamily proteins, including osteogenic proteins, such as bone morphogenetic proteins (BMPs), are disclosed.

Description

NEW METHODS FOR THE PURIFICATION OF PROTEIN TGF-β Field of the Invention This invention relates generally to new methods for the recovery and purification of proteins from the transforming growth factor-ß (TGF-β) superfamily of proteins. More particularly, this invention relates to novel methods of purifying such proteins, including bone morphogenetic proteins (BMPs).

BACKGROUND OF THE INVENTION The superfamily of transforming growth factor ß proteins, including BMPs and other osteogenic proteins, can be produced in cultures (eg, yeast, E. coli and mammalian cells) transformed with an expression vector containing the corresponding DNA. The cloning and expression of the transforming growth factor ß superfamily of proteins, including bone morphogenetic proteins (also called osteogenic proteins), has previously been described. See, for example, U.S. Patent Nos. 4,877,864; 5,108,922; 5,013,649; 5,116,738; 5,106,748; 5,187,076; 5,141, 905; 5,688,678; 5,661, 007; 5,637,480; 5,639,638; 5,658,882 and 5,635,372. Other compositions that may also be useful include Vgr-2 and any of the growth and differentiation factors (GDFs), including those described in PCT publications WO94 / 15965; WO94 / 15949; WO95 / 01801; WO95 / 01802; W094 / 21681; W094 / 15966 and others. The BIP, described in WO94 / 01557, may also be useful in the present invention; and MP52, described in PCT publication WO93 / 16099. Descriptions of all of the mentioned publications are incorporated herein by reference. The use of properly transformed host cells allows the recombinant production of high levels of protein. For proteins that are secreted from the host cell, purification of the protein of interest generally involves the isolation and purification of the culture medium of the host cell. Typically, the culture medium contains selected nutrients (e.g., vitamins, amino acids, co-factors, minerals) and may contain additional growth factors / supplements, including insulin and possibly additional exogenous proteins. In addition, the conditioned medium frequently contains not only the secreted protein of interest, but also significant amounts of additional secreted host cell and other host cell substances (e.g., nucleic acids, membrane vesicles). Thus, even when expressed at high levels, the product of interest may represent only a minority of all the proteins present in the conditioned medium. Not unexpectedly, the proteins secreted by transformed host cells have characteristics quite similar to those of the product of interest (eg, charge, molecule size, amino acid composition), significantly complicating the process used for purification. Certain purification conditions that are effective in preventing denaturation of the product of interest are not effective in distinguishing minor differences between the secreted proteins, thus making it extremely difficult to separate the product of interest from all other proteins present in the host cell. In addition to the secreted host cell proteins that are not desired as described above, the conditioned medium may also contain products derived from the gene expressed in heterologous form and which codes for the product of interest. These are not desirable for the final drug substance and include, for example, product forms lacking certain post-translational modifications such as glycosylation, sulfation, gamma carboxylation, or other modifications potentially necessary for biological activity (such as processing of precursor forms). further, proteolytically degraded forms of the product of interest may be present in the conditioned medium and also require to be removed during purification, but which closely resemble the product of interest. Unfortunately, most approaches, such as ion exchange chromatography, hydrophobic interaction chromatography, and size exclusion chromatography, do not provide the degree of resolution necessary to distinguish the product of interest from the undesired forms of the product. To take full advantage of the minor differences between the desired product and the contaminants (for example, small differences in charge, small differences in the size of the molecule), the use of strong denaturizers is often required. Such denaturing agents, however, can lead to loss of biological activity, expression of neo-antigenic sites, and can potentially improve the chemical decomposition of selected post-translational modifications. Typically, researchers have used combinations of traditional chromatographic techniques to purify the desired products. Often, such techniques are insufficient for the purification of a product at the level of purity and consistency that is desired for a human therapeutic product. Researchers have tried to overcome this difficulty by using affinity chromatography where a protein of interest is linked to an immobilized ligand with which it acts specifically. Following an appropriate wash, the desired product can be eluted by breaking the ligand-protein interaction, often resulting in a significantly purer eluate. However, in the instance of separation of a desired product from modified forms present in the conditioned medium, the single-step affinity chromatography techniques are frequently ineffective, and must be used in conjunction with other affinity resin techniques. and / or traditional separation. Unfortunately, the use of multiple stages to achieve higher resolution can also result in unacceptably low yields. Still high resolution affinity chromatography steps (e.g., immunoaffinity purification using immobilized monoclonal antibody) may not provide sufficient resolution of the desired product of the other components present in the culture medium due to common sites of interaction. For example, where an epitope that is present in the product of interest is also present in a degraded form in the proteolytic form of the product or a precursor form of the desired product, both will compete for the same site. In addition, to separate the product of interest from between molecules with similar properties (eg, modified forms of the expressed gene) it is also important to separate the desired product from the components present in the conditioned medium with which it specifically interacts. Where the protein of interest is positively charged, it will tend to bind to any negatively charged and present molecules, thus making it very difficult to purify the protein by traditional methods. For example, certain proteins, once expressed and secreted, actually "cling" to the host cell and remain recalcitrantly associated with the host cell, making purification virtually impossible without concomitant denaturation. Accordingly, there continues to exist in the current art a need for protein purification methods that effectively overcome all these difficulties.

Summary of the Invention The methods of the present invention are directed to protein purification, which comprises the steps of applying cell culture medium to a heparin or heparin-like resin, eluting with salt to displace the protein of interest, application of the first eluate to a hydrophobic interaction resin, elution with solvents with decreased or non-polar ionic strength to decrease hydrophobic interactions, and subsequent and optionally applying the second eluate to an anion exchange resin, used in the non-adsorbent mode, and used in tandem with a cation exchange resin and subjected to salt elution. Also, optionally, this third eluate is diafiltered and / or concentrated using, for example, a spiral wound membrane cartridge or other suitable device.

More specifically, conditioned medium containing cell culture is filtered through a filter and loaded into a column of chromatography Cellufine Sulfate. Heparin or heparin-like resins include those resins that have a negatively charged group such as heparin, sulfated cellulose esters, sulphylpropyl (SP), carboxyl, and carboxymethyl and include Matrex Cellufine Sulfate, Heparin Sepharose, Heparin Toyopearl, Carboxy Sulfon, Fractogel EMD-SO3 and Fractogel-EMD COO, being the preferred Matrex Cellufine Sulfate. The column is washed and subsequently eluted to collect, for example, BMP. A suitable first wash comprises a saline solution such as sodium chloride, potassium chloride, sodium sulfate, sodium phosphate, or potassium phosphate, and optionally, may contain a suitable regulating agent. Suitable concentration ranges are those which are effective for washing without eluting BMP and include, for example, 5 mM to 600 mM salt, and preferably is 50 mM Tris, 500 mM sodium chloride. The first eluent comprises 50 mM Tris, 0.5 M NaCl, 0.5 M arginine; suitable concentration ranges are those which are effective in eluting BMP, including for example a solution containing a buffer at a pH of 8.0, such as Tris, in the range of 5 to 100 mM, preferably about 50 mM, such a salt as NaCl in the range of 200 to 1000 mM, preferably 500 mM, and arginine in the range of 0 to 1000 mM, preferably 500 mM. The first eluate is applied to a column of Butyl Sepharose (Butyl Sepharose) which is washed with an appropriate second wash and which comprises a saline solution such as sodium chloride, ammonium sulfate, potassium chloride, sodium sulfate, phosphate sodium or potassium phosphate and, optionally, may contain a suitable regulatory agent. Suitable concentration ranges are those which are effective for washing the column without eluting BMP, and include, for example, 750 mM to 1,250 mM salt, and preferably is 50 mM Tris, 1000 mM sodium chloride. The second eluent is one that is sufficient to elute the protein of interest, for example, one comprising 50 mM Tris, 0.5 M arginine, 20% propylene glycol; suitable concentration ranges are those which are effective in eluting BMP, and include, for example, a solution containing a regulatory agent at a pH of about 7.0, such as Tris or its equivalent, in the concentration range of 5 to 100 mM , preferably about 50 mM, arginine or its equivalent in the range of 250 mM to 1000 mM, preferably about 500 mM, and a non-polar solvent, such as propylene glycol or its equivalent, in the range of 10% to 50%, preferably approximately twenty%. The eluate of the Butyl Sepharose column (referred to herein as the second column) is optionally pumped through a DEAE anion exchange resin; the unbound flow is pumped into a Carboxy Sulfon cation exchange resin connected in tandem to the DEAE resin.

The columns of DEAE and Carboxy Sulfon are washed, disconnected, and subsequently the Carboxy Sulfon column is eluted with salt to collect the BMP i (referred to here as the third eluate). Suitable anion exchange resins include those resins having a positively charged group such as diethylaminoethane (DEAE), polyethyleneimine (PEI) and quaternary aminoethane (QAE) and include Q-Sepharose Fast Flow, DEAE-Sepharose Fast Flow, POROS-Q , Fractogel-TMAE. Fractogel-DMAE, QAE-Toyopearl and DEAE-Toyopearl, the preferred resin being DEAE-Toyopearl (Tosohaas). Suitable cation exchange resins include those that possess a negatively charged group, such as heparin, sulfated cellulose esters, sulphylpropyl (SP), carboxyl, and carboxymethyl and include Matrex Cellufine Sulfate, SP-Sepharose Fast Flow, Mono S, Resource-S, Source S, Carboxy Sulfon, Fractogel EMD-SO3 and Fractogel-EMD COO, with Carboxy Sulfon being preferred. A third suitable wash comprises a saline solution such as sodium chloride, potassium chloride, sodium sulfate or ammonium sulfate, and may contain a suitable regulating agent and, optionally, arginine. Suitable concentration ranges are those which are effective in washing without eluting BMP, and include, for example, salt from 0mM to 250mM and, preferably, 50mM potassium phosphate, 250mM arginine. The third eluent comprises 50 mM potassium phosphate, 400 mM NaCl and 500 mM arginine; suitable concentration ranges are those which are effective for eluting BMP, including, for example, a solution containing a regulating agent at a pH of about 7.5, such as potassium phosphate, in the concentration range of 5 to 100 mM, preferably about 50 mM, a salt such as NaCl in the range of 200 mM to 1000 mM, preferably 400 mM or higher, and arginine in the range of 0 to 1000 mM, preferably 500 mM. Optionally, a spiral wound membrane cartridge is used to exchange the Carboxy Sulfon elution buffer within a suitable formulation buffer. Immediately after this diafiltration step, the BMP can be concentrated to > 2.4 absorbance units / mL (at 280 nm) using the spiral wound cartridge, if necessary. The concentrated BMP is then filtered, sampled, labeled and stored at -80 ° C. The effectiveness of the process in the purification of BMP is demonstrated by SDS-PAGE analysis. After the Cellufine Sulfate stage, BMP is clearly visible as two major bands in the 15-20 kd region in a reduced gel, although other contaminating proteins are still present. These contaminating proteins are extensively eliminated by Butyl Sepharose and are further eliminated by the DEAE / Carboxy Sulfon stage. BMP compositions purified and produced by the methods of the invention are also provided by the present invention.

Brief Description of the Drawings Figure 1 is a block diagram illustrating the steps of the purification process of the present invention.

Detailed Description of Preferred Modalities As used herein, the term "BMP" includes, but is not limited to, proteins from the transforming growth factor ß superfamily of proteins, including BMPs, isolated from a variety of tissue sources (including , but not limited to, epidermis, tendon, bone, cartilage, blood, fetal tissue, neuronal tissue, liver, ligament, muscle, pancreas, lung, heart, spleen, kidney), derived from transformed cell lines, and recombinantly produced proteins that they are isolated from the host cell culture medium or microbial sources (including, but not limited to fermentation broth, E. Coli lysate, yeast lysate and the like). As used herein, the terms "heparin" resin and "heparin-like" resin are used interchangeably and include, but are not limited to, resins containing an immobilized and negatively charged moiety such as heparin, sulfated cellulose esters , sulphylpropyl (SP), carboxyl, and carboxymethyl and include Fractogel EMD-SO3, Carboxy Sulfon, Fractogel-EMD-COO, Heparin Sepharose, Matrex Cellufine Sulfate and equivalents thereof, Matrex Cellufine Sulfate being currently the most preferred. One skilled in the art will readily appreciate that the "first wash" can be with any saline solution and include, for example, sodium chloride, potassium chloride, sodium sulfate, sodium phosphate or potassium phosphate, and can be regulated in appropriate form. Typically, concentrations range from low (5 mM salt) to high (600 mM salt), with 500 mM sodium chloride currently being preferred. As used herein, the term "first eluent" includes, but is not limited to, solutions composed of a regulatory agent (e.g., Tris) at a concentration of about 50 mM, salt (e.g., NaCl) at a concentration that it is sufficient for the elution of the resin (for example, approximately 500 mM), at approximately pH 8.0, and approximately 500 mM arginine; suitable concentration ranges are those which are effective in the elution of BMP, including for example a solution containing a buffer at a pH of about 8.0 such as Tris, in the range of 5 to 100 mM, preferably about 50 mM, a salt such as NaCl in the range of 200 to 1000 mM, preferably 500 mM, and arginine in the range of 0 to 1000 mM, preferably 500 mM.

As used herein, the term "similar to Butyl Sepharose" includes, but is not limited to, Butyl Sepharose 4B, Butyl Sepharose Fast Flow, Butyl-Toyopearl, and other hydrophobic interaction media including Phenyl Sepharose Fast Flow, Phenyl Toyopearl, Phenyl Fractogel, Butyl Fractogel and suitable equivalents, being currently the most preferred is Butyl Sepharose 4B. As used herein, the "second wash" can be any salt solution and includes, for example, sodium chloride, potassium chloride, sodium sulfate, ammonium sulfate, sodium phosphate or potassium phosphate, and can be suitably regulated . Typically, the concentrations range from low (750 mM salt) to high (1250 mM salt), with 50 mM Tris, 1000 mM sodium chloride currently being preferred. As used herein, the term "second agent" includes, but is not limited to solutions comprising a regulatory agent (e.g., Tris) at a concentration of about 5 to 100 mM, preferably 50 mM, a solubility-promoting agent ( for example, arginine, urea or other equivalent chaotropic agent), preferably arginine at a concentration range of about 250 mM to 1000 mM, preferably about 500 mM, and a non-polar solvent (eg, propylene glycol, ethylene glycol, glycerol and equivalents) at a sufficient concentration to break the interaction of BMP with Butyl Sepharose, at about pH 7.0, at a concentration range of about 10% to 50%, and preferably propylene glycol or its equivalent, at about 20%. As used in this process, the second eluent is preferably compatible with the subsequent process step, including pre-loading dilution or diafiltration within the next step. As used herein, the term "anion exchange resin" includes, but is not limited to, resins having a positively charged moiety (at neutral pH), such as diethylaminoethane (DEAE), polyethyleneimine (PEI) and quaternary aminoethane (QAE) ) and include, for example, Q-Sepharose Fast Flow (Pharmacia), DEAE-Sepharose Fast Flow, DEAE-Toyopearl, QAE-Toyopearl, POROS-Q, Fractogel-DMAE, Fractogel EEMD-TMAE, Matrex Cellufine DEAE and the like, being currently the preferred DEAE. As used herein, the term "cation exchange resin" includes, but is not limited to, resins having a negatively charged group such as heparin, sulfated cellulose esters, sulphylpropyl (SP), carboxyl and carboxymethyl, and include Matrex Cellufine Sulfate, SP-Sepharose Fast Flow, Mono S, Resource-S, Source S, Carboxy Sulfon, Fractogel EMD-SO3 and Fractogel-EMD COO, Carboxy Sulfon being currently preferred. As used herein, the term "third wash" may be any salt solution and includes, for example, sodium chloride, potassium chloride, sodium sulfate or ammonium sulfate, and may be suitably regulated (e.g., Tris, phosphate or sulfate) and optionally may contain arginine. Typically, salt concentrations range from low (0 mM salt) to high (250 mM salt), with 0 mM sodium chloride currently being preferred. The "third wash" that is currently preferred comprises phosphate buffer of approximately 50 mM and approximately 250 mM arginine. As used herein, the term "third eluent" includes, but is not limited to, solutions comprising a regulatory agent (e.g., Tris, phosphate or sulfate) at a concentration range of about 5 to 100 mM, preferably 50 mM, a solubility promoting agent (eg, arginine, urea or other chaotropic agent), preferably arginine at a concentration range of 0mM to 1000mM, preferably a concentration of about 500mM, and salt (eg, sodium chloride, potassium chloride) at a concentration sufficient to break the interaction of the BMP with the resin (for example in the range of 200 to 1000 mM, and preferably, approximately 400 mM or higher) Figure 1 provides an overview of the process. While the established order of the steps is that of the currently preferred embodiment, it will be appreciated by one skilled in the art that numerous variations and modifications are possible and that such modifications are of the present invention. For example, if the order is desired it can be re-configured and some stages can be omitted.

Genes encoding recombinant osteogenic proteins can be expressed in mammalian cell lines such as CHO (Chinese Hamster Ovary), COS, BHK, Balb / c 3T3, 293, and similar cell lines that are known in the art. Mammalian cells can be grown in any suitable medium that is known in the art. Suitable culture media for cells may contain amino acids, vitamins, inorganic salts, glucose, sodium pyruvate, thioctic acid, linoleic acid, hydrocortisone, putrescine, recombinant insulin, dextran sulfate and methotrexate. For example, a suitable medium is a cell culture medium, based on DME / F12 (50:50), supplemented with hydrocortisone, putrescine, recombinant insulin, dextran sulfate and methotrexate. Other means, such as a-MEM, Dulbecco MEM, RPMI 1640, may also be appropriate, with suitable complements, as necessary. (Freshney, R.l. Culture of Animal Cells, A Manual of Basic Technique, Alan R. Liss, Inc., New York (1983)). The cells can be grown in the presence or absence of a supplemented serum such as fetal bovine serum (FBS). The cells can be grown in suspension culture or monolayer and, additionally, can be grown in large-scale production batches. Incorporated as references are to the descriptions of WO 95/12664 (Gl 5217-PCT) related to methods and nutrient media useful for the adaptation of mammalian cell lines to culture densities, and of the pending patent application USSN 08 / 481,774 (Gl 5233) related to a cell culture medium for the production of dimeric proteins. Any cell capable of producing a protein of the TGF-β superfamily of proteins can be used in the method of the present invention. Transformed CHO cells are the preferred host cells that are used to produce an osteogenic protein, such as BMPs, particularly BMP-2, according to the present invention. The cell growth medium can be supplemented with FBS to enhance the growth of transformed CHO cells in the culture. If it is desired to add FBS, FBS concentrations as low as 0.5% (v / v) can be added. However, the addition of proteins of animal origin always presents the risk of harboring viruses and other harmful agents. The addition of FBS is not necessary for the practice of the present invention. Serum-free media are preferred for use in the production of recombinant osteogenic proteins according to the present invention. It is known that CHO cells release lipids, carbohydrates, nucleic acids and type C particles (similar to defective retrovirals) into the conditioned medium. Therefore, the ability of a purification process to eliminate and / or inactivate contaminants derived from the host and that may be present, is an important aspect of the process. CHO cell protein removal is confirmed by intentionally mixing radiolabelled CHO cell protein with loading material and quantifying the reduction at each step. A reduction factor for contaminants of the host cell protein at each purification stage, and a global one, are estimated by introducing protein concentrations of CHO cells that are higher than that expected during normal production. It has never been shown that type C particles present in CHO cells are infectious. However, the removal or inactivation of these particles during the purification process is still considered desirable. A virus consensus group is used to estimate the removal / inactivation potential of the purification steps. These viruses have been chosen to represent different types and ranges of size (eg, encapsulated / unencapsulated, containing DNA / containing RNA). It includes a murine retrovirus (Xenotropic Murine Leukemia virus) and others that are human pathogens for which CHO cells are permissive (Parafluenza 3 and Retrovirus 3). The Simian virus 40 is also included to investigate a more resistant virus. In these studies, the virus is introduced into the process in each chromatographic step and the removal / inactivation is determined. Most of the components of the medium are minor chemicals, including salts, amino acids and sugars that are not generally purified together with the protein of interest in the chromatographic columns and are generally not retained by a diafiltration membrane. However, large polymers, such as dextran sulfate and polyvinyl alcohol, which are useful additives for the medium, can interact specifically with the product of interest and often be purified together. These components must therefore be purified and eliminated from the protein of interest. For example, a dextran sulfate useful in the medium for producing recombinant proteins such as BMP has a molecular weight of 5,000 and a sulfur content of 18% (catalog Sigma # D-7037). Another dextran sulfate has a molecular weight of 500,000 and a sulfur content of 17% (Pharmacia). Incorporated herein as reference is found in U.S. Patent No. 5,516,654 (Gl 5180A), which refers to a method for the production of protein wherein dextran sulfate is added to the culture medium. According to the present invention, dextran sulfate can be added to the growth medium in a range of from about 1 to about 500 μg / mL, preferably about 200 μg / mL dextran sulfate. Methotrexate and other markers susceptible to selection, which are frequently used in a small volume in the early production of cell cultures, can be toxic. Its removal from the protein preparation is an important step (for example, the Matrex Cellufine Sulfate Stage that provides a 3,540 fold removal) of the purification process (See Table 8). The expression of an osteogenic protein, such as BMP-2, can be achieved by inserting a suitable gene into an expression vector, inserting this vector into a mammalian cell and selecting cells that express the osteogenic protein. For example, vectors encoding BMP-2 are described in U.S. Pat. No. 5,013,649, the content of which is incorporated herein by reference. The production of recombinant osteogenic protein, such as BMP-2, from mammalian cells expressing the BMP-2 gene can be measured by known methods such as radioactive labeling of cells with [35 S] -methionine and analyzing secreted proteins by polyacrylamide gel electrophoresis (PAGE) and autoradiography. For the measurement of BMP-2 expression from batch production, the amount of secreted functional BMP-2 can be quantified by bioassay or chromatographic methodologies. Any suitable assay can be employed, for example, the assay of the induction of alkaline phosphatase activity in a cell line responsive to BMP-2, or the assay of ectopic bone formation in a mammal such as the rat, rabbit, cat or dog. Any chromatographic assay that separates the product of interest from contaminants, including RP-HPLC, may be employed. Although the following examples describe the present invention being carried out with a cell line coding for BMP-2, these examples are not limiting. The present invention can also be used with similar results for other protein members of the transforming growth factor beta superfamily, particularly the bone morphogenetic proteins, including BMP-1 to BMP-15. The osteogenic proteins of the BMP family are a promising development in the bone and cartilage field. The BMP family of proteins includes BMPs 1 to 15, and proteins that are encoded by DNA sequences that anneal them under stringent conditions. The following examples illustrate the practice of the invention. These examples are for illustrative purposes only and are not intended in any way to limit the scope of the claimed invention. Example 1 describes the affinity stage of heparin / heparin-like; Example 2 refers to the step of hydrophobic interaction chromatography; Example 3 describes the purification steps using an anion-cation exchange tandem in Matrex Cellufine Sulfate; Example 4 refers to an additional purification using the diafiltration / concentration step; and Example 5 describes the purity test.

Example 1: Heparin Affinity Stage / Heparin-like Upon secretion of the host cell, the secreted protein is positively charged and binds strongly to the outer surface of the host cell that is negatively charged. A preferred way to break this binding to the outside of the host cell, without destroying the protein of interest and / or without breaking and subsequent leakage of the content of the host cell into the culture medium, is to add dextran sulfate to the culture medium. . Although this has the desired effect of breaking the interaction with the host cell, it creates another problem, which is the binding of the protein of interest to the dextran sulfate, which further complicates the purification process. Surprisingly, it has been found that heparin or heparin-like resin will effectively compete with dextran sulfate for binding to BMP so that the resin can be effectively used to separate BMP from dextran sulfate. Matrex Cellufine Sulfate (Amicon) is used as an affinity matrix for the purification of rhBMP-2 from the conditioned medium. This resin is composed of spheroidal cellulose beads activated with sulfate esters and which functions as an analogue of heparin for the purification of heparin binding proteins.

The resin efficiently competes with the dextran sulfate present in the culture medium to bind rhBMP-2 at a pH of 8.0. Elution of bound rhBMP-2 is achieved using 0.5 M L-arginine added to 50 mM Tris plus 0.5 M NaCl. A chromatographic column of Cellufine Sulfate, or equivalent, is the first step in the purification of BMP. The conditioned medium is filtered at a pH of 8.0 ± 0.2. The titled material is loaded into a balanced column of Cellufine Sulfate at a linear flow rate < 3 cm / min. The column is then washed (50 mM Tris, 0.5 M NaCl, pH 8.0) and can be eluted in reverse (50 mM Tris, 0.5 M NaCl, 0.5 M L-arginine HCl, pH 8.0). The eluate of the column is collected as a single elution peak, approximately one column volume. The appropriate operating parameters are described in Table 1.

Table 1 Example 2: Hydrophobic Interaction Chromatography Stage The heparin-like step effectively eliminates several species of protein contaminants, methotrexate, dextran sulfate and DNA. Still present in the first eluate, together with the protein of interest, are several forms of BMP in various stages of the proteolytic process, including the high molecular weight precursor forms (approximately 110 kD and 80 kD in non-reducing SDS-PAGE analysis) as well as the desired product (15 kD - 20 kD in reducing SDS-PAGE analysis). Because these diverse species differ only slightly in their hydrophobicity, it is difficult to purify these species from the others by conventional methods. Surprisingly, it has been found that Butyl Sepharose allows the separation of several forms of BMP-2 by an unconventional use of the displacement chromatography. The precursor species of BMP-2 competes with the desired product for binding to the resin. Due to the slight differences in hydrophobicity, the desired product, which is more hydrophobic, binds more lightly to the resin; this allows the other species to be removed in the column loading and washing. Butyl Sepharose 4B (Pharmacia) is a resin used for the purification of BMP in a hydrophobic interaction chromatography (HIC) mode. This resin is composed of butylamine coupled to Sepharose 4B activated with CNBr. Butyl Sepharose, or an equivalent column, is the second stage of the present invention. The proteins are linked to HIC resins in high conductivities, which promotes hydrophobic interactions. "High conductivity" is a minimum value of approximately 50 mS / cm. The elution is carried out by lowering the ionic strength and / or by the addition of non-polar solvents to reduce the hydrophobic interactions. The decreased ionic strength is defined as the decrease in conductivity to a value of below about 20 mS / cm. Non-polar solvents include propylene glycol, ethylene glycol, glycerol and their equivalents. The elution peak of Matrex Cellufine Sulfate is adjusted to pH 7.0 ± 0.2 and 1000 mM NaCl with 200 mM MES, 4000 mM NaCl, 500 mM L-arginine HCl, pH 6.8. The MES is 2- [N-morpholino] ethanesulfonic acid. This material is then loaded into the balanced Butyl Sepharose, or an equivalent column, at a flow rate of <; 30 cm / hr. The column is then washed (50 mM Tris, 1000 mM NaCl, 500 mM L-arginine-HCl, pH 7.0) and the bound rhBMP-2 is optionally eluted in reverse with 50 mM Tris, 20% propylene glycol, L-arginine -HCl 500 mM, pH 7.0. The eluate of the column is collected as a single peak of the elution, approximately 1.5 column volumes. The appropriate operating parameters of the column are detailed in Table 2. Table 2 Example 3: Tandem Anion-Cation Exchange The Butyl Sepharose stage shows the removal of contaminants from the CHO protein, DNA and BMP-related species, other than the defined product. It has been found that the inclusion of an anion exchange chromatography step results in increased removal of DNA and other non-proteinaceous contaminants. An additional step of cation exchange chromatography provides additional removal of the contaminants from the CHO protein and the concentration of the BMP. Toyopearl-DEAE (TosoHaas) is a weak anion exchange resin that binds to negatively charged proteins and other contaminants based on charge. It is used in the non-adsorbent mode for the purification of BMP, such that it does not bind to the resin, but the negatively charged contaminants are able to bind to the DEAE resin. Carboxy Sulfon (JT Baker, Inc.) is a functionalized silica-based matrix with mixed weak and strong cation exchange groups (eg, carboxy and sulfone groups) BMP binds to the Carboxy Sulfon via charge interactions and is eluted by the breaking of these interactions using regulators with increased ionic strength. The final chromatographic step in the purification of BMP is composed of these two ion exchange columns, or their equivalents, operated in tandem: Toyopearl-DEAE anion exchange column (or its equivalents followed by a Carboxy Sulfon cation exchange column ( or its equivalents.) The entry to the DEAE / Carboxy Sulfon system penetrates the DEAE column first, the DEAE column outlet is then installed towards the entrance of the Carboxy Sulfon column, the peak of the Butyl Sepharose is diluted with 9 -11 volumes of (50 mM potassium phosphate, 0.25 M L-arginine-HCl, pH 7.6) This solution is pumped through the DEAE column and onto the Carboxy Sulfon column at a rate of flow. < 300 cm / hr. The columns are then washed (50 mM potassium phosphate, L arginine-HCl 0.25 M, pH 7.6). The two columns are disconnected and the BMP attached to the Carboxy Sulfon column is eluted with 50 mM potassium phosphate, 0.5 M L-arginine-HCl, 0.4 M NaCl, pH 7.5. The eluate of the column is collected as a single elution peak, approximately 1 column volume. The appropriate operating parameters for these columns are detailed in Table 3.

Table 3 Example 4: Diafiltration / Concentration Stage This is an optional "finishing" step. Tangential flow filtration is used for concentration and regulator exchange of the protein solutions. Membrane cuts of specified molecular weight are used to retain high molecular weight components (e.g., BMPs) while low molecular weight components are removed (e.g., salts). By continuously adding new regulator to the retentate, at a speed similar to that the solution is passing through the filter, the components of the original regulator will gradually be diluted. In this continuous diafiltration mode, the replacement of 5 retentate volumes of a new regulator will effectively replace > 98% of the original regulator. Without the addition of a new regulator, a protein solution is concentrated without altering the composition of the regulator. The final stage in the purification process involves the exchange of the Carboxy Sulfon elution regulator within a formulation buffer suitable for BMP. This is followed by the concentration of the material a > 2.4 absorbance units (at 280 nm), as necessary. This step can be performed using a membrane of a cut of 10,000 molecular weight, spirally wound, or equivalent. The Carboxy Sulfone eluate is placed in a clean container, autoclaved and sealed. The material in the container is then pumped through the membrane, at a positive transmembrane pressure and recirculated back into the container. The positive transmembrane pressure forces the low molecular weight solutes across the membrane. The regulatory solution (L-histidine 0.01 M, L-arginine-HCL 0.5M or other suitable buffer) enters the vessel at approximately the same velocity as the material flows through the membrane, thereby diluting the elution buffer of Carboxy Sulfon. This process of dialfiltration is continued until at least 5 volumes of the buffer have flowed into the container. After the diafiltration has been completed, the valve that allows the regulatory solution to penetrate the container is closed. To concentrate the BMP, the system pump is restarted and the material is filtered until a concentration is obtained >; 2.4 absorbance units (at 280 nm). The BMP regulator is pumped out of the container through a 0.2 μm filter and into Teflon bottles of appropriate dimensions. The material is sampled, weighed, labeled and stored at -80 ° C. The appropriate operating parameters for this process are detailed in Table 4.

Table 4 Example 5: Purity Test Studies were carried out that investigated the effectiveness of the purification process referred to above to eliminate DNA, virus, dextran sulfate and methotrexate, with the results described in the following tables: Table 5 DNA clearance studies Table 6 MuLV Virus Removal / Inactivation Studies Table 7 Dextran Sulfate Removal Studies Table 8 Methotrexate Elimination Studies While the above examples are not limiting, it can be appreciated that the above process results in a high removal of potential contaminants, including DNA, virus, dextran sulfate and methotrexate, from the recombinant osteogenic protein produced from transfected CHO cells. Although the present method of the invention is exemplified by the purification of BMP produced in recombinant form from transformed host cells, the method is also applicable to the purification of BMP that occurs naturally within a cell and can be used to purify proteins. from a solution or from various tissue types, cell homogenates, cell culture supernatants, or isolated cell sub-fractions. Although the present invention has been described in terms of specific methods and compositions, it will be understood that variations and modifications will occur to those skilled in the art upon consideration of the present invention.

It is expected that numerous modifications and variations in the invention, as described in the above illustrative examples, occur to those skilled in the art and consequently, only the limitations appearing in the dependent claims should be placed on them. Accordingly, it is intended that the appended claims cover all equivalent variations that fall within the scope of the invention as claimed.

Claims (23)

1. Novelty of the Invention i 1.. A method for the purification of a protein of the TGF-β superfamily in a solution, which comprises the steps of: applying said solution to a heparin-like resin, eluting said heparin-like resin with a first eluent to form a first eluate , applying said first eluate to a resin similar to Butyl Sepharose,. eluting said resin similar to Butyl Sepharose with a second eluent to form a second eluate containing said protein of the TGF-β superfamily.
2. 2. The method of claim 1, further comprising the steps of: applying said second eluate to an ion exchange resin, and eluting said ion exchange resin with a third eluent to form a third eluate.
3. 3. The method of claim 2., wherein said ion exchange resin is a resin selected from the group consisting of a resin of - Anion exchange and a cationic intercarbonate resin. The method of claim 1, wherein said heparin-like resin has a negatively charged group, which is a member selected from the group consisting of heparin, sulfated cellulose esters, sulphylpropyl (SP), carboxyl and carboxymethyl. 5. The method of claim 4, wherein said heparin-like resin is Matrex Cellufine Sulfate. 6. The method of claim 1, wherein said first eluent comprises a salt. The method of claim 6, wherein said first eluent comprises 50 mM Tris, 0.5 M NaCl and 0.5 M L-arginine. The method of claim 1, wherein said Butyl Sepharose-like resin is a selected member from the group consisting of Butyl Sepharose 4B, Butyl Sepharose Fast Flow and Butyl-Toyopearl. 9. The method of claim 8, wherein said resin similar to Butyl Sepharose is Butyl Sepharose 4B. The method of claim 1, wherein said second eluent comprises a regulatory agent, a chaotropic agent and a non-polar solvent. The method of claim 10, wherein said second eluent is about 50 mM Tris, 500 mM arginine and 20% propylene glycol. The method of claim 3, wherein said anion exchange resin has a positively charged group, which is a member selected from the group consisting of diethylaminoethane (DEAE), polyethyleneimine (PEI) and quaternary aminoethane (QAE). The method of claim 12, wherein said anion exchange resin is DEAE. The method of claim 3, wherein said cation exchange resin has a negatively charged group, which is a member selected from the group consisting of heparin, sulfated cellulose esters, sulphylpropyl (SP), carboxyl and carboxymethyl. 15. The method of claim 14, wherein said cation exchange resin is Carboxy Sulfon. The method of claim 1, wherein said third eluent comprises a regulatory agent, a solubility promoting agent and a salt. The method of claim 16, wherein said third eluent comprises approximately 50 mM Tris, 500 mM arginine and 400 mM sodium chloride. 18. The method of claim 1, wherein said TGF-β superfamily protein is a BMP. The method of claim 18, wherein said BMP is BMP-2. 20. A BMP produced by the method of claim 18. 21. A method of purifying BMP-2 in a solution comprising the steps of: applying said solution to a Celiufine Sulfate resin, eluting said Cellufine Sulfate resin with a first eluent to form a first eluate, apply said first eluate to Butyl Sepharose 4B resin, elute said Butyl Sepharose 4B resin with a second eluent to form a second eluate containing BMP-2. The method of claim 21, further comprising the steps of: applying said second eluate containing said BMP-2 to a DEAE resin, and washing said DEAE resin to form a third wash, applying said wash to a Carboxy Sulfon resin, and eluting said Carboxy Sulfon resin with a third eluent to form a third eluate containing said BMP-2. 23. The method of claim 22, wherein: said first eluent comprises approximately 50 mM Tris, 500 mM NaCl and 500 mM arginine.; said second eluent comprises approximately 50 mM Tris, 500 mM arginine and 20% propylene glycol; said third wash comprises approximately 50 mM potassium phosphate and 250 mM arginine; and said third eluent comprises about 50 mM Tris, 500 mM arginine and 400 mM sodium chloride.