WO2009027284A1 - Purification of osteopontin - Google Patents

Abstract

The invention relates to a process for the purification of osteopontin. The process according the invention employs three different purification principles: immobilized metal ion affinity chromatography (IMAC), hydrophobic interaction chromatography (HIC) and ion exchange chromatography (IEC). The invention relates further to method of manufacturing a pharmaceutical composition comprising osteopontin using the process for the purification of osteopontin.

Description

PURIFICATION OF OSTEOPONTIN

FIELD OF THE INVENTION

The present invention is in the field of protein purification. More specifically, it relates to a process for the purification of osteopontin (OPN), in particular recombinant osteopontin.

BACKGROUND OF THE INVENTION

Proteins have become commercially important as drugs that are also generally called "biologicals".

The large-scale, economic purification of proteins is increasingly an important problem for the biotechnology industry. Generally, proteins are produced by cell culture, using either mammalian or bacterial cell lines engineered to produce the protein of interest by insertion of a recombinant plasmid containing the gene for that protein. Since the cell lines used are living organisms, they must be fed with a complex growth medium, containing sugars, amino acids, and growth factors. The standards set by health authorities for proteins intended for human administration are very high. Many purification methods for proteins known in the art contain steps requiring the application e.g. of low or high pH, high salt concentration or other extreme conditions that may irreversibly jeopardize the biological activity of the protein to be purified and are therefore not suitable. Thus, separation of the desired protein from the mixture of compounds fed to the cells and from the by-products of the cells themselves to a purity sufficient for use as a human therapeutic indeed poses a formidable challenge. Historically, protein purification schemes have been predicated on differences in the molecular properties of size, charge and solubility between the protein to be purified and undesired protein contaminants. Protocols based on these parameters include size exclusion chromatography, ion exchange chromatography, differential precipitation and the like. Ion exchange chromatography involves the interaction of charged functional groups in the sample with ionic functional groups of opposite charge on an adsorbent surface in a buffer with low ionic strength. Elution is generally achieved by increasing the ionic strength (i.e. conductivity) of the buffer to compete with the solute for the charged sites of the ion exchange matrix. Changing the pH and thereby altering the charge of the solute is another way to achieve elution of the solute. The change in conductivity or pH may be gradual (gradient elution) or stepwise (step elution). Two general types of interaction are known: Anionic exchange chromatography mediated by negatively charged amino acid side chains (e.g. aspartic acid and glutamic acid) interacting with positively charged surfaces and cationic exchange chromatography mediated by positively charged amino acid residues (e.g. lysine and arginine) interacting with negatively charged surfaces. Anion exchangers can be classified as either weak or strong. The charge group on a weak anion exchanger is a weak base, which becomes de-protonated and, therefore, looses its charge at high pH. Diethylaminoethyl (DEAE)-cellulose is an example of a weak anion exchanger, where the amino group can be positively charged below pH~9 and gradually loses its charge at higher pH values. DEAE or diethyl-(2-hydroxy-propyl)aminoethyl (QAE) have chloride as counter ion, for instance.

A strong anion exchanger, on the other hand, contains a strong base, which remains positively charged throughout the pH range normally used for ion exchange chromatography (pH 1-14). Q-sepharose (Q stands for quaternary ammonium) is an example for a strong anion exchanger.

More recently hydrophobic interaction chromatography techniques have been developed to supplement the more traditional size exclusion and ion exchange chromatographic protocols.

Chromatographic systems having a hydrophobic stationary phase have also been widely employed in the purification of proteins. Included in this category are hydrophobic interaction chromatography (HIC) (Queiroz et al. 2001 ) and reversed phase liquid chromatography (RPLC). The physicochemical basis for separation by HIC and RPLC is the hydrophobic effect; proteins are separated on a hydrophobic stationary phase based on differences in hydrophobicity. In HIC, generally, sample molecules in a high salt buffer are loaded on the

HIC column. The salt in the buffer interacts with water molecules to reduce the solvation of the molecules in solution, thereby exposing hydrophobic regions in the sample molecules which are consequently adsorbed by the HIC column. The more hydrophobic the molecule, the less salt needed to promote binding. Usually, a decreasing salt gradient is used to elute samples from the column. As the ionic strength decreases, the exposure of the hydrophilic regions of the molecules increases and molecules elute from the column in order of increasing hydrophobicity. Sample elution may also be achieved by the addition of mild organic modifiers or detergents to the elution buffer. Commonly used hydrophobic ligands are phenyl-, butyl- or octyl- residues.

Hydrophobic charge-induction chromatography is a subset of HIC using resins carrying ligands such as 4-mercaptotheylpyridine derivatives (Burton and Harding 1998). Hydrophobic interaction chromatography is performed in aqueous solvent conditions and changes in ionic strength are used to elute the column. The protein typically binds in the native state via hydrophobic groups located on the surface of the protein, and the native state is retained during the elution conditions.

A further type of chromatography widely used for protein purification is called immobilized metal ion affinity chromatography (IMAC) (Porath 1992). IMAC consists of derivatizing a resin with iminodiacetic acid (IDA) and chelating metal ions to the

IDA-derivatized resin. The proteins are separated on the basis of their affinity for metal ions, which have been immobilized by chelation. Proteins bind to the metal ions through unoccupied coordination sites and are immobilized on the column. Since then, other ligands than IDA were used to chelate metal ions to resins.

Studies with serum proteins have shown IMAC to be an extremely specific and selective separation technique (Porath and ONn 1983). The adsorbent is prepared by coupling a metal chelate forming ligand, such as, iminodiacetic acid, to

Sepharose or Superose, which are bothcrosslinked, beaded-forms of agarose, and treating it with a solution of one or more divalent metal ions such as Zn²⁺, Cu²⁺,

Cd²⁺, Hg²⁺, Co²⁺, Ni²⁺, or Fe²⁺. The binding reaction is pH dependent and elution is carried out by reducing the pH and increasing the ionic strength of the buffer or by including EDTA in the buffer.

The actual mechanisms which give rise to the binding of proteins to free metal ions are not well understood and are dependent upon a number of factors, not the least of which is the conformation of the particular protein. However, when the metal ions are immobilized, at least three limiting factors come into play, namely reduced number of available coordination sites on the metal, restricted accessibility of the tethered metal to the binding sites on the protein, and, depending upon the characteristics of the resin, limited protein access to the immobilized metal ion. Thus, it is extremely difficult a priori to state which proteins will and which will not exhibit an affinity for immobilized metal ions.

Osteopontin (secreted phosphoprotein 1/SPP-1 , early T-lymphocyte activation protein-1/Eta-1 , 2ar, uropontin, OPN) is a secreted glycoprotein with pleiotropic developmental and immunological functions (Denhardt et al. 2001 ). It contains a polyaspartate sequence, an Arg-Gly-Asp (RGD) cell attachment motif, an N- and several O-glycosylation sites and 2 heparin-binding domains. There is also evidence for an intracellular form of OPN.

OPN is highly acidic and exhibits variation in glycosylation, phosphorylation and sulfatation that give rise to different functional forms. Human OPN is cleaved in vivo by thrombin between amino acids Arg¹⁶⁹-Ser¹⁷⁰ (Kon et al. 2000). A further conserved thrombin cleavage site is at residues Arg¹⁶⁰-Gly¹⁶¹ (Smith et al. 1996). It has been well documented that OPN is very susceptible to proteolytic degradation leading to various fragments and truncated forms of OPN (Maeda et al. 2001 ;Maeda et al. 2001 ). Human and rat OPN are cleaved by Matrix-Metalloproteinase-3 (Stomelysin-1 ) and Matrix-Metalloproteinase-7 (Matrilysin). Human OPN cleavage sites for Stomelysin-1 and Matrilysin are between residues Gly¹⁶⁶-Leu¹⁶⁷ (GIy¹⁵¹- Leu¹⁵² in rat OPN), Ala^-Tyr²⁰² and Asp²¹⁰-Leu²¹¹ (Agnihotri et al. 2001 ). Furthermore, OPN is a substrate for enterokinase (Giachelli et al. 1995). In humans, alternative splicing of the OPN mRNA has been documented to give rise to 3 isoforms of the OPN protein designated OPN-a, OPN-b and OPN-c. OPN-a corresponds to the protein encoded by all known exons of the OPN gene, while OPN-b is missing exon IV of the OPN gene (corresponding to residues 59-73 of the OPN-a protein) and OPN-c is missing exon III of the OPN gene (corresponding to residues 30-58 of the OPN-a protein) (He et al. 2006).

OPN appears to be involved in many physiological processes, in particular in bone metabolism, and has also been implicated in the pathophysiology of different disorders such as cancer (Rangaswami et al. 2006), autoimmune disorders such as arthritis, inflammatory bowel disease (Sato et al. 2005) or multiple sclerosis (Kadi et al. 2006), as well as other neurological disorders (Schroeter et al. 2006). WO 02/092122 relates to the use of osteopontin for the treatment of neurological disorders, such as disorders of the peripheral or central nervous system, in particular demyelinating disorders, such as multiple sclerosis, and also peripheral neuropathies, such as diabetic neuropathy or chemotherapy induced neuropathy. The multifarious functions of OPN are mediated via several types of integrin receptor heterodimers, including the α₄βi, α_vβ3, α_vβ5, α_vβi, α₈βi and α₉βi receptors, with high specificity and affinity. Interactions between OPN and its α_vβ3 and α_vβs integrin partners are mediated by the RGD motif of the OPN protein. OPN also interacts with the v6 and v7 splice isoforms of the CD44 transmembrane receptor.

OPN is thus of particular interest in many medical conditions. There is a demand for purified OPN, such as recombinant OPN for therapeutic but also research purposes.

The purification of rat OPN produced by a rat cell line (Senger et al. 1989), human OPN produced by a human kidney cell line (MH2) (Denhardt et al. 1995) or marrow stromal fibroblasts (Fedarko et al. 2000) has been performed. Purification of

OPN from human breast milk (Philip et al. 2001 ) and human urine (Min et al. 1998;

Shiraga et al. 1992) has also been described. Bovine OPN was purified from bovine milk (Sorensen and Petersen 1993). The work referred to supra relates to the purification of small quantities; i.e. in the range of milligrams, of OPN polypeptide.

However, no process for the purification of OPN, in particular recombinant OPN, suitable also for the production of large quantities; i.e. in the range of grams or even kilograms of OPN polypeptide, is currently available.

The methods for the purification of OPN known in the art are, however, not applicable for the purification of large quantities of OPN, e.g. for administration to humans. One reason for this is the lack of orthogonality, meaning that the different steps in the purification process do not employ separation mechanisms that are sufficiently distinct from one another. The purification schemes are mainly based on ion exchange resins (Philip et al. 2001 ) (Fedarko et al. 2000). The lack of orthogonality impacts on the efficacy to clear impurities and especially viruses. This is of particular relevance since for therapeutic applications in humans minimal viral clearance must be demonstrated for a purification process. Furthermore, impurities in a biological product frequently lead to an increased immune reaction against the protein (in this case OPN) after administration to a human.

Furthermore, the methods for purification of OPN described in the art are also not amenable to up-scaling for commercial production in order to produce gram or kilogram amounts of OPN polypeptide. The prior art methods comprise steps; like e.g. precipitation and gel filtration (Denhardt et al. 1995), (Senger et al. 1989), (Min et al. 1998). Precipitation at large scale is very expensive and poses environmental issues to an extent that the process would not be commercially viable due to the requirement for a certain equipment (e.g. tanks and centrifuges) as well as chemicals and salts. Gel filtration methods are complex and cannot be run at large scale due to excessive processing time, which increases the risk of degradation of the molecule of interest.

Consequently there is a need for an efficient purification process of OPN, in particular recombinant OPN. There is particular need for a purification process of

OPN, in particular recombinant OPN, which can provide OPN in sufficient amounts and with sufficient productivity such that the process is suitable for the isolation of

OPN for therapeutic applications in humans.

Clinical use and regulatory approval for therapeutic applications of OPN in humans requires well characterized and homogenous preparations of OPN, that also exhibit batch-to-batch consistency. In view of these requirements that are posed on proteins for human application there is consequently a particular need for a purification process of recombinant OPN, which process consistently provides homogenous OPN preparations with low amounts of truncated forms of OPN.

SUMMARY OF THE INVENTION

The present invention is based on the development of a purification process for osteopontin (OPN), such as such as recombinant OPN, from a solution.

One embodiment of the invention is a process for the purification of OPN, preferably recombinant OPN, from cell culture supernatant.

Another embodiment of the invention relates to a process for the purification of OPN, such as recombinant OPN, comprising: a) subjecting a solution containing OPN, such as recombinant OPN, to immobilized metal ion affinity chromatography (IMAC) to produce a first eluate; b) subjecting the first eluate to Hydrophobic interaction chromatography (HIC) to produce a second eluate; and c) subjecting the second eluate to ion exchange chromatography (IEC) to produce a third eluate. Another embodiment of the invention relates to a process for purification of OPN, such as recombinant OPN, wherein before the process for purification according to the above embodiment a capture step is performed wherein the cell culture supernatant is subjected to IEC to produce an eluate, wherein the IEC comprises using a column comprising quaternary ammonium, diethylaminoehtyl (DEAE) or triethylaminoethyl (TMAE), preferably quaternary ammonium or DEAE, more preferably DEAE.

Another embodiment of the invention is a method of manufacturing a pharmaceutical composition comprising OPN, such as recombinant OPN, comprising the steps of: a) purifying OPN according to the embodiment described herein; and b) formulating said purified OPN into a pharmaceutical composition, optionally with a pharmaceutically acceptable carrier or excipient.

Another embodiment of the invention is pharmaceutical composition comprising osteopontin obtainable by a method of manufacturing a pharmaceutical composition comprising OPN, such as recombinant OPN, according to the embodiments described herein.

Even another embodiment of the invention is a pharmaceutical composition comprising OPN, wherein the pharmaceutical composition is obtainable by a method of manufacturing according to the embodiments described herein, and wherein not more than 40%, more preferred not more than 35%, 30% or 25%, even more preferred not more than 22% and most preferred not more than 20% of the OPN is truncated OPN.

Even another embodiment of the invention is a pharmaceutical composition obtainable by a method of manufacturing according to the embodiments described herein, wherein at least 60%, or 65%, or 70%, or 75%, or 78% is full length osteopontin.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 : Flow diagram showing the OPN purification process.

Fig. 2: A RP-HLPC chromatogram of purified OPN is shown. DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a process for the purification of OPN, preferably recombinant OPN, more preferred human recombinant OPN, and most preferred human recombinant OPN from cell culture supernatant. A preferred cell culture supernatant is from CHO, PER.C6, NSO, BHK or Sp2/0 cell culture containing recombinant OPN, preferably recombinant OPN. The process according the invention employs three different purification principles and by that overcomes the limitations of the methods for purification of osteopontin known in the art.

In a first embodiment, the invention relates to a process for the purification of OPN, such as recombinant OPN, comprising: a) subjecting a solution containing OPN, such as recombinant OPN, to immobilized metal ion affinity chromatography (IMAC) to produce a first eluate; b) subjecting the first eluate to hydrophobic interaction chromatography (HIC) to produce a second eluate; and c) subjecting the second eluate to ion exchange chromatography (IEC) to produce a third eluate.

The immobilized metal ion affinity chromatography can be done using a column comprising any divalent metal ion, such as Zn²⁺, Cu²⁺, Cd²⁺, Hg²⁺, Co²⁺, Ni²⁺, or Fe²⁺, coupled to a resin through any suitable chelator known in the art. The divalent metal ion can be coupled to suitable material known in the art, including but not limited to crosslinked, beaded-forms of agarose (e.g. Sepharose or Superose), wherein the beads preferably have a diameter of 20 to 100 μm, more preferred a diameter of 30 to 90 μm; modified methacrylate polymers (e.g. tentacle, hydroxylated); silica; ceramic and styrene divinylbenzene. A preferred column comprises Ni²⁺ coupled to any of the above resins, preferably to crosslinked, beaded-forms of agarose, more preferred Sepharose. Particularly preferred is a column comprising Ni²⁺ coupled to a crosslinked, beaded-form of agarose of 90 μm diameter. In a second embodiment, the invention relates to a process for the purification of OPN, such as recombinant OPN, comprising: a) subjecting a solution containing OPN, such as recombinant OPN, to immobilized metal ion affinity chromatography (IMAC) on a resin comprising divalent metal ion to produce a first eluate; b) subjecting the first eluate to hydrophobic interaction chromatography (HIC) to produce a second eluate; and c) subjecting the second eluate to ion exchange chromatography (IEC) to produce a third eluate.

In a third embodiment, the invention relates to a process for the purification of OPN, such as recombinant OPN, comprising: a) subjecting a solution containing OPN, such as recombinant OPN, to immobilized metal ion affinity chromatography (IMAC) on a resin comprising Ni²⁺, Zn²⁺ or Cu²⁺ to produce a first eluate; b) subjecting the first eluate to hydrophobic interaction chromatography (HIC) to produce a second eluate; and c) subjecting the second eluate to ion exchange chromatography (IEC) to produce a third eluate.

In a fourth embodiment, the invention relates to a process for the purification of OPN, such as recombinant OPN, comprising: a) subjecting a solution containing OPN, such as recombinant OPN, to immobilized metal ion affinity chromatography (IMAC) on a resin of a crosslinked, beaded-form of agarose comprising Ni²⁺, Zn²⁺ or Cu²⁺ to produce a first eluate; b) subjecting the first eluate to hydrophobic interaction chromatography (HIC) to produce a second eluate; and c) subjecting the second eluate to ion exchange chromatography (IEC) to produce a third eluate.

Hydrophobic interaction chromatography (HIC) can be done using a column comprising hexyl, butyl or phenyl residues coupled to a resin of suitable material known in the art, including but not limited to crosslinked, beaded-forms of agarose (e.g. Sepharose or Superose), wherein the beads preferably have a diameter of 20 to 100 μm, more preferred a diameter of 30 to 90 μm; modified methacrylate polymers (e.g. tentacle, hydroxylated); silica; ceramic and styrene divinylbenzene. Preferred is a column comprising butyl or phenyl.

In a fifth embodiment, the invention relates to a process for the purification of OPN, such as recombinant OPN, according to the first, second, third or fourth embodiment of the invention, wherein the hydrophobic interaction chromatography (HIC) is performed on a resin comprising butyl or phenyl, preferably butyl, residues.

Ion exchange chromatography (IEC) can be done using a column comprising quaternary ammonium, diethylaminoehtyl (DEAE) or triethylaminoethyl (TMAE) coupled to a resin of suitable material known in the art, including but not limited to crosslinked, beaded-forms of agarose (e.g. Sepharose or Superose), wherein the beads preferably have a diameter of 20 to 100 μm, more preferred a diameter of 30 to 90 μm; modified methacrylate polymers (e.g. tentacle, hydroxylated); silica; ceramic and styrene divinylbenzene. Preferred is a column comprising quaternary ammonium or DEAE, more preferred is quaternary ammonium. In a sixth embodiment, the invention relates to a process for the purification of OPN, such as recombinant OPN, according to the first, second, third, fourth or fifth embodiment of the invention wherein the ion exchange chromatography (IEC) is performed on quaternary ammonium or DEAE, more preferred quaternary ammonium. It has been determined by the present inventors that OPN is more stable in a solution having a pH of equal or above 3.0. Whereas the purification may be done at pH at low pH such as 2.0, the novel process for purification preferably features washing and elution during the different purification steps at a pH equal or above 3.0. In a seventh embodiment, the invention relates to a process for the purification of OPN, such as recombinant OPN, according to the first, second, third, fourth, fifth or sixth embodiment of the invention, wherein the elution step during ion affinity chromatography (IMAC) is done in imidazol, or is done in a sodium phosphate, sodium acetate or sodium citrate buffer having a pH of 2.0 to 6.0, preferably 3.0 to 5.0, more preferred 3.5 to 4.5, and even more preferred 3.9 to 4.1 ; more preferred in a sodium phosphate buffer (50 mM) having a pH of 2.0 to 6.0, preferably 3.0 to 5.0, more preferred 3.5 to 4.5, and even more preferred 3.9 to 4.1. In an eighth embodiment, the invention relates to a process for the purification of OPN, such as recombinant OPN, according to the seventh embodiment wherein step (a) comprises i) washing with a sodium or kalium phosphate or TRIS buffer at a pH of 6.5 to 7.5; more preferred with a buffer containing sodium phosphate at a molarity of 5 to 50.5 mM, preferably 49.5 to 50.5 mM, and sodium chloride at a molarity of 272 to 278 mM, the buffer having a conductivity of 29-33 mS/cm and a pH of 6.5 to 7.5; and ii) eluting with an acetate, citrate or phosphate buffer at a pH of 2.0 to

6.0, preferably 3.0 to 5.0, more preferred 3.5 to 4.5, and even more preferred 3.9 to 4.1 ; the most preferred buffer contains sodium phosphate at a molarity of 5 to 50.5 mM, preferably 49.5 to 50.5 mM, the buffer having a conductivity of 3-4 mS/cm and a pH of 2.0 to 6.0, preferably 3.0 to 5.0, more preferred 3.5 to 4.5, and even more preferred 3.9 to 4.1 ; and wherein step (b) comprises i) washing with an ammonium, sodium or kalium sulphate or TRIS buffer at pH of 6.0 to 8.0, preferably 6.9 to 7.1 ; more preferred with a buffer containing sodium phosphate at a molarity of 5 to 50.5 mM, preferably 19.8 to 20.2 mM, and ammonium sulphate at a molarity of 990 to 1500 mM, more preferred 990 to 1010 mM, the buffer having a conductivity of 130-140 mS/cm and a pH of 6.0 to 8.0, more preferred 6.9 to 7.1 ; and ii) eluting with a sodium phosphate or TRIS buffer at a pH of 6.0 to 8.0, preferably 6.9 to 7.1 ; more preferred with a buffer containing sodium phosphate at a molarity of 5 to 20.2 mM, preferably 19.8 to 20.2 mM, the buffer having a conductivity of 2-3 mS/cm and a pH of 6.0 to 8.0, more preferred a pH of 6.9 to 7.1 ; and wherein step (c) comprises i) washing with a sodium or kalium phosphate or TRIS buffer at a pH of 6.9 to 7.1 ; more preferred with a buffer containing sodium phosphate at a molarity of 49.5 to 50.5 mM and sodium chloride at a molarity of 198 to 202 mM, the buffer having a conductivity of 23-27 mS/cm and a pH of 6.9 to 7.1 ; and ii) eluting with a sodium or kalium phospate or TRIS buffer at a pH of

6.9 to 7.1 ; more preferred with a buffer containing sodium phosphate at a molarity of 49.5 to 50.5 mM and sodium chloride at a molarity of 396 to 404 mM, the buffer having a conductivity of 39-45 mS/cm and a pH of 6.9 to 7.1.

In a ninth embodiment, the invention relates to a process for purification of OPN, such as recombinant OPN, wherein before the process for purification according to the first, second, third, fourth, fifth, sixth or eighth embodiment an IEC capture step is performed comprising subjecting cell culture supernatant, such as cell culture supernatant from mammalian cell culture, e.g. from CHO, PER.C6, NSO, BHK or Sp2/0 cell culture, to IEC to produce an eluate using a column comprising quaternary ammonium, diethylaminoehtyl (DEAE) or triethylaminoethyl (TMAE) coupled to a resin of suitable material known in the art, including but not limited to crosslinked, beaded-forms of agarose (e.g. Sepharose or Superose), wherein the beads preferably have a diameter of 20 to 100 μm, more preferred a diameter of 30 to 90 μm; modified methacrylate polymers (e.g. tentacle, hydroxylated); silica; ceramic and styrene divinylbenzene.

Preferably a column comprising quaternary ammonium or DEAE, more preferably DEAE, is used.

In a tenth embodiment, the invention relates to a process for purification of OPN, such as recombinant OPN, according to the ninth embodiment wherein the capture step comprises i) washing with a sodium or natrium phosphate or TRIS buffer; preferably with a buffer containing sodium phosphate at a molarity of 49.5 to 50.5 mM, the buffer having a conductivity of 1 1-15 mS/cm and a pH of 6.8 to 7.4, preferably a pH of 7.1 to 7.3 and ii) eluting with a buffer containing sodium phosphate at a molarity of 5 to 50.5 mM, preferably 49.5 to 50.5 mM, and sodium chloride at a molarity of 272 to 278 mM, the buffer having a conductivity of 29-33 mS/cm and a pH of 6.8 to 7.4, preferably a pH of 7.1 to 7.3. An eleventh embodiment of the invention is a method of manufacturing a pharmaceutical composition comprising OPN as active ingredient, such as recombinant OPN, comprising the steps of: a) purifying OPN according to the process of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth or tenth embodiment; and b) formulating said purified OPN into a pharmaceutical composition as active ingredient, optionally with a pharmaceutically acceptable carrier or excipient.

A twelfth embodiment of the invention is pharmaceutical composition comprising osteopontin as active ingredient, which pharmaceutical composition is obtainable by a method of manufacturing a pharmaceutical composition comprising OPN, such as recombinant OPN, according to the eleventh embodiment.

Even another embodiment of the invention is a pharmaceutical composition comprising osteopontin as active ingredient, wherein the pharmaceutical composition is obtainable by a method of manufacturing according to the ninth, tenth of eleventh embodiment, and wherein not more than 40%, more preferred not more than 35%, 30% or 25%, even more preferred not more than 22% and most preferred not more than 20% of the OPN is truncated OPN.

Even another embodiment of the invention is a pharmaceutical composition comprising osteopontin as active ingredient, wherein the pharmaceutical composition is obtainable by a method of manufacturing according to the ninth, tenth of eleventh embodiment, and wherein at least 60%, or 65%, or 70%, or 75%, or 78% is full length osteopontin.

Even another embodiment of the invention is a pharmaceutical composition comprising OPN as active ingredient, wherein not more than 40%, more preferred not more than 35%, 30% or 25%, even more preferred not more than 22% and most preferred not more than 20% of OPN is truncated OPN, and further comprising a pharmaceutically acceptable carrier, solvent or excipient.

Even another embodiment of the invention is a pharmaceutical composition comprising OPN as active ingredient, wherein at least 60%, or 65%, or 70%, or 75%, or 78% of OPN is full length OPN, and further comprising a pharmaceutically acceptable carrier, solvent or excipient. The term "purified" as it is used herein denotes that the indicated molecule is present in the substantial absence of other biological macromolecules, e.g., polynucleotides, proteins, and the like. The term contemplates preferably that full length OPN is present in a solution or composition, wherein (a) at least 80% by weight; preferably, at least 85% by weight; more preferably, at least 95% by weight; and, most preferably, at least 97% by weight of said solution or composition is OPN. Preferably said OPN comprises not more than 40%, more preferred not more than 35%, 30% or 25%, even more preferred not more than 22% and most preferred not more than 20% truncated OPN. Water, buffers, and other small molecules, especially molecules having a molecular weight of less than about one kDa, can be present.

The term "purification" refers to a process of obtaining a purified molecule.

The term "solution containing OPN", as used herein, refers to a liquid composition of matter comprising OPN at least partially in solution. The liquid composition may comprise substances other than OPN, wherein the substances are either in solution or not. A solution containing OPN may be any body fluid, including but not limited to blood, serum, urine, liquor, synovial fluid or milk of an animal, preferably a mammal and most preferred a human. One particular solution containing OPN is cell culture supernatant containing recombinant OPN, including but not limited to cell culture supernatant from yeast (e.g. Candida boidinii, Hansenula polymorpha, Pichia methanolica, Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis and other Kluyveromyces spp., Yarrowia lipolytica), Myxomycete (e.g. Dictyostelium discoideum), filamentous fungi (e.g. Trichoderma reesei and other Trichoderma spp., Aspergillus spp.), moss (e.g. Physcomitrella patens, Atrichum undulatum), insect or mammalian cell culture. Cell culture supernatant containing OPN refers to cell culture supernatant, wherein OPN is at least partially in solution. Cell culture supernatant containing OPN may be from mammalian cell culture. Mammalian cell culture systems are known in the art and make use, e.g. of NSO, SP2.0, 3T3 cells, COS cells, human osteosarcoma cells, MRC-5 cells, BHK cells, VERO cells, CHO cells, rCHO-tPA cells, rCHO-Hep B Surface Antigen cells, CHO-S cells, HEK 293 cells, rHEK 293 cells, rC127-Hep B Surface Antigen cells, human fibroblast cells, Stroma cells, hepatocyte cells or PER.C6 cells. The term "OPN" refers to human osteopontin-a (OPN-a) polypeptide. The human OPN-a polypeptide is the polypeptide according to SEQ ID NOs: 2 or the polypeptide encoded by the polynucleotide according to SEQ ID NOs: 1.

The terms "full length OPN" and "full length OPN polypeptide" refer to the osteopontin-a (OPN-a) mature full length polypeptide according to SEQ ID NO: 2, wherein the mature full length OPN polypeptide lacks the signal peptide of amino acids no. 1 to 16 of SEQ ID NO: 2. The terms "full length OPN" and "full length OPN polypeptide" also refer to the mature full length OPN-a polypeptide encoded by the polynucleotide according to SEQ ID NO: 1 , wherein the mature full length OPN polypeptide lacks the signal peptide of amino acids no. 1 to 16 encoded by the polynucleotides no. 166 to 213 of SEQ ID NO: 1.

The term "truncated OPN" refers to any fragment of full-length OPN. Said fragment of full-length OPN can be of any length, whereby the fragment has at least one amino acid, or at least five amino acids, or at least ten amino acids, or at least 15 amino acids, or at least 20 amino acids, or at least 30 amino acids or at least 50 amino acids less than mature full length OPN polypeptide according to SEQ ID NO: 2 or a mature full-length OPN polypeptide encoded by the polynucleotide according to SEQ ID NO: 1. Truncated OPN may result, e.g. from hydrolysis due to proteolytic degradation; e.g. during cell culture or during harvest of the cell culture supernatant. Proteolytic degradation may result; e.g. from enzymatic activity of a protease. Said protease may be a serine protease (e.g. thrombin), a threonine protease, a cysteine protease, an aspartic acid protease (e. g., plasmepsin), a metalloprotease or a glutamic acid protease. A method for determining the truncation of OPN is described in Example 1. The OPN purified according to the process of the invention may be intended for therapeutic use, i.e. for administration to patients. If OPN is administered to patients, it is preferably administered systemically, preferably parenteral, and more preferably subcutaneously or intramuscularly. Rectal or intrathecal administration may also be suitable, depending on the specific use of OPN. The definition of "pharmaceutically acceptable" is meant to encompass any carrier, which does not interfere with effectiveness of the biological activity of the active ingredient and that is not toxic to the host to which it is administered. For example, for parenteral administration, the active protein(s) may be formulated in a unit dosage form for injection in vehicles such as saline, dextrose solution, serum albumin and Ringer's solution. The OPN or pharmaceutical composition according to the embodiments of the invention can be administered systemically or locally

Systemic administration is, for example, achieved by administration through the digestive tract (enteral administration) or through other routes (parenteral administration). Parenteral administration routes are, for example, intravenous, intraarterial, subcutaneous, transdermal, intradermal, intramuscular, intraperitoneal, nasal, intracranial, intrathecal, intracardiac, intraosseous or transmucosal routes. Enteral administration routes are, for example, oral, rectal, sublingual, or buccal routes. Local administration is achieved, for example, through topical, epidural, epicutaneous, inhalational, nasal, intraarticular, vaginal, auricular or intravitreal routes. Local administration is also achieved by intracranial or intratumoral injection.

The OPN or pharmaceutical compositions of the present invention can also be administered in sustained or controlled release dosage forms, including depot injections, osmotic pumps, and the like, for the prolonged administration of the polypeptide at a predetermined rate, preferably in unit dosage forms suitable for single administration of precise dosages.

Parenteral administration can be by bolus injection or by gradual perfusion over time. Preparations for parenteral administration include sterile aqueous or non- aqueous solutions, suspensions, and emulsions, which may contain auxiliary agents or excipients known in the art, and can be prepared according to routine methods. In addition, suspension of the active compounds as appropriate oily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides. Aqueous injection suspensions that may contain substances increasing the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension may also contain stabilizers. Pharmaceutical compositions include suitable solutions for administration by injection, and contain from about 0.01 to 99.99 percent, preferably from about 20 to 75 percent of active compound together with the excipient.

For parenteral (e.g. intravenous, subcutaneous, intramuscular) administration, the active protein(s) can be formulated as a solution, suspension, emulsion or lyophilized powder in association with a pharmaceutically acceptable parenteral vehicle (e.g. water, saline, dextrose solution) and additives that maintain isotonicity (e.g. mannitol) or chemical stability (e.g. preservatives and buffers). The formulation is sterilized by commonly used techniques.

The therapeutically effective amounts of the active protein(s) will be a function of many variables, including but without limitation, the route of administration, the clinical condition of the patient, the pharmacokinetics of the active protein(s) in a patient.

A "therapeutically effective amount" is amount of OPN that when administered to a patient in need of treatment with OPN, such as e.g. a patient suffering from a neurological disorder, the neurological disorder including without limitation multiple sclerosis, stroke, a neurodegenerative disorder or a peripheral nervous system such as peripheral neuropathy including without limitation diabetic neuropathy, the amount of OPN results in an improvement of the disorder un that patient vis-a-vis a patient who did not receive a therapeutically effective amount of OPN. An improvement of the disorder can be measured by methods known in the art, the methods including the measurement of laboratory parameters taken from blood, urine or cerebrospinal fluid, the measurement of the functional status, pain, or disability; e.g. of a patient suffering from multiple sclerosis, stroke, a neurodegenerative disorder or a peripheral nervous system such as peripheral neuropathy including without limitation diabetic neuropathy; the methods also including imaging such as magnetic resonance imaging (MRI) or X-ray.

The dosage administered, as single or multiple doses, to an individual will vary depending upon a variety of factors, including OPN pharmacokinetic properties, the route of administration, patient conditions and characteristics (sex, age, body weight, health, size), extent of symptoms, concurrent treatments, frequency of treatment and the effect desired. Adjustment and manipulation of established dosage ranges are well within the ability of those skilled in the art, as well as in vitro and in vivo methods of determining the effect of OPN in an individual.

OPN may be used in amounts in the ranges of 0.001 to 100 mg/kg or 0.01 to 10 mg/kg or body weight, or 0.1 to 5 mg/kg of body weight or 1 to 3 mg/kg of body weight or 2 mg/kg of body weight.

OPN may be administered daily or every other day or three times per week or once per week, at similar doses, or at doses increasing or decreasing with the time. The daily doses are usually given in divided doses or in sustained release form effective to obtain the desired results. Second or subsequent administrations can be performed at a dosage which is the same, less than or greater than the initial or previous dose administered to the individual. A second or subsequent administration can be administered during or prior to onset of the disease.

OPN may be administered prophylactically or therapeutically to an individual prior to, simultaneously or sequentially with other therapeutic regimens or agents (e.g. multiple drug regimens), in therapeutically effective amounts.

OPN purified in accordance with the present invention may be used for preparation of a medicament for treatment and/or prevention of disorders, in particular human disorders . For instance but without limitation, OPN may be used for treatment and/or prevention of neurological disorders, including peripheral and central nervous system disorders, in particular multiple sclerosis, stroke and peripheral neuropathies such as diabetic neuropathy.

Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations and conditions without departing from the spirit and scope of the invention and without undue experimentation. While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows in the scope of the appended claims.

All references cited herein, including journal articles or abstracts, published or unpublished U.S. or foreign patent application, issued U.S. or foreign patents or any other references, are entirely incorporated by reference herein, including all data, tables, figures and text presented in the cited references. Additionally, the entire contents of the references cited within the references cited herein are also entirely incorporated by reference.

Reference to known method steps, conventional methods steps, known methods or conventional methods is not any way an admission that any aspect, description or embodiment of the present invention is disclosed, taught or suggested in the relevant art.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein), readily modify and/or adapt for various application such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.

EXAMPLES

Example 1 : Determination of truncated forms of OPN

The level of truncated forms of OPN was assessed using a RP-HPLC technique. In this assay, the culture supernatant containing OPN was loaded on a Vydac 5 μm column and eluted with a gradient of Trifluoroacetic acid (TFA) in water and TFA in acetonitrile. The quantification of OPN was performed by running a standard composed of purified OPN-a at a known concentration (measured using a reference technique such as amino-acid analysis). An example of RP-HPLC chromatogram of purified OPN according to Example 3 is shown in Fig. 2. The truncated forms were eluted as the shoulder prior to the main peak which represents intact OPN.

Example 2: Harvest of cell culture supernatant containing OPN

This example describes the capture of OPN from cell culture harvest. The goal of the capture is to stabilize the OPN molecule by removing contaminants that may impact the integrity of the molecule and to reduce the volume.

The cell culture suspension of a high cell density culture of CHO cells transfected to express OPN that was grown in a stirred tank bioreactor was first clarified on a depth filter (Millipore Millistack or equivalent) and loaded onto a chromatographic column filled with a weak anion exchange resin (DEAE Sepharose Fast Flow, G. E. Biosciences).

In one set of experiment, the column was loaded at maximum capacity

(determined at 13 g of intact OPN per liter resin) in a 8 ml column in order to optimize the clearance of truncated forms. In another set of experiment, the same column was loaded below capacity (1 O g intact OPN per liter resin). In a third set, a 3.1 liter column was loaded with 10 g intact OPN per liter resin.

Loading is done without pH or conductivity adjustment.

The column was washed with a sodium phosphate buffer (5OmM) the buffer with a conductivity of about 13 mS/cm and a pH of 7.2.

OPN is eluted from the column with a sodium phosphate buffer (50 mM) containing 275 mM NaCI with a conductivity of about 31 mS/cm at pH 7.2.

Table 1 : Summary of 3 captures of OPN harvests performed at both small and production scale

Results:

The capture step used in this example has shown a reproducible yield in intact OPN of almost 100%. Furthermore, the level of truncated forms in the eluate is decreased from 49-52% to 34-43%.

No differences were observed between the small-scale and the production scale.

Summary and conclusion:

This example describes the use of a weak anion exchange resin to capture OPN from cell culture supernatant comprising serum-free medium and cell-derived contaminants. The chromatographic step presents several advantages: ■ a high capacity of at least 13 grams of OPN per liter of resin;

^■ direct load of harvest without pH, conductivity adjustment nor ultrafiltration;step;

^■ clearance of truncated forms as shown in Table 1 ; and

^■ high recovery of intact OPN during elution. Example 3: Purification of recombinant OPN

This example describes the purification of OPN from the capture step (as described in Example 2), OPN is purified to a degree that is suitable for injection into human. In one set of experiments, 3 purification runs were performed at 150 mg scale using the eluate from the capture step. In a second set of experiments, the average of 6 runs performed at production scale is shown. The purification of OPN is composed of 3 chromatographic steps and one concentration/ultrafiltration using tangential flow systems.

In the first step the eluate from the capture step of Example 2 is further purified by immobilized metal ion affinity chromatography (IMAC). The eluate from the capture step (composed of about 50 to 100 grams of OPN) is loaded onto a BPG200/500 column packed with 3.5 liters of Nickel Chelating Sepharose Fast Flow (Ni SFF, G. E. Biosciences). The column is washed with a sodium phosphate buffer (50 mM) containing 275 mM NaCI with a conductivity of about 31 mS/cm at pH 7.2. OPN is then eluted in sodium phosphate buffer (50 mM) at acidic pH (4.0). In the second step the eluate from the first step is further purified by hydrophobic interaction chromatography (HIC). The eluate from the IMAC step is adjusted to a conductivity of 130-140 mS/cm using ammonium sulfate then loaded into a column with 6.6 L of resin Super Butyl 550C (Tosoh Biosciences). The column is washed with a sodium phosphate buffer (20 mM) containing ammonium sulphate (1 M) at pH 7.0. OPN is eluted in sodium phosphate buffer (20 mM) without salts at pH 7.0.

In the third step the eluate from the second step is further purified by ion exchange chromatography (IEC). The eluate from the Super Butyl 550C resin is loaded directly into an ion exchange resin Q Sepharose Fast Flow (G. E. Biosciences). The column is washed with a sodium phosphate buffer (50 mM) containing sodium chloride (200 mM) at pH 7.0. OPN is eluted in sodium phosphate buffer (50 mM) with 0.4 M NaCI at pH 7.0.

The eluate from the QSFF is concentrated and the buffer exchanged to remove NaCI. Typical final concentrations ranged from 10 to 100 mg/ml.

Results:

The results of OPN purification performed at 150-mg scale is shown in Table 2 (average of 3 purification runs). The cumulated yield was 55% and the level of truncated forms decreased from 25% to 14%. The level of host cell proteins of the purified material was 392 ppm.

The results of OPN purification performed at 50-100 g scale are shown in Table 3 (for 6 production runs). The average cumulated yield was 59.5% and the level of truncated forms in purified OPN bulk was 28.9%. The level of host cell proteins, i.e. impurities, of the purified material was 55 ppm.

κ> Table 2: Average data of 3 purifications runs performed at 150 mg scale.

Table 3: Comparison of 6 runs performed at production scale

Example 4: Purification of milk OPN

OPN is purified from cow milk. OPN is first captured and stabilized by removing contaminants that may impact the integrity of the molecule and to reduce the volume. Casein and whey proteins can be removed from skimmed milk by adjusting pH to 4.3 and adding calcium chloride (CaCI₂-2 H₂O) to a final concentration of 60 mM followed by precipitate removal by centrifugation or filtration.

Pre-treated milk will then be clarified on a depth filter (Millipore Millistack ) and loaded onto a chromatographic column filled with a weak anion exchange resin (DEAE Sepharose Fast Flow, G. E. Biosciences).

Loading will be done with or without pH or conductivity adjustment.

The column was washed with a sodium phosphate buffer (5OmM) with a conductivity of about 13 mS/cm and a pH of 7.2.

OPN is eluted from the column with a sodium phosphate buffer (50 mM) containing 275 mM NaCI with a conductivity of about 31 mS/cm at pH 7.2.

The eluate from the capture step is loaded onto a column packed with Nickel

Chelating Sepharose Fast Flow (Ni₆SFF, G. E. Biosciences). The column is washed with a sodium phosphate buffer (50 mM) containing 275 mM NaCI with a conductivity of about 31 mS/cm at pH 7.2. OPN is then eluted in sodium phosphate buffer (50 mM) at acidic pH (e.g pH 4.0).

The eluate from the IMAC step is adjusted to a conductivity of 130-140 mS/cm using ammonium sulfate and then loaded onto a column with Super Butyl

550C (Tosoh Biosciences). The column is washed with a sodium phosphate buffer

(20 mM) containing ammonium sulphate (1 M) at pH 7.0. OPN is eluted in sodium phosphate buffer (20 mM) without salts at pH 7.0.

The eluted material is loaded directly onto a ion exchange resin Q Sepharose Fast Flow (G. E. Biosciences). The column is washed with a sodium phosphate buffer (50 mM) containing sodium chloride (200 mM) at pH 7.0. OPN is eluted in sodium phosphate buffer (50 mM) with 0.4 M NaCI at pH 7.0. Post-QSFF is concentrated and buffer exchange to remove NaCI. Typical final concentrations ranged from 10 to 100 mg/ml. Example 5: Purification of OPN from urine

OPN is purified from human urine. OPN is first captured and stabilized by removing contaminants that may impact the integrity of the molecule and to reduce the volume. Peferably, a salt-precipitation step is performed to remove some major contaminant proteins (e.g. Tamm-Horsfall protein) prior to capture and purification.

Pre-treated urine will then be clarified on a depth filter (Millipore Millistack) and loaded onto a chromatographic column filled with a weak anion exchange resin (DEAE Sepharose Fast Flow, G. E. Biosciences). Loading will be done with or without pH or conductivity adjustment. The column was washed with a sodium phosphate buffer (5OmM) the buffer with a conductivity of about 13 mS/cm and a pH of 7.2.

OPN is eluted from the column with a sodium phosphate buffer (50 mM) containing 275 mM NaCI with a conductivity of about 31 mS/cm at pH 7.2. The eluate (treated or not) is then loaded onto a column packed with Nickel

Chelating Sepharose Fast Flow (Ni₆SFF, G. E. Biosciences). The column is washed with a sodium phosphate buffer (50 mM) containing 275 mM NaCI with a conductivity of about 31 mS/cm at pH 7.2. OPN is then eluted in sodium phosphate buffer (50 mM) at acidic pH (e.g. pH 4.0). The eluate is adjusted to a conductivity of 130-140 mS/cm using ammonium sulfate then loaded onto a column with SuperButyl 550C (Tosoh Biosciences). The column is washed with a sodium phosphate buffer (20 mM) containing ammonium sulphate (1 M) at pH 7.0. OPN is eluted in sodium phosphate buffer (20 mM) without salts at pH 7.0. The eluted material is loaded directly onto a ion exchange resin Q

Sepharose Fast Flow (G. E. Biosciences). The column is washed with a sodium phosphate buffer (50 mM) containing sodium chloride (200 mM) at pH 7.0. OPN is eluted in sodium phosphate buffer (50 mM) with 0.4M NaCI at pH 7.0.

The eluate from the QSFF is concentrated and buffer exchange to remove NaCI. Typical final concentrations ranged from 10 to 100 mg/ml. REFERENCES

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Claims

1. A process for the purification of osteopontin comprising: a) subjecting a solution containing osteopontin to immobilized metal ion affinity chromatography to produce a first eluate; b) subjecting the first eluate to hydrophobic interaction chromatography to produce a second eluate; and c) subjecting the second eluate to ion exchange chromatography.

2. The process according to claim 1 , wherein the immobilized metal ion affinity chromatography (IMAC) of step (a) is performed on a resin comprising a divalent metal ion.

3. The process according to claim 2, wherein the IMAC resin comprises the divalent ion Ni²⁺, Cu²⁺ or Zn²⁺.

4. The process according to claim 3, wherein the IMAC resin comprises a crosslinked, beaded-form of agarose.

5. The process according to any one of claims 1 to 4, wherein the hydrophobic interaction chromatography of step (b) is performed on a resin comprising butyl or phenyl residues.

6. The process according to any one of claims 1 to 5, wherein the ion exchange chromatography is performed on a resin comprising quaternary ammonium or

DEAE.

7. The process according to any one of claims 1 to 6, wherein the elution step during immobilized metal ion affinity chromatography is done in a) imidazol, b) sodium phosphate buffer having a pH of 2.0 to 6.0; or c) sodium acetate buffer having a pH of 2.0 to 6.0; or d) sodium citrate buffer having a pH of 2.0 to 6.0.

8. The process according to claim 7, wherein the elution step during immobilized metal ion affinity chromatography is done in a) sodium phosphate buffer having a pH 3.0 to 5.0; or b) sodium acetate buffer having a pH of 3.0 to 5.0; or c) sodium citrate buffer having a pH of 3.0 to 5.0.

9. The process according to claim 7 or 8, wherein the immobilized metal ion affinity chromatography (IMAC) is performed on a resin comprising a crosslinked, beaded-form of agarose comprising Ni²⁺, Zn²⁺ or Cu²⁺, and the IMAC comprises a) washing with a buffer containing 50 mM sodium phosphate and 275 mM sodium chloride 6.5 to 7.5; and b) eluting with a buffer containing 20 mM sodium phosphate at a pH of 3.0 to 5.0; and wherein the hydrophobic interaction chromatography (HIC) is performed on a resin comprising butyl or phenyl residues, and the HIC comprises a) washing with a buffer containing 20 mM sodium phosphate and 1 M ammonium sulphate, the buffer having a conductivity of 130-140 mS/cm and a pH of 6.0 to 8.0; and b) eluting with a buffer containing 20 mM sodium phosphate, the buffer having a conductivity of 2-3 mS/cm and a pH of 6.0 to 8.0; and wherein the ion exchange chromatography (IEC) is performed on a resin comprising quaternary ammonium or DEAE, and the IEC comprises a) washing with a buffer containing 50 mM sodium phosphate and 200 mM sodium chloride at a pH of 6.9 to 7.1 ; and b) eluting with a buffer containing 50 mM sodium phosphate and 400 mM sodium chloride at a pH of 6.9 to 7.1.

10. The process according to any one of claims 1 to 9, wherein prior to the process for the purification a capture step of ion exchange chromatography is performed, the capture step comprising using a resin comprising quaternary ammonium or DEAE.

1 1. A method of manufacturing a pharmaceutical composition comprising osteopontin as active ingredient, the method comprising the steps of: a) purifying osteopontin according to the process according to any one of claims 1 to 10; and b) formulating said purified osteopontin as active ingredient into a pharmaceutical composition, optionally with a pharmaceutically acceptable carrier or excipient.

12. A pharmaceutical composition comprising osteopontin as active ingredient, wherein not more than 35% of osteopontin is truncated osteopontin, and further comprising a pharmaceutically acceptable carrier, solvent or excipient.

13. A pharmaceutical composition comprising osteopontin as active ingredient, wherein at least 65% of osteopontin-a is full length osteopontin-a, and further comprising a pharmaceutically acceptable carrier, solvent or excipient.