WO2011018515A1

WO2011018515A1 - Method of purifying pegylated proteins

Info

Publication number: WO2011018515A1
Application number: PCT/EP2010/061832
Authority: WO
Inventors: Haleh Ahmadian
Original assignee: Novo Nordisk Health Care Ag
Priority date: 2009-08-14
Filing date: 2010-08-13
Publication date: 2011-02-17

Abstract

The invention relates to a method of purifying PEGylated proteins ( which comprises anion exchange chromatography with an elution buffer comprising MgC12), in particular, but not exclusively vitamin K-dependent blood coagulation factors such as Factor VII (FVII), to proteins purified by said method and to the use of said purified proteins in therapy, in particular but not exclusively, for the treatment of diseases alleviated by blood coagulation factors such as the prophylactic treatment of hemophilia.

Description

METHOD OF PURIFYING PEGYLATED PROTEINS

FIELD OF THE INVENTION

The invention relates to a method of purifying PEGylated proteins, in particular, but not exclusively vitamin K-dependent blood coagulation factors such as Factor VII (FVII), to proteins purified by said method and to the use of said purified proteins in therapy, in particular but not exclusively, for the treatment of diseases alleviated by blood coagulation factors such as the prophylactic treatment of hemophilia. BACKGROUND OF THE INVENTION

Prolongation of circulating half-life of proteins can be achieved by modification of the native structure of the proteins. PEGylation is an established method for prolonging the circulating half-life of proteins. GlycoPEGylation of Factor VII (FVII) results in various PEGylated species such as mono-, di and tri-PEGylated species. GlycoPEGylation occurs at the termi- nal galactose residues on Asn145 and/or Asn322 of FVII. Such an arrangement therefore provides the possibility for formation of various mono-PEGylated and di-PEGylated species. Mono-PEGylated forms have been identified to possess a desirable pharmacological profile and have therefore been chosen as the preferred drug candidate. It is thus desirable to isolate the mono-PEGylated forms from a mixture of PEGylated and non-PEGylated species.

Vitamin K-dependent proteins are distinguished from other proteins by sharing a common structural feature in their amino terminal part of the molecule. The N-terminal of these proteins, also referred to as the Gla-domain, is rich in the unusual amino acid γ- carboxy glutamic acid which is synthesized from glutamate in a Vitamin K dependent reac- tion catalysed by the enzyme γ-glutamyl carboxylase. Because of the presence of about 9 to 12 GIa residues, the Gla-domain is characterised by being capable of binding divalent cations such as Ca²⁺. Upon binding of metal ions, these proteins undergo conformational changes which can be measured by several techniques such as circular dichroism and fluorescence emission.

The discovery of metal induced conformational changes of GIa- containing proteins (Nelsestuen et. a/., J. Biol. Chem. 1976; 251 , 6886-6893) together with identification of conformation specific polyclonal antibodies (Furie et al., J. Biol. Chem. 1978; 253, 8980-8987) opened the way for the introduction of conformation specific immunoaffinity chromatography. These antibodies could recognise and bind the Gla-domain in the presence of Ca²⁺ ions but released the protein upon removal of Ca²⁺ ions using a Ca²⁺ chelator such as EDTA or citrate.

In 1986, Bjørn and Thim reported purification of rFVII on an anion exchange material taking advantage of the Ca²⁺-binding property of Gla-domain of FVII (Bjørn S. and Thim L., Research Dislosure, 1986, 26960-26962.). Adsorption was achieved in a buffer without Ca²⁺and elution of FVII was possible using a Ca²⁺ containing buffer with low ionic strength and under mild conditions. While the presence of Gla-domain provides an advantage for separation of GIa containing proteins from other proteins, it is difficult to separate PEGylated species of a GIa containing protein from each other and from the nonPEGylated protein, since the same GIa domain is present in all species. WO 2007/022512 (Neose Technologies, Inc) describes a process of resolving PE¬

Gylated FVIIa from unPEGylated FVIIa comprising anion exchange chromatography with a 10 column volume gradient of MgCI₂. This process makes use of a linear-gradient elution using MgCI₂ as eluting salt. Mg²⁺ ions interact with the Gla-domain of FVII and facilitate differential elution of PEG species and nonPEG species.

PEG is a hydrophilic polymer that may shield the surface charge of proteins. Thus the binding of PEGylated proteins to anion exchange material is weaker than the nonPEGylated proteins. Although in GlycoPEGylated rFVIIa the site of PEG attachment is away from GIa domain, it affects the binding of various PEGylated rFVIIa species to the anion ex- changer.

In addition to separation of PEGylated species from each other and from nonPEGylated species, the purification process must provide sufficient reduction of reagents used in the reaction. It is required to develop a method that ensures the desired product quality and it will be advantageous to develop a single step of purification that can provide sufficient reduction of process related impurities (such as PEGylating reagents, enzymes, by products from reagents) as well as product related impurities (such as nonPEGylated species).

SUMMARY OF THE INVENTION According to a first aspect of the invention, there is provided a method of purifying a PEGylated protein which comprises anion exchange chromatography with an elution buffer comprising a Mg salt, characterized in that said chromatography comprises step-gradient elution with a plurality of Mg²⁺ concentrations.

According to a second aspect of the invention, there is provided a purified PEGylated Factor VII blood coagulation factor obtainable by a method as herein defined.

The invention also provides a method for selective elution of PEG species by appli- cation of stepwise elution followed by step-gradient elution of nonPEG species. Impurities such as the PEGylating enzyme ST3Gal3 are sufficiently reduced. Furthermore, by using the method of invention there is no need for pH adjustment of the pooling fraction, which is advantageous in large scale productions. As in the preferred embodiment of the method of invention, monoPEGylated species are eluted by step-gradient elution, the collected pool is more concentrated, which is advantageous in large scale productions.

DETAILED DESCRIPTION OF THE INVENTION

According to a first aspect of the invention, there is provided a method of purifying a

PEGylated protein which comprises anion exchange chromatography with an elution buffer comprising a Mg salt, characterized in that said chromatography comprises step-gradient elution with a plurality of Mg²⁺ concentrations. The purification method of the invention provides a number of advantages over previously described purification processes. For example, it has been surprisingly found that the content of ST3Gal3 in the monoPEGylated fractions was reduced by about 30 times in the present invention. ST3Gal3 is a PEGylating enzyme used during the GlycoPEGylation process. The presence of such an enzyme in the monoPEGylated fractions is clearly undesirable because further PEGylation may occur following the purification process. Furthermore, residual enzyme is considered as an impurity and there is a desire to reduce the amount of residual enzyme to very low levels in pharmaceutical preparations of mono-PEGylated rFVIIa. The content of ST3Gal3 in the desired monoPEGylated fractions was measured to be about 50 ng/ml in the step-gradient elution method of the invention. By contrast, the content of ST3Gal3 in the desired monoPEGylated fractions was measured to be about 1500 ng/ml in the known linear-gradient elution method.

Certain embodiments of the invention have the further advantage of allowing purification in a shorter time duration. For example, the linear-gradient elution methods previously known must be performed at relatively low flow rates, such as 12 column volumes per hour.

PEGylated proteins can be obtained by one of the known methods in the literature and known by the person skilled in the art. WO 2007/022512 describes a method for PEGylation of rFVIIa where the PEG group is attached enzymatically to terminal galactose residues of the N-glycans. The PEGylation reaction mixture subjected to purification by the method of invention can be obtained by GIy- coPEGylation or by other known PEGylation methods.

Although purification of PEGylated proteins constitutes a particular aspect of the invention, the method can also be equally applied to purification of proteins that are attached to polymers other than PEG, such as polysialic acid.

In one embodiment, the size of the attached PEG group varies from about 2 to about 40 KD. References herein to "step-gradient elution" refer to a step between an elution buffer containing a first concentration of Mg salt to a second concentration of Mg salt. The Mg salt can be chosen from any commercial and available salts such as MgCI2, Mgacetate, Mgsulphate, Mgascorbat and Mgaspartate. Step-gradient elution denotes a chromatography method wherein e.g. the concentration of a substance causing elution, i.e. the dissolution of a bound substance from a chromatography material, is raised or lowered at once, i.e. directly from one value/level to the next value/level. In this "step elution" one or more conditions, for example the ionic strength, concentration of a salt, and/or the flow of a chromatography method, is/are changed all at once from a first, e.g. starting, value to a second, e.g. final, value. "Step elution" denotes that the conditions are changed promptly, i.e. stepwise, in contrast to a linear change. After each increase the conditions are maintained and the elution is performed is- ocratically until the next step in the elution method. This is in distinct contrast to a "linear-gradient elution" wherein the percentage of one buffer gradually decreases to 0% and the percentage of another buffer gradually increases to 100%.

One of the buffers used during linear-gradient elution is normally the last washing buffer prior to elution start. The percentage of this buffer usually decreases from 100% to 0% during elution. The second buffer is then elution buffer, the percentage of which usually increases during the elution.

The method of invention comprises a stepwise elution of PEGylated species followed by a step-gradient elution intended to elute nonPEGylated species.

The number of steps in stepwise elution of PEGylated species can vary from one to four. Each step might be performed by a step-gradient elution or by a linear- gradient elution.

The term of elution is used herein to indicate release of all protein species related to the protein of interest from the chromatographic resin. Although release and elution of unwanted species such as di- and tri-PEGylated species can be consid- ered as a wash step, we refer to all the steps, where release of PEGylated and non-PEGylated species of the protein of interest occurs as elution steps.

In one embodiment, the method comprises a first step-gradient elution at a concentration of between 5 and 20 mM of a Mg salt. In a further embodiment, the first step-gradient elution comprises a concentration of between 10 and 15 mM of a Mg salt. In a yet further embodiment, the first step-gradient elution comprises a concentration of 12.5 mM of a Mg salt. The first step-gradient elution at such Mg²⁺ concentrations is intended to reduce or even remove the content of the multiPEGylated species, such as di- and tri-PEGylated species.

In one embodiment, the first step-gradient elution comprises between 2 and 10 column volumes of elution buffer. In a further embodiment, the first step- gradient elution comprises between 5 and 10 column volumes of elution buffer.

Alternatively, removal of multiPEGylated species can be accomplished during two steps, where combination of the two steps reduces or even removes multiPEGy- lated species.

In one embodiment, the method comprises a second step-gradient elution at a concentration of between 15 and 45 mM of a Mg salt, such as between 25 and 45 mM MgCI₂. In a further embodiment, the second step-gradient elution comprises a concentration of between 30 and 40 mM of a Mg salt. In a yet further embodiment, the second step-gradient elution comprises a concentration of 35 mM of a Mg salt. If the first step-gradient has provided sufficient reduction of multiPEGylated species, then the second step-gradient elution at Mg²⁺ concentrations of between 30 and 45 mM is intended to isolate the desired monoPEGylated spe- cies. If the first step-gradient is intentionally used for partial removal of multiPEGylated species, then the second step is used to further elute these species.

In one embodiment, the second step-gradient elution comprises between 2 and 10 column volumes of elution buffer. In a further embodiment, the second step- gradient elution comprises between 5 and 10 column volumes of elution buffer.

In one embodiment, the method of the invention comprises a third step-gradient elution. In such an embodiment, elution comprises a concentration of greater than 15 mM Mg²⁺such as greater 45 mM MgCI₂. In a further embodiment, the third step-gradient elution comprises a concentration of 50 mM MgCI₂. If the second step-gradient elution is used for elution of monoPEGylated species, then the third step-gradient elution at such MgCI₂ concentrations is intended to isolate the nonPEGylated species. NonPEGylated species are not desirable in the therapeutic preparations of monoPEGIyated rFVIIa due to their short in vivo half life compared to PEGylated species. Furthermore, the fractions containing the nonPEGy- lated species can be pooled, recycled and subjected to PEGylation in order to increase the overall yield of the PEGylation process. If the second step-gradient elution is used for elution of multiPEGylated species, then the third step-gradient is used for elution of monoPEGylated species. In this case, concentration of MgCI₂ between 30-45 mM is used.

In one embodiment, a final step-gradient elution can be carried out with an eluting salt other than Mg salt for isolation of non-PEGylated species.

In another embodiment, the final step-gradient elution can be carried out with the same buffer as used in PEGylation reaction, thus enabling isolation of nonPE- Gylated species for direct rePEGylation without a need for further processing such as buffer exchange.

In an alternative embodiment, the final step-gradient elution comprises a Ca salt containing buffer. In a further embodiment, the final step-gradient elution comprises a buffer containing 20 mM CaCI₂ (such as 10 mM histidine, 20 mM CaCI₂ and 50 mM NaCI).

In one embodiment, the final step-gradient elution comprises between 2 and 20 column volumes of elution buffer.

The binding of proteins with GIa- domain to anion exchangers is facilitates by re- moval of metal ions that interact with GIa- domain such as Ca²⁺ . Removal of metal ions in loading solution can be achieved by addition of a metal chelator substance such as EDTA and citrate. In one embodiment, the purification method additionally comprises the step of removing EDTA prior to elution. Such a step may typically comprise washing the anion exchange column with a buffer which is substantially free of EDTA. Thus, in a further embodi- ment, the washing buffer is substantially free of EDTA. By "substantially free" it is meant that the washing buffer contains less than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 , 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 , 0.05 or θ.01 % w/v of EDTA or other chelating agents. Without being bound by theory, it is believed that the presence of EDTA during elution results in chelation of the Mg²⁺ ions which replace the protons attached to acetic acid residues of EDTA. A consequence of the release of protons into the solution causes a reduction in pH which facilitates faster release of PEGylated species from the anion exchanger material. Surprisingly, it has been found that removal of EDTA from the washing buffer in linear-gradient elution causes poorer separation of PEGylated species. By contrast, removal of EDTA from the washing buffer prior to step-gradient elution did not detrimentally impact the separation of PEGylated species but furthermore overcame the detrimental effect of reduction in pH.

When gradient elution is started by mixing the elution buffer with an EDTA containing buffer, the pH in the pooled fractions is lower than 5.5 and has to subsequently be ad- justed to 5.7-7.9. Additional adjustment of pH is not necessary when EDTA is not used in the washing buffer.

In one embodiment, linear-gradient elution may be used in combination with the step-gradient elution methods of the invention.

In one embodiment, the PEGylated protein is a vitamin K-dependent protein, such as a vitamin K-dependent blood coagulation factor. In a further embodiment, the vitamin K- dependent blood coagulation factor comprises a galactose containing blood coagulation factor. In a yet further embodiment, the vitamin K-dependent blood coagulation factor is se- lected from Factor Il (FII), Factor VII (FVII), Factor IX (FIX), Factor X (FX), protein S and protein C. In a yet further embodiment, the vitamin K-dependent blood coagulation factor is Factor VII (FVII).

Thus, according to a further aspect of the invention there is provided a method of isolating monoPEGylated Factor VII which comprises anion exchange chromatography, characterized in that said chromatography comprises step-gradient elution.

In one embodiment of any of the aforementioned aspects of the invention, the Factor VII (FVII) is selected from the following non-limiting examples: PEGylated human Fac- tor Vila, cysteine-PEGylated human Factor Vila and variants thereof. Non-limiting examples of Factor VII derivatives includes GlycoPegylated FVII derivatives as disclosed in WO 03/31464 and US Patent applications US 2004/0043446, US 2004/0063911 , US

2004/0142856, US 2004/0137557 and US 2004/0132640 (Neose Technologies, Inc.); FVII conjugates as disclosed in WO 01/04287, US patent application 2003/0165996, WO

01/58935, WO 03/93465 (Maxygen ApS) and WO 02/02764, US patent application 2003/0211094 (University of Minnesota). In a further embodiment, the Factor VII is Factor Vlla-SA-PEG-40kDa, as described in Example 12 of WO 2007/022512.

It will be appreciated that anion exchange chromatography can be performed in ac- cordance with procedures known to the skilled person. Examples of suitable anion exchange materials include: Q-resin, a Quaternary amine, and DEAE resin, DiEthylAminoEthane. Anion exchange resins are commercially available, e.g. Mono Q Source 15Q or 3OQ (GE-health care), Poros 20HQ or 50HQ (Applied Biosystems), Toyopearl Q650S (Toso Haas) and others.

In one embodiment, the anion exchange material comprises HQ, such as Poros® HQ, for example, Poros® 50 HQ. Poros® HQ is available from Applied Biosystems and is based on a quaternized polyethyleneimine functional group yielding a high capacity. In another embodiment, the anion exchange material comprises a Source , such as

Source 3OQ or 15Q, which is available from GE Healthcare.

According to a second aspect of the invention, there is provided a purified PEGy- lated protein obtainable by a method as herein defined.

In one embodiment, the purified protein, such as a mono-PEGylated vitamin K- dependent protein is substantially free of multiPEGylated species. By "substantially free" it is meant that the mono-PEGylated vitamin K-dependent protein contains less than 20% of multiPEGylated species, such as less than 15%, or less than 10% or less than 5%, less than 3%, less than 2% or less than 1%. In one embodiment, the purified protein, such as a mono-PEGylated vitamin K- dependent protein is substantially free of ST3Gal3. By "substantially free" it is meant that the mono-PEGylated vitamin K-dependent protein contains less than 100 ng/mL of ST3Gal3. According to a further aspect of the invention there is provided a pharmaceutical composition comprising a purified PEGylated protein as herein defined, such as a mono- PEGylated vitamin K-dependent protein, such as purified Factor VII, e.g. mono-PEGylated Factor Vl I. The purified blood coagulation factors and pharmaceutical compositions comprising the blood coagulation factors may be used in the treatment of diseases alleviated by administration of blood coagulation factors (e.g. FVII), such as a bleeding disorder e.g. hemophilia, a blood disease, hemarthrosis, hematomas, mucocutaneous bleeding, inherited blood disease, familial bleeding disorder, familial blood disease or factor replacement therapy. In one embodiment, the disease alleviated by administration of a blood coagulation factor is hemophilia, such as hemophilia B or Christmas disease.

Thus according to a further aspect of the invention there is provided a method of treating hemophilia which comprises administering to a patient a therapeutically effective amount of a purified blood coagulation factor as defined hereinbefore.

There is also provided a purified blood coagulation factor as defined hereinbefore for use in the treatment of hemophilia. There is also provided the use of a purified blood coagulation factor as defined hereinbefore in the manufacture of a medicament for the treatment of hemophilia.

There is also provided a pharmaceutical composition comprising a purified blood coagulation factor as defined hereinbefore for use in the treatment of hemophilia.

It is to be understood, that therapeutic and prophylactic (preventive) regimes represent separate aspects of the present invention. In particular, it should be understood that the present invention provides purified blood coagulation factors with increased plasma half-lives which make them desirable for the prophylactic treatment of hemophilia. Such prophylactic treatment of hemophilia constitutes a preferred embodiment of the invention. The formulation may further comprise a buffer system, preservative(s), tonicity agent(s), chelating agent(s), stabilizers and surfactants. In one embodiment of the invention the pharmaceutical formulation is an aqueous formulation, i.e. formulation comprising water. Such formulation is typically a solution or a suspension. In one embodiment of the invention the pharmaceutical formulation is an aqueous solution.

In one embodiment the pharmaceutical formulation is a freeze-dried formulation, whereto the physician or the patient adds solvents and/or diluents prior to use.

In one embodiment the pharmaceutical formulation is a dried formulation (e.g.

freeze-dried or spray-dried) ready for use without any prior dissolution.

In one embodiment the invention relates to a pharmaceutical formulation comprising an aqueous solution of a purified blood coagulation factor of the present invention, and a buffer, wherein said purified blood coagulation factor is present in a concentration from 0.1- 100 mg/ml, and wherein said formulation has a pH from about 2.0 to about 10.0.

In one embodiment of the invention the pH of the formulation is selected from the list consisting of 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7,

3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8,

5.9, 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1 , 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1 , 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, and 10.0.

In one embodiment of the invention the buffer is selected from the group consisting of sodium acetate, sodium carbonate, citrate, glycylglycine, (2-hydroxyethyl)-1 - piperazineethanesulfonic acid (HEPES), 3-(N-morpholino)propanesulfonic acid (MOPS); 2- (N-morpholino)ethanesulfonic acid (MES); N-cyclohexyl-3-aminopropanesulfonic acid (CAPS); N-Cyclohexyl-2-aminoethanesulfonic acid (CHES); histidine, glycine, lysine, argin- ine, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, and tris(hydroxymethyl)-aminomethan, bicine, tricine, malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid or mixtures thereof. Each one of these specific buffers constitutes an alternative embodiment of the invention. In one embodiment of the invention the formulation further comprises an active site inhibitor.

In one embodiment of the invention the formulation further comprises a pharmaceu- tically acceptable preservative. In one embodiment of the invention the preservative is selected from the group consisting of phenol, o-cresol, m-cresol, p-cresol, methyl p- hydroxybenzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol, butyl p-hydroxybenzoate, 2- phenylethanol, benzyl alcohol, chlorobutanol, and thiomerosal, bronopol, benzoic acid, imidurea, chlorohexidine, sodium dehydroacetate, chlorocresol, ethyl p-hydroxybenzoate, benzethonium chloride, chlorphenesine (3p-chlorphenoxypropane-1 ,2-diol) or mixtures thereof.

In one embodiment of the invention the preservative is present in a concentration from 0.1 mg/ml to 20 mg/ml. In one embodiment of the invention the preservative is present in a concentration from 0.1 mg/ml to 5 mg/ml. In one embodiment of the invention the preservative is present in a concentration from 5 mg/ml to 10 mg/ml. In one embodiment of the invention the preservative is present in a concentration from 10 mg/ml to 20 mg/ml. Each one of these specific preservatives constitutes an alternative embodiment of the invention. The use of a preservative in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 20^th edition, 2000.

In one embodiment of the invention the formulation further comprises an isotonic agent. In one embodiment of the invention the isotonic agent is selected from the group con- sisting of a salt (e.g. sodium chloride), a sugar or sugar alcohol, an amino acid (e.g. L- glycine, L-histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine), an aldi- tol (e.g. glycerol (glycerine), 1 ,2-propanediol (propyleneglycol), 1 ,3-propanediol, 1 ,3- butanediol) polyethyleneglycol (e.g. PEG400), or mixtures thereof. Any sugar such as mono-, di-, or polysaccharides, or water-soluble glucans, including for example fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch and carboxymethylcellulose-Na may be used. In one embodiment the sugar additive is sucrose. Sugar alcohol is defined as a C4-C8 hydrocarbon having at least one -OH group and includes, for example, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol. In one embodiment the sugar alcohol additive is mannitol. The sugars or sugar alcohols mentioned above may be used individually or in combination. There is no fixed limit to the amount used, as long as the sugar or sugar alcohol is soluble in the liquid preparation and does not adversely affect the stabilizing effects achieved using the methods of the invention. In one embodiment, the sugar or sugar alcohol concentration is between about 1 mg/ml and about 150 mg/ml. In one embodiment of the in- vention the isotonic agent is present in a concentration from 1 mg/ml to 50 mg/ml. In one embodiment of the invention the isotonic agent is present in a concentration from 1 mg/ml to 7 mg/ml. In one embodiment of the invention the isotonic agent is present in a concentration from 8 mg/ml to 24 mg/ml. In one embodiment of the invention the isotonic agent is present in a concentration from 25 mg/ml to 50 mg/ml. Each one of these specific isotonic agents constitutes an alternative embodiment of the invention. The use of an isotonic agent in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 20^th edition, 2000.

In one embodiment of the invention the formulation further comprises a chelating agent. In one embodiment of the invention the chelating agent is selected from salts of ethyl- enediaminetetraacetic acid (EDTA), citric acid, and aspartic acid, and mixtures thereof. In one embodiment of the invention the chelating agent is present in a concentration from 0.1 mg/ml to 5mg/ml. In one embodiment of the invention the chelating agent is present in a concentration from 0.1 mg/ml to 2mg/ml. In one embodiment of the invention the chelating agent is present in a concentration from 2mg/ml to 5mg/ml. Each one of these specific chelating agents constitutes an alternative embodiment of the invention. The use of a chelating agent in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 20^th edition, 2000. In one embodiment of the invention the formulation further comprises a stabilizer.

The use of a stabilizer in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 20^th edition, 2000. More particularly, compositions of the invention are stabilized liquid pharmaceutical compositions whose therapeutically active components include a polypeptide that possibly exhibits aggregate formation during storage in liquid pharmaceutical formulations. By "aggregate formation" is intended a physical interaction between the polypeptide molecules that results in formation of oligomers, which may remain soluble, or large visible aggregates that precipitate from the solution. By "during storage" is intended a liquid pharmaceutical compo- sition or formulation once prepared, is not immediately administered to a subject. Rather, following preparation, it is packaged for storage, either in a liquid form, in a frozen state, or in a dried form for later reconstitution into a liquid form or other form suitable for administration to a subject. By "dried form" is intended the liquid pharmaceutical composition or formulation is dried either by freeze drying (i.e., lyophilization; see, for example, Williams and PoIIi (1984) J. Parenteral Sci. Technol. 38:48-59), spray drying (see Masters (1991) in Spray-Drying Handbook (5th ed; Longman Scientific and Technical, Essez, U.K.), pp. 491-676; Broadhead et al. (1992) Drug Devel. Ind. Pharm. 18:1 169-1206; and Mumenthaler et al. (1994) Pharm. Res. 11 :12-20), or air drying (Carpenter and Crowe (1988) Cryobiology 25:459-470; and Roser (1991) Biopharm. 4:47-53).

Aggregate formation by a polypeptide during storage of a liquid pharmaceutical composition can adversely affect biological activity of that polypeptide, resulting in loss of therapeutic efficacy of the pharmaceutical composition. Furthermore, aggregate formation may cause other problems such as blockage of tubing, membranes, or pumps when the polypeptide-containing pharmaceutical composition is administered using an infusion system.

The pharmaceutical compositions of the invention may further comprise an amount of an amino acid base sufficient to decrease aggregate formation by the polypeptide during storage of the composition. By "amino acid base" is intended an amino acid or a combination of amino acids, where any given amino acid is present either in its free base form or in its salt form. Where a combination of amino acids is used, all of the amino acids may be present in their free base forms, all may be present in their salt forms, or some may be present in their free base forms while others are present in their salt forms. In one embodiment, amino acids to use in preparing the compositions of the invention are those carrying a charged side chain, such as arginine, lysine, aspartic acid, and glutamic acid. Any stereoisomer (i.e., L, D, or mixtures thereof) of a particular amino acid (e.g. methionine, histidine, imidazole, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine and mixtures thereof) or combinations of these stereoisomers, may be present in the pharmaceutical compositions of the invention so long as the particular amino acid is present either in its free base form or its salt form. In one embodiment the L-stereoisomer is used.

Compositions of the invention may also be formulated with analogues of these amino acids. By "amino acid analogue" is intended a derivative of the naturally occurring amino acid that brings about the desired effect of decreasing aggregate formation by the polypeptide during storage of the liquid pharmaceutical compositions of the invention. Suitable arginine analogues include, for example, aminoguanidine, ornithine and N-monoethyl L- arginine, suitable methionine analogues include ethionine and buthionine and suitable cysteine analogues include S-methyl-L cysteine. As with the other amino acids, the amino acid analogues are incorporated into the compositions in either their free base form or their salt form. In one embodiment of the invention the amino acids or amino acid analogues are used in a concentration, which is sufficient to prevent or delay aggregation of the protein.

In one embodiment of the invention methionine (or other sulphuric amino acids or amino acid analogous) may be added to inhibit oxidation of methionine residues to methionine sulfoxide when the polypeptide acting as the therapeutic agent is a polypeptide comprising at least one methionine residue susceptible to such oxidation. By "inhibit" is intended minimal accumulation of methionine oxidized species over time. Inhibiting methionine oxidation results in greater retention of the polypeptide in its proper molecular form. Any stereoi- somer of methionine (L, D, or mixtures thereof) or combinations thereof can be used. The amount to be added should be an amount sufficient to inhibit oxidation of the methionine residues such that the amount of methionine sulfoxide is acceptable to regulatory agencies. Typically, this means that the composition contains no more than about 10% to about 30% methionine sulfoxide. Generally, this can be achieved by adding methionine such that the ratio of methionine added to methionine residues ranges from about 1 : 1 to about 1000: 1 , such as 10: 1 to about 100: 1.

In one embodiment of the invention the formulation further comprises a stabilizer selected from the group of high molecular weight polymers or low molecular compounds. In one embodiment of the invention the stabilizer is selected from polyethylene glycol (e.g. PEG 3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone, carboxy-/hydroxycellulose or derivates thereof (e.g. HPC, HPC-SL, HPC-L and HPMC), cyclodextrins, sulphur-containing substances as monothioglycerol, thioglycolic acid and 2-methylthioethanol, and different salts (e.g. sodium chloride). Each one of these specific stabilizers constitutes an alternative em- bodiment of the invention.

The pharmaceutical compositions may also comprise additional stabilizing agents, which further enhance stability of a therapeutically active polypeptide therein. Stabilizing agents of particular interest to the present invention include, but are not limited to, methion- ine and EDTA, which protect the polypeptide against methionine oxidation, and a nonionic surfactant, which protects the polypeptide against aggregation associated with freeze- thawing or mechanical shearing.

In one embodiment of the invention the formulation further comprises a surfactant. In one embodiment of the invention the surfactant is selected from a detergent, ethoxylated castor oil, polyglycolyzed glycerides, acetylated monoglycerides, sorbitan fatty acid esters, polyoxypropylene-polyoxyethylene block polymers (eg. poloxamers such as Pluronic^® F68, poloxamer 188 and 407, Triton X-100), polyoxyethylene sorbitan fatty acid esters, poly- oxyethylene and polyethylene derivatives such as alkylated and alkoxylated derivatives (tweens, e.g. Tween-20, Tween-40, Tween-80 and Brij-35), monoglycerides or ethoxylated derivatives thereof, diglycerides or polyoxyethylene derivatives thereof, alcohols, glycerol, lectins and phospholipids (eg. phosphatidyl serine, phosphatidyl choline, phosphatidyl etha- nolamine, phosphatidyl inositol, diphosphatidyl glycerol and sphingomyelin), derivates of phospholipids (eg. dipalmitoyl phosphatidic acid) and lysophospholipids (eg. palmitoyl lyso- phosphatidyl-L-serine and 1 -acyl-sn-glycero-3-phosphate esters of ethanolamine, choline, serine or threonine) and alkyl, alkoxyl (alkyl ester), alkoxy (alkyl ether)- derivatives of lyso- phosphatidyl and phosphatidylcholines, e.g. lauroyl and myristoyl derivatives of lysophos- phatidylcholine, dipalmitoylphosphatidylcholine, and modifications of the polar head group, that is cholines, ethanolamines, phosphatidic acid, serines, threonines, glycerol, inositol, and the positively charged DODAC, DOTMA, DCP, BISHOP, lysophosphatidylserine and lyso- phosphatidylthreonine, and glycerophospholipids (eg. cephalins), glyceroglycolipids (eg. ga- lactopyransoide), sphingoglycolipids (eg. ceramides, gangliosides), dodecylphosphocholine, hen egg lysolecithin, fusidic acid derivatives- (e.g. sodium tauro-dihydrofusidate etc.), long- chain fatty acids and salts thereof C6-C12 (eg. oleic acid and caprylic acid), acylcarnitines and derivatives, N^α-acylated derivatives of lysine, arginine or histidine, or side-chain acylated derivatives of lysine or arginine, NT-acylated derivatives of dipeptides comprising any combination of lysine, arginine or histidine and a neutral or acidic amino acid, N^α-acylated derivative of a tripeptide comprising any combination of a neutral amino acid and two charged amino acids, DSS (docusate sodium, CAS registry no [577-11-7]), docusate calcium, CAS registry no [128-49-4]), docusate potassium, CAS registry no [7491-09-0]), SDS (sodium do- decyl sulphate or sodium lauryl sulphate), sodium caprylate, cholic acid or derivatives thereof, bile acids and salts thereof and glycine or taurine conjugates, ursodeoxycholic acid, sodium cholate, sodium deoxycholate, sodium taurocholate, sodium glycocholate, N- Hexadecyl-N,N-dimethyl-3-ammonio-1 -propanesulfonate, anionic (alkyl-aryl-sulphonates) monovalent surfactants, zwitterionic surfactants (e.g. N-alkyl-N,N-dimethylammonio-1- propanesulfonates, S-cholamido-i-propyldimethylammonio-i-propanesulfonate, cationic surfactants (quaternary ammonium bases) (e.g. cetyl-trimethylammonium bromide, cetylpyridin- ium chloride), non-ionic surfactants (eg. Dodecyl β-D-glucopyranoside), poloxamines (eg. Tetronic's), which are tetrafunctional block copolymers derived from sequential addition of propylene oxide and ethylene oxide to ethylenediamine, or the surfactant may be selected from the group of imidazoline derivatives, or mixtures thereof. Each one of these specific surfactants constitutes an alternative embodiment of the invention.

The use of a surfactant in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 20^th edition, 2000.

It is possible that other ingredients may be present in the pharmaceutical formulation of the present invention. Such additional ingredients may include wetting agents, emulsifiers, antioxidants, bulking agents, tonicity modifiers, chelating agents, metal ions, oleaginous vehicles, proteins (e.g., human serum albumin, gelatine or proteins) and a zwitterion (e.g., an amino acid such as betaine, taurine, arginine, glycine, lysine and histidine). Such additional ingredients, of course, should not adversely affect the overall stability of the pharmaceutical formulation of the present invention.

Pharmaceutical compositions containing a purified blood coagulation factor of the present invention may be administered to a patient in need of such treatment at several sites, for example, at topical sites, for example, skin and mucosal sites, at sites which bypass absorption, for example, administration in an artery, in a vein, in the heart, and at sites which involve absorption, for example, administration in the skin, under the skin, in a muscle or in the abdomen.

Administration of pharmaceutical compositions according to the invention may be through several routes of administration, for example, lingual, sublingual, buccal, in the mouth, oral, in the stomach and intestine, nasal, pulmonary, for example, through the bronchioles and alveoli or a combination thereof, epidermal, dermal, transdermal, vaginal, rectal, ocular, for examples through the conjunctiva, uretal, and parenteral to patients in need of such a treatment. Compositions of the current invention may be administered in several dosage forms, for example, as solutions, suspensions, emulsions, microemulsions, multiple emulsion, foams, salves, pastes, plasters, ointments, tablets, coated tablets, rinses, capsules, for example, hard gelatine capsules and soft gelatine capsules, suppositories, rectal capsules, drops, gels, sprays, powder, aerosols, inhalants, eye drops, ophthalmic ointments, ophthalmic rinses, vaginal pessaries, vaginal rings, vaginal ointments, injection solution, in situ transforming solutions, for example in situ gelling, in situ setting, in situ precipitating, in situ crystallization, infusion solution, and implants. Compositions of the invention may further be compounded in, or attached to, for example through covalent, hydrophobic and electrostatic interactions, a drug carrier, drug delivery system and advanced drug delivery system in order to further enhance stability of the peptide of the present invention, increase bioavailability, increase solubility, decrease adverse effects, achieve chronotherapy well known to those skilled in the art, and increase pa- tient compliance or any combination thereof. Examples of carriers, drug delivery systems and advanced drug delivery systems include, but are not limited to, polymers, for example cellulose and derivatives, polysaccharides, for example dextran and derivatives, starch and derivatives, polyvinyl alcohol), acrylate and methacrylate polymers, polylactic and polyglycolic acid and block co-polymers thereof, polyethylene glycols, carrier proteins, for example albu- min, gels, for example, thermogelling systems, for example block co-polymeric systems well known to those skilled in the art, micelles, liposomes, microspheres, nanoparticulates, liquid crystals and dispersions thereof, L2 phase and dispersions there of, well known to those skilled in the art of phase behaviour in lipid-water systems, polymeric micelles, multiple emulsions, self-emulsifying, self-microemulsifying, cyclodextrins and derivatives thereof, and dendrimers.

Compositions of the current invention are useful in the formulation of solids, semisolids, powder and solutions for pulmonary administration of a peptide of the present invention, using, for example a metered dose inhaler, dry powder inhaler and a nebulizer, all being de- vices well known to those skilled in the art.

Compositions of the current invention are specifically useful in the formulation of controlled, sustained, protracting, retarded, and slow release drug delivery systems. More specifically, but not limited to, compositions are useful in formulation of parenteral controlled release and sustained release systems (both systems leading to a many-fold reduction in number of administrations), well known to those skilled in the art. Even more preferably, are controlled release and sustained release systems administered subcutaneous. Without limiting the scope of the invention, examples of useful controlled release system and compositions are hydrogels, oleaginous gels, liquid crystals, polymeric micelles, microspheres and nanoparticles.

Methods to produce controlled release systems useful for compositions of the current invention include, but are not limited to, crystallization, condensation, co-crystallization, precipitation, co-precipitation, emulsification, dispersion, high pressure homogenisation, en- capsulation, spray drying, microencapsulating, coacervation, phase separation, solvent evaporation to produce microspheres, extrusion and supercritical fluid processes. General reference is made to Handbook of Pharmaceutical Controlled Release (Wise, D. L., ed. Marcel Dekker, New York, 2000) and Drug and the Pharmaceutical Sciences vol. 99: Protein Formulation and Delivery (MacNally, E. J., ed. Marcel Dekker, New York, 2000).

Parenteral administration may be performed by subcutaneous, intramuscular, intraperitoneal or intravenous injection by means of a syringe, optionally a pen-like syringe. Alternatively, parenteral administration can be performed by means of an infusion pump. A further option is a composition which may be a solution or suspension for the administration of the peptide of the present invention in the form of a nasal or pulmonal spray. As a still further option, the pharmaceutical compositions containing the peptide of the present invention can also be adapted to transdermal administration, e.g. by needle-free injection or from a patch, optionally an iontophoretic patch, or transmucosal, e.g. buccal, administration. The term "stabilized composition" refers to a composition with increased physical stability, increased chemical stability or increased physical and chemical stability.

The term "physical stability" of the protein composition as used herein refers to the tendency of the protein to form biologically inactive and/or insoluble aggregates of the protein as a result of exposure of the protein to thermo-mechanical stresses and/or interaction with interfaces and surfaces that are destabilizing, such as hydrophobic surfaces and interfaces. Physical stability of the aqueous protein compositions is evaluated by means of visual inspection and/or turbidity measurements after exposing the composition filled in suitable containers (e.g. cartridges or vials) to mechanical/physical stress (e.g. agitation) at different tern - peratures for various time periods. Visual inspection of the compositions is performed in a sharp focused light with a dark background. The turbidity of the composition is characterized by a visual score ranking the degree of turbidity for instance on a scale from 0 to 3 (a composition showing no turbidity corresponds to a visual score 0, and a composition showing visual turbidity in daylight corresponds to visual score 3). A composition is classified physical unstable with respect to protein aggregation, when it shows visual turbidity in daylight. Alternatively, the turbidity of the composition can be evaluated by simple turbidity measurements well-known to the skilled person. Physical stability of the aqueous protein compositions can also be evaluated by using a spectroscopic agent or probe of the conformational status of the protein. The probe is preferably a small molecule that preferentially binds to a non-native conformer of the protein. One example of a small molecular spectroscopic probe of protein structure is Thioflavin T. Thioflavin T is a fluorescent dye that has been widely used for the detection of amyloid fibrils. In the presence of fibrils, and perhaps other protein configurations as well, Thioflavin T gives rise to a new excitation maximum at about 450 nm and enhanced emission at about 482 nm when bound to a fibril protein form. Unbound Thioflavin T is essen- tially non-fluorescent at the wavelengths.

Other small molecules can be used as probes of the changes in protein structure from native to non-native states. For instance the "hydrophobic patch" probes that bind preferentially to exposed hydrophobic patches of a protein. The hydrophobic patches are gener- ally buried within the tertiary structure of a protein in its native state, but become exposed as a protein begins to unfold or denature. Examples of these small molecular, spectroscopic probes are aromatic, hydrophobic dyes, such as antrhacene, acridine, phenanthroline or the like. Other spectroscopic probes are metal-amino acid complexes, such as cobalt metal complexes of hydrophobic amino acids, such as phenylalanine, leucine, isoleucine, methion- ine, and valine, or the like.

The term "chemical stability" of the protein composition as used herein refers to chemical covalent changes in the protein structure leading to formation of chemical degradation products with potential less biological potency and/or potential increased immunogenic properties compared to the native protein structure. Various chemical degradation products can be formed depending on the type and nature of the native protein and the environment to which the protein is exposed. Elimination of chemical degradation can most probably not be completely avoided and increasing amounts of chemical degradation products is often seen during storage and use of the protein composition as well-known by the person skilled in the art. Most proteins are prone to deamidation, a process in which the side chain amide group in glutaminyl or asparaginyl residues is hydrolysed to form a free carboxylic acid. Other degradations pathways involves formation of high molecular weight transformation products where two or more protein molecules are covalently bound to each other through transami- dation and/or disulfide interactions leading to formation of covalently bound dimer, oligomer and polymer degradation products (Stability of Protein Pharmaceuticals, Ahern. T.J. & Manning M. C, Plenum Press, New York 1992). Oxidation (of for instance methionine residues) can be mentioned as another variant of chemical degradation. The chemical stability of the protein composition can be evaluated by measuring the amount of the chemical degradation products at various time-points after exposure to different environmental conditions (the for- mation of degradation products can often be accelerated by for instance increasing temperature). The amount of each individual degradation product is often determined by separation of the degradation products depending on molecule size and/or charge using various chromatography techniques (e.g. SEC-HPLC and/or RP-HPLC). Hence, as outlined above, a "stabilized composition" refers to a composition with increased physical stability, increased chemical stability or increased physical and chemical stability. In general, a composition must be stable during use and storage (in compliance with recommended use and storage conditions) until the expiration date is reached. In one embodiment of the invention the pharmaceutical composition comprising the purified protein of the invention is stable for more than 6 weeks of usage and for more than 3 years of storage.

In another embodiment of the invention the pharmaceutical composition comprising the purified protein of the invention is stable for more than 4 weeks of usage and for more than 3 years of storage.

In a further embodiment of the invention the pharmaceutical composition comprising the purified protein of the invention is stable for more than 4 weeks of usage and for more than two years of storage.

In an even further embodiment of the invention the pharmaceutical composition comprising the purified protein of the invention is stable for more than 2 weeks of usage and for more than two years of storage. In an even further embodiment of the invention the pharmaceutical composition comprising the purified protein of the invention is stable for more than 1 week of usage and for more than six months of storage. The invention will now be described with reference to the following non-limiting examples.

Example 1

Isolation of monoPEGylated Factor VII

(a) Determination of PEGylated lsoforms by Reversed phase HPLC analysis

PEGylated Factor Vila was analyzed by HPLC on a reversed-phase column

10 (Zorbax 300SB-C3, 5 μm particle size, 2.1 x 150 mm). The eluants were A) 0.1 TFA in water and B) 0.09 % TFA in acetonitrile. Detection was at 214 nm. The gradient, flow rate, and column temperature depended on the PEG length (40 KDa, 20 KDa, and 10 KDa PEG: 35-65 %B in 30 min, 0.5 mL/min, 45°C; 10 KDa PEG: 35-60 %B in 30 min, 0.5 mL/min, 45°C; 5 KDa: 40-50 %B in 40 min, 0.5 mL/min, 45°C; 2 15 KDa: 38-43 %B in 67 min, 0.6 mL/min, 55°C). The identity of each peak was assigned based on two or more of four different pieces of evidence: the known retention time of native Factor Vila, the SDS-PAGE migra- tion of the isolated peak, the MALDI-TOF mass spectrum of the isolated peak, and the orderly progression of the retention time of each peak with increasing number of attached PEG.

(b) Purification of 4OK PEG-rFVIIa by three step gradient elution

PEGylated rFVIIa was prepared as described in WO 2007/022512. The GlycPEGy- lation reaction mixture, containing 10 mg rFVIIa species in total, was diluted 1 +2 with 10 mM histidine, pH 6.0 and added EDTA solution to a final concentration of 15 mM. A column, containing 2.7 mL anion exchange material was equilibrated with 10 mM histidine, 50 mM NaCI, 10 mM EDTA, pH 6.0. After loading, the column was washed with 10 mM histidine, 50 mM NaCI, pH 6.0 for 10 CV. The first step elution was performed 1O mM histidine, 15 mM MgCI₂, pH 6.0 for 6 CV, then changed to the second step by eluting with 10 mM histidine, 35 mM MgCI₂, pH 6.0 for 5,7 CV and then changed to 10 mM histidine, 50 mM MgCI₂, pH 6.0 for 4.3 CV. The pooled fractions were analysed by RP-HPLC and the results are shown in table 1 : Table 1

The results in Table 1 showed that the peak from the 2nd elution step comprised 94.4% monoPEGylated rFVIIa. The content of ST3Gal3 was measured to be 54.5 ng/mL in the collected peak from the 2^nd step.

(c) Purification of 4OK PEG-rFVIIa by four step gradient elution

PEGylated rFVIIa was prepared as described in WO 2007/022512. The GlycPEGy- lation reaction mixture, containing 10 mg rFVIIa species in total, was diluted 1 +2 with 10 mM histidine, pH 6.0 and added EDTA solution to a final concentration of 15 mM. A column, containing 2.7 ml. anion exchange material was equilibrated with 10 mM histidine, 50 mM NaCI, 10 mM EDTA, pH 6.0. After loading, the column was washed with 10 mM histidine, 50 mM NaCI, pH 6.0 for 10 CV. The first step elution was performed 1O mM histidine, 12.5 mM MgCI₂, pH 6.0 for 4 CV, then changed to the second step by eluting with 10 mM histidine, 15 mM MgCI₂, pH 6.0 for 3 CV and then changed to 10 mM histidine, 35 mM MgCI₂, pH 6.0 for 5 CV. The fourth step was performed by eluting with 10 mM histidine, 50 mM MgCI₂, pH 6.0 for 5 CV The pooled fractions were analysed by RP-HPLC and the results are shown in table 2:

Table 2

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference in their entirety and to the same extent as if each refer- ence were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein (to the maximum extent permitted by law), regardless of any separately provided incorporation of particular documents made elsewhere herein.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. For example, the phrase "the compound" is to be understood as referring to various "compounds" of the invention or particular described aspect, unless otherwise indicated. Unless otherwise indicated, all exact values provided herein are representative of corresponding approximate values (e.g., all exact exemplary values provided with respect to a particular factor or measurement can be considered to also provide a corresponding approximate measurement, modified by "about," where appropriate). The description herein of any aspect or aspect of the invention using terms such as

"comprising", "having," "including," or "containing" with reference to an element or elements is intended to provide support for a similar aspect or aspect of the invention that "consists of", "consists essentially of", or "substantially comprises" that particular element or elements, unless otherwise stated or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context).

Claims

1. A method of purifying a PEGylated protein which comprises anion exchange chromatography with an elution buffer comprising a Mg salt, characterized in that said chromatography comprises step-gradient elution with a plurality of Mg salt concentrations.

2. A method as defined in claim 1, which comprises a first step-gradient elution at a concentration of between 5 and 20 mM of a Mg salt, such as between 10 and 15 mM of a Mg salt, in particular, 12.5 mM of a Mg salt.

3. A method as defined in claim 1 or claim 2, which comprises a second step-gradient elution at a concentration of between 15 and 45 mM of a Mg salt, for example between 25 and 40 mM of a Mg salt, such as between 30 and 40 mM of a Mg salt, in particular, 35 mM of a Mg salt.

4. A method as defined in any preceding claims, which comprises a third step-gradient elution at a concentration of greater than 15 mM of a Mg salt, such as greater than 45 mM of a Mg salt, in particular 50 mM of a Mg salt.

5. A method as defined in any preceding claims, which comprises a final step- gradient elution comprising a Ca salt containing buffer, such as a buffer containing 20 mM CaCI₂.

6. A method as defined in claim 5, where the non-PEGylated protein is collected in order to be recycled in a new PEGylation reaction.

7. A method as defined in any preceding claims, which additionally comprises the step of removing EDTA or other chelating agents prior to elution.

8. A method as defined in claim 7, wherein said EDTA removing step comprises washing the anion exchange column with a buffer which is substantially free of EDTA.

9. A method as defined in any preceding claims, wherein the PEGylated protein is a vitamin K-dependent protein, such as a vitamin K-dependent blood coagulation factor, for example a galactose containing blood coagulation factor.

10. A method as defined in claim 9, wherein the vitamin K-dependent blood coagulation factor is selected from Factor Il (FII), Factor VII (FVII), Factor IX (FIX), Factor X (FX), protein S and protein C, such as Factor VII (FVII).

11. A method as defined in claim 10, wherein the Factor VII is Factor VIIa-SA- PEG-40kDa.

12. A method as defined in any preceding claims wherein the anion exchange material comprises HQ, such as Poros® HQ, for example, Poros® 50 HQ.

13. A purified PEGylated protein, such as purified Factor VII, e.g. mono-

PEGylated Factor VII obtainable by a method as defined in any preceding claims.

14. A purified PEGylated protein as defined in claim 13, which is substantially free of multiPEGylated species.

15. A purified PEGylated protein as defined in claim 13 or claim 14, which is substantially free of ST3Gal3.

16. A pharmaceutical composition comprising a purified PEGylated protein as defined in any of claims 13 to 15.