HK1181661B - Method for purifying pegylated erythropoietin - Google Patents

Abstract

Herein is reported a method for the purification of a protein comprising erythropoietin and a single poly (ethylene glycol) residue from reaction by-products or not reacted starting material by a cation exchange chromatography method. It has been found that by employing a cation exchange SP Sephacryl S 500 HR chromatography material conditioned to a conductivity of 21 mS/cm and a linear gradient elution a fusion protein of erythropoietin and a single poly (ethylene glycol) residue can be obtained in a single step with high purity and yield.

Description

Method for purifying pegylated erythropoietin

Technical Field

Herein is reported a method for the purification of pegylated erythropoietin using a linear gradient elution method on a SP Sephacryl S500HR column.

Background

Proteins play an important role in today's medical products. For human use, each therapeutic protein must meet unique criteria. In order to ensure the safety of biopharmaceuticals to humans, it is necessary in particular to remove by-products accumulated during the production process. In order to meet regulatory specifications, one or more purification steps must be performed after the production process. Among other things, purity, throughput and yield play an important role in determining a suitable purification process.

Conjugates of the following therapeutic proteins have been reported: for example, polyethylene glycol (PEG) and interleukin-6 (EP0442724), PEG and erythropoietin (WO01/02017), chimeric molecules comprising endostatin and immunoglobulins (US2005/008649), secreted antibody-based fusion proteins (US2002/147311), fusion polypeptides comprising albumin (US 2005/0100991; human serum albumin US5,876,969), pegylated polypeptides (US2005/0114037) and erythropoietin fusions.

Necina, R., et al (Biotechnol. Bioeng.60(1998)689-698) report the direct capture of human monoclonal antibodies from cell culture supernatants using ion exchange media exhibiting high charge density. In WO89/05157 a method for purifying immunoglobulin products by subjecting cell culture media directly to cation exchange treatment is reported. Danielsson, a., et al, j.immun.meth.115(1988)79-88, describe the one-step purification of monoclonal IgG antibodies from mouse ascites. In WO2004/024866 a method for purifying a polypeptide by ion exchange chromatography is reported, wherein a gradient elution is used to dissociate the polypeptide of interest from one or more contaminants. In EP0530447 a method for purifying IgG monoclonal antibodies by a combination of three chromatographic steps is reported. Yu, G.et al, Process Biotechnol.42(2007)971-977 reported the easy purification of mono-pegylated interleukin-1 receptor antagonists. Wang, h, et al, Peptides26(2005)1213-1218 reported the purification of hTFF3 expressed in e.coli (e.coli) by two-step cation exchange chromatography. Yun, q., et al (Yun, q., et al, j. biotechnol.118(2005)67-74) report purification of pegylated rhG-CSF by two sequential ion exchange chromatography steps.

Disclosure of Invention

Herein is reported a method for purifying a protein conjugate comprising erythropoietin and a single poly (ethylene glycol) residue from reaction by-products or unreacted starting materials by cation exchange chromatography. It has been found that by using the cation exchange chromatography material SP Sephacryl S500HR and linear gradient elution, a conjugated protein comprising erythropoietin and a single poly (ethylene glycol) residue can be obtained in high purity and yield in a single step, wherein a buffer solution with a defined conductivity has been pre-applied in the column.

Thus, in one aspect, herein is reported a method for obtaining a fusion protein comprising erythropoietin and a single poly (ethylene glycol) residue, said method comprising the steps of:

a) a solution having a conductivity of about 21mS/cm was applied to a chromatography column containing SP Sephacryl S500HR chromatography material,

b) applying a solution to the column of a), said solution comprising a mixture of free erythropoietin and fusion proteins of erythropoietin and poly (ethylene glycol) wherein each erythropoietin molecule has one or more poly (ethylene glycol) residues,

c) applying a solution having a conductivity of about 21mS/cm to the column and recovering therefrom a fusion protein comprising 2 or more poly (ethylene glycol) residues,

d) applying a solution to the column having a continuously and linearly increasing conductivity up to a final value of at least 60mS/cm and recovering the fusion protein comprising erythropoietin and a single poly (ethylene glycol) residue and free erythropoietin, respectively, therefrom, wherein the fusion protein comprising erythropoietin and a single poly (ethylene glycol) residue is obtained first.

In one embodiment, the solution having a conductivity of about 21mS/cm is a solution having a pH of from pH2.5 to pH 3.5. In one embodiment, the solution having a conductivity of about 21mS/cm is a phosphate buffer solution having a pH value of from pH2.5 to pH 3.5.

In one embodiment, the pH of the solution applied in step d) is between pH2.5 and pH 3.5. In one embodiment, applying a solution having a continuously and linearly increasing conductivity is up to a final conductivity value of about 70.0 mS/cm.

In one embodiment, the solution having a continuously and linearly increasing conductivity is a solution having a continuously and linearly increasing sodium chloride concentration.

In one embodiment, the erythropoietin is human erythropoietin. In one embodiment, the human erythropoietin has the amino acid sequence of SEQ ID NO: 01 or SEQ ID NO: 02, or a pharmaceutically acceptable salt thereof.

In one embodiment, the single poly (ethylene glycol) residue has a molecular weight of 20kDa to 40 kDa.

In one embodiment, a solution comprising a mixture of free erythropoietin and fusion proteins of erythropoietin and poly (ethylene glycol), wherein each erythropoietin molecule has one or more poly (ethylene glycol) residues, is applied to 1ml of chromatographic material in a manner that 1mg up to 4mg of fusion protein is applied to the chromatographic material.

Description of the invention

Herein is reported a method for purifying a protein comprising one erythropoietin molecule and one poly (ethylene glycol) residue using a gradient elution method, wherein said gradient is a linear conductivity gradient on a SPSephacryl S500HR column, wherein a solution with a defined conductivity has been applied on a chromatography column before applying a solution comprising said protein.

General chromatographic methods and their use are known to the person skilled in the art. See, e.g., Heftmann, e., (ed.), Chromatography, 5 th edition, Part a: fundamentals and dtechniques, Elsevier Science Publishing Company, New York (1992); deyl, Z., (eds.), Advanced chromatography and electrochemistry Methods in Biosciences, Elsevier Science BV, Amsterdam, The Netherlands (1998); poole, c.f., and Poole, s.k., Chromatography Today, Elsevier Science Publishing Company, new york (1991); scopes, Protein Purification: principles and Practice, Springer Verlag (1982); sambrook, j, et al, (eds), Molecular Cloning: a Laboratory Manual, 2 nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); or Ausubel, F.M., et al, (eds.), Current Protocols in molecular biology, John Wiley & Sons, Inc., New York (1987-.

The term "applied to" denotes a partial step of the purification process in which the solution is contacted with the chromatographic material. This means that: a) adding the solution to a chromatographic apparatus containing a chromatographic material, or b) adding a chromatographic material to the solution. In the case of a), the solution is passed through the device, allowing the chromatographic material to interact with the substances contained in the solution. Depending on the conditions (e.g. pH, conductivity, salt concentration, temperature and/or flow rate), some of the substances in the solution bind to the chromatography material and can thus be recovered from the chromatography material in a further step. The material remaining in solution can be found in a flow-through. By "flow-through" is meant the solution obtained after passing through the device, which may be an applied solution or a buffer solution used to wash the column or to cause elution of the substance bound to the chromatographic material. In one embodiment, the device is a column or cassette. In the case of b), the chromatographic material (e.g. as a solid) may be added, for example, to a solution containing the target substance to be purified, thereby allowing interaction between the chromatographic material and the substance in the solution. After the interaction, the chromatographic material is removed, for example by filtration, and the substances bound to the chromatographic material are also subsequently removed from the solution, while the substances not bound to the chromatographic material remain in the solution.

The term "binding and elution mode" denotes an operating mode of a chromatography step, wherein a solution containing a target substance to be purified is applied to a chromatography material, thereby binding the target substance to the chromatography material. Thus, the target substance remains on the chromatographic material, while non-target substances are removed with the flow-through or supernatant. Then, in a second step, the target substance is recovered from the chromatography material with an elution solution. In one embodiment, the method as reported herein works in binding and elution mode.

The solution used in the process as reported herein is a crude solution or a buffered solution. The term "buffer solution" denotes a solution: wherein pH changes caused by the addition or release of acidic or basic substances are leveled by the dissolved buffer substance. Any buffer substance having such properties may be used. Typically, pharmaceutically acceptable buffer substances are used. In one embodiment, the buffer solution is selected from: a phosphate buffer solution composed of phosphoric acid and/or a salt thereof, or an acetic acid buffer solution composed of acetic acid and a salt thereof, or a citric acid buffer solution composed of citric acid and/or a salt thereof, or a morpholine buffer solution, or a 2- (N-morpholino) ethanesulfonic acid buffer solution, or a histidine buffer solution, or a glycine buffer solution, or a TRIS (hydroxymethyl) aminomethane (TRIS) buffer solution. In one embodiment, the buffer solution is selected from a phosphate buffer solution, or an acetate buffer solution, or a citrate buffer solution, or a histidine buffer solution. Optionally, the buffer solution may comprise additional salts, such as sodium chloride, sodium sulfate, potassium chloride, potassium sulfate, sodium citrate, or potassium citrate.

The terms "continuous elution" and "continuous elution method" are used interchangeably in this application to denote a method in which: wherein the conductivity of the solution causing elution (i.e. recovery of bound compounds from the chromatographic material) is continuously varied (i.e. increased or decreased), i.e. the concentration is varied by a series of small steps, each small step being no more than a 2% or 1% variation in the concentration of the substance causing elution. In this "continuous elution" one or more conditions (e.g. pH, ionic strength, salt concentration and/or chromatographic flow rate) may vary linearly or exponentially or asymptotically. In one embodiment, the variation is linear.

The term "ion exchange chromatography material" denotes an immobilized high molecular weight matrix with covalently bound charged substituents, which is used as stationary phase in ion exchange chromatography. To achieve overall charge neutrality, non-covalently bound counterions are bound thereto. An "ion exchange chromatography material" has the ability to exchange its non-covalently bound counter-ions for similarly charged ions in a surrounding solution. An "ion exchange resin" is referred to as a cation exchange resin or an anion exchange resin, depending on the charge of its exchangeable counter ions. Depending on the nature of the charged groups (substituents), "ion exchange resins" are referred to as: for example, in the case of cation exchange resins, sulfonic acid resins (S), sulfopropyl resins (SP), or carboxymethyl resins (CM).

The steps and methods for converting an amino acid sequence (e.g., of a polypeptide) into a corresponding nucleic acid sequence encoding the amino acid sequence are well known to those skilled in the art. Thus, a nucleic acid is characterized by its nucleic acid sequence consisting of individual nucleotides, as is the amino acid sequence of the polypeptide encoded by it.

The term "poly (ethylene glycol)" or "poly (ethylene glycol) residue" refers to a non-proteinaceous residue containing poly (ethylene glycol) as a substantial moiety. Such poly (ethylene glycol) residues may contain other chemical groups necessary for the binding reaction, resulting from chemical synthesis of the molecule, or be spacers for optimal distance of parts of the molecule. These other chemical groups are not used to calculate the molecular weight of the poly (ethylene glycol) residue. In addition, such poly (ethylene glycol) residues may be composed of one or more poly (ethylene glycol) chains that are covalently linked together. Poly (ethylene glycol) residues having more than one PEG chain are referred to as multi-arm or branched poly (ethylene glycol) residues. For example, branched poly (ethylene glycol) residues can be prepared by adding polyethylene oxide to various polyols, including glycerol, pentaerythritol, and sorbitol. Branched poly (ethylene glycol) residues are reported, for example, in EP0473084, US5,932,462. In one embodiment, the poly (ethylene glycol) residue has a molecular weight of 20kDa to 35kDa and is a linear poly (ethylene glycol) residue. In another embodiment, the poly (ethylene glycol) residue is a branched poly (ethylene glycol) residue having a molecular weight of 35kDa to 40 kDa.

The term "fusion of erythropoietin with poly (ethylene glycol) residues" denotes the covalent attachment of a poly (ethylene glycol) residue chemically introduced at the N-terminal or internal lysine residue of erythropoietin. The fusion results in a protein conjugate comprising one erythropoietin molecule and one or more poly (ethylene glycol) residues. The fusion process is also known as pegylation and the product is known as pegylated erythropoietin. Fusion/conjugation of polypeptides to poly (ethylene glycol) residues is widely known in the art and is reviewed, for example, in Veronese, f.m., Biomaterials22(2001) 405-. Poly (ethylene glycol) residues can be attached using different functional groups. Poly (ethylene glycol) s having different molecular weights, different forms, and different linking groups may be used (see also Francis, g.e., et al, int.j.hematol.68(1998) 1-18; Delgado, c., et al, crit.rev.ther. drug carrier Systems9(1992) 249-304). The fusion of erythropoietin and poly (ethylene glycol) residues can be carried out in an aqueous solution of a reagent containing poly (ethylene glycol) residues, see, for example, WO 00/44785. The fusion can also be performed on a solid phase, see Lu, Y., et al, reactive polymers22(1994) 221-229. According to WO94/01451, it is also possible to generate nonrandom N-terminal fusions.

The terms "fusion of erythropoietin with poly (ethylene glycol)" and "pegylation" mean that a covalent bond is formed between a poly (ethylene glycol) residue and an N-terminal and/or internal lysine residue of erythropoietin, so as to obtain a protein conjugate comprising one erythropoietin molecule and one poly (ethylene glycol) residue. In one embodiment, the pegylation of erythropoietin is performed in aqueous solution using NHS-activated linear or branched PEG molecules with a molecular weight between 5kDa and 40 kDa.

The term "under conditions suitable for binding" and grammatical equivalents thereof as used herein means that the substance of interest (e.g., pegylated erythropoietin) binds to the stationary phase (e.g., ion exchange material) when contacted therewith. This does not necessarily mean that 100% of the target substance is bound to the stationary phase, but means that essentially 100% of the target substance is bound to the stationary phase, i.e. at least 50% of the target substance is bound to the stationary phase, at least 75% of the target substance is bound to the stationary phase, at least 85% of the target substance is bound to the stationary phase, or more than 95% of the target substance is bound to the stationary phase.

Chemical fusion or conjugation of erythropoietin and poly (ethylene glycol) typically results in a mixture of different compounds, such as polypegylated erythropoietin, monopegylated erythropoietin, non-pegylated erythropoietin, hydrolysis products of activated PEG esters, and hydrolysis products of erythropoietin itself. In order to obtain the mono-pegylated erythropoietin in a substantially homogeneous form, these substances must be separated.

Thus, in one aspect as reported herein, a method is provided for producing a protein conjugate comprising one erythropoietin molecule and a single poly (ethylene glycol) residue in a substantially homogeneous form, wherein said method comprises the steps of:

a) conjugating erythropoietin to poly (ethylene glycol) using an activated poly (ethylene glycol) ester having a molecular weight of 20kDa to 40kDa,

b) applying the conjugate obtained in step a) to a SP Sephacryl S500HR chromatography material to which a solution having a conductivity of about 21mS/cm has been applied,

c) recovering the protein comprising one erythropoietin molecule and a single poly (ethylene glycol) residue in a substantially homogeneous form (mono-pegylated erythropoietin) by linear conductivity gradient elution, and thereby producing a protein conjugate comprising erythropoietin and a single poly (ethylene glycol) residue.

In one embodiment, the SP Sephacryl S500HR chromatography material is in a chromatography column. The method is particularly suitable for the purification of pegylated recombinant erythropoietin, which is glycosylated, i.e. which has been produced by mammalian cells, in one embodiment by CHO cells, or HEK293 cells, or BHK cells, orCells, or HeLaCells are produced and then chemically pegylated.

In the first step of the method, erythropoietin is pegylated. The poly (ethylene glycol) (PEG) polymer molecules used in the pegylation reaction have a molecular weight of about 20kDa to 40kDa (the term "molecular weight" as used herein should be understood to mean the average molecular weight of PEG, since PEG is not obtained as a polymeric compound at a defined molecular weight, but rather actually has a molecular weight distribution; the term "about" means that in the PEG preparation some molecules have a weight greater than the indicated molecular weight and some molecules have a weight less than the indicated molecular weight, i.e., the term "about" refers to a molecular weight distribution in which 95% of the PEG molecules have a molecular weight within +/-10% of the indicated molecular weight; e.g., a molecular weight of 30kDa means a range of 27kDa to 33 kDa).

The term "erythropoietin" and its abbreviation "EPO" refer to a polypeptide having the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a protein or polypeptide substantially homologous thereto, the biological properties of which are associated with the stimulation of erythropoiesis and the stimulation of division and differentiation of committed erythroid progenitors in the bone marrow. Recombinant erythropoietin can be prepared as follows: expression in eukaryotic cells (e.g.in CHO cells, or BHK cells, or HeLa cells) by recombinant DNA techniques or by endogenous gene activation (that is to say expression of the erythropoietin glycoprotein by endogenous gene activation), see for example U.S. Pat. No. 5,733,761, U.S. Pat. No. 5,641,670, U.S. Pat. No. 5,733,746, WO93/09222, WO94/12650, WO95/31560, WO90/11354, WO91/06667 and WO 91/09955. In one embodiment, the erythropoietin is human EPO. In one embodiment, the human erythropoietin has the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof. In one embodiment, the human erythropoietin has the amino acid sequence set forth in SEQ ID NO: 1. The term "erythropoietin" also means, SEQ ID NO: 1 or SEQ ID NO: 2 in which one or more amino acid residues have been altered, deleted or inserted, and which have a biological activity comparable to that of the unmodified protein (such as those reported in EP1064951 or US6,583,272). The variant may have the amino acid sequence of human erythropoietin with 1-6 additional glycosylation sites. The specific activity of pegylated erythropoietin can be determined by a variety of assays known in the art. The biological activity of purified pegylated erythropoietin is such that administration of the protein by injection to a human patient results in bone marrow cells that increase production of reticulocytes and red blood cells as compared to non-injected or control subjects. The biological activity of pegylated Erythropoietin obtained and purified according to the methods reported herein can be tested by the method of Bristow, A.A., Pharmeuropa spec.

Amino acid sequence variants of erythropoietin may be prepared by introducing appropriate modifications into the nucleotide sequence encoding erythropoietin, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into, and/or substitutions of, residues within the amino acid sequence of erythropoietin. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct has comparable biological activity to human erythropoietin.

Conservative amino acid substitutions are shown in table 1 under the heading of "preferred substitutions". Further substantial variations are provided in table 1 under the heading "exemplary substitutions" and are described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into human erythropoietin and the product screened for retention of the biological activity of human erythropoietin.

TABLE 1

Original residues	Exemplary permutations	Preference is given to substitution
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Asp，Lys；Arg	Gln
Asp(D)	Glu；Asn	Glu
			Cys(C)	Ser；Ala	Ser
Gln(Q)	Asn；Glu	Asn
			Glu(E)	Asp；Gln	Asp
Gly(G)	Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu; val; met; ala; phe; norleucine	Leu
			Leu(L)	Norleucine; ile; val; met; ala; phe (Phe)	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Trp；Leu；Val；Ile；Ala；Tyr	Tyr
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Val；Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile; leu; met; phe; ala; norleucine	Leu

Amino acids can be grouped according to common side chain properties:

(1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: cys, Ser, Thr, Asn, Gln;

(3) acidic: asp and Glu;

(4) basic: his, Lys, Arg;

(5) residues that influence chain orientation: gly, Pro;

(6) aromatic: trp, Tyr, Phe.

Non-conservative substitutions are those that replace a member of one of these classes with another.

Chemical pegylation of erythropoietin typically results in a protein preparation comprising erythropoietin pegylated at one or more-amino groups of a lysine residue and/or at the N-terminal amino group. Selective pegylation at the N-terminal amino acid was performed according to Felix, A.M., et al, ACS Symp.Ser.680(Poly (ethylene glycol)) (1997) 218-238. By making N^α-pegylated ammoniaThe coupling of the amino acid derivative to the N-1 terminal amino acid of the peptide chain allows for selective N-terminal pegylation during solid phase synthesis. By making NCoupling of pegylated lysine derivatives to growing chains allows side chain pegylation during solid phase synthesis. Combined N-terminal and side chain pegylation is possible within solid phase synthesis as described above, or by solution phase synthesis by adding an activated PEG reagent to the amino deprotected peptide.

Suitable PEG derivatives are activated PEG molecules: in one embodiment, it has an average molecular weight of about 5kDa to about 40kDa, in another embodiment, it has an average molecular weight of about 20kDa to about 40kDa, and in another embodiment, it has an average molecular weight of about 30kDa to about 35 kDa. The PEG derivative may be a linear or branched PEG. A wide variety of PEG derivatives are available that are suitable for use in the preparation of PEG-protein and PEG-peptide conjugates.

Activated PEG derivatives are known in the art and are described, for example, in morphugo, m., et al, j.bioconjugate.chem.7 (1996) 363-. Linear chain and branched PEG species are suitable for preparing pegylated fragments. Examples of reactive PEG reagents are iodo-acetyl-methoxy-PEG, or methoxy-PEG-vinyl sulfone (in one embodiment, m is an integer from about 450 to about 900, and R is a linear or branched lower alkyl group having 1-6 carbon atoms, such as methyl, ethyl, isopropyl, etc., with methyl being preferred):

the use of these iodine-activated substances is known in the art, see for example Hermanson, G.T., Bioconjugate Techniques, Academic Press, San Diego (1996) p 147-148.

In one embodiment, the PEG species is an activated PEG ester, for example, N-hydroxysuccinimidyl propionate, or N-hydroxysuccinimidyl butyrate, or N-hydroxysuccinimidyl such as PEG-NHS (Monfardini, C., et al, Bioconjugate chem.6(1995) 62-69). In one embodiment, an alkoxy-PEG-N-hydroxysuccinimide, such as methoxy-PEG-N-hydroxysuccinimide (MW30000), wherein R and m are as defined above, is used to activate PEG with N-hydroxysuccinimide ester.

In one embodiment, the PEG species is N-hydroxysuccinimide ester of methoxypoly (ethylene glycol) -butyric acid. The term "alkoxy" refers to an alkyl ether group, wherein the term "alkyl" means a straight or branched chain alkyl group containing up to 4 carbon atoms, such as methoxy, ethoxy, n-propoxy, and the like, preferably methoxy.

The term "substantially homogeneous form" means that the erythropoietin protein fusions or conjugates obtained, contained or used are those having a defined number of additional PEG residues. In one embodiment, the pegylated erythropoietin is a monopegylated erythropoietin. The preparation may contain unreacted (i.e., lacking PEG groups) erythropoietin, polypegylated erythropoietin, and polypeptide fragments produced during the pegylation reaction. The term "substantially homogeneous form" means that a mono-pegylated erythropoietin preparation contains at least 50% (w/w) mono-pegylated erythropoietin, or at least 75% mono-pegylated erythropoietin, or at least 90% mono-pegylated erythropoietin, or more than 95% mono-pegylated erythropoietin. The percentage values are based on the area% of a chromatogram corresponding to the chromatographic method used to obtain the monopegylated erythropoietin.

Herein is reported a method for purifying pegylated erythropoietin in order to obtain monopegylated erythropoietin in a substantially homogeneous form. It has been found that prior to applying the pegylated erythropoietin preparation, the chromatographic material must be conditioned (conditioning) with a solution having a conductivity of about 21 mS/cm. The separation efficiency of different substances of pegylated erythropoietin preparations is lower if the chromatographic material is adjusted with a lower conductivity. Also, it is not advantageous to adjust the conductivity of a solution of a pegylated erythropoietin preparation prior to application to a chromatographic material. Furthermore, the use of a step gradient method is less effective because non-pegylated erythropoietin cannot be quantitatively recovered from the chromatographic material. Recovery of unreacted starting materials is beneficial because it can be reused in the pegylation reaction.

Accordingly, the present invention provides a method for obtaining monopegylated erythropoietin using SPSephacryl S500HR chromatography material in a single step, wherein a solution having a conductivity of about 21mS/cm is first applied onto the chromatography material, and then a solution comprising a pegylated erythropoietin preparation is applied onto the chromatography material. It has been found that the conductivity of the first solution must be precisely controlled in order to ensure separation of the individual components of the crude protein preparation.

Thus, the method as reported herein for obtaining a protein conjugate comprising erythropoietin and a single poly (ethylene glycol) residue comprises the following steps:

a) a solution having a conductivity of about 21mS/cm was applied to a chromatography column containing SP Sephacryl S500HR chromatography material,

b) applying a solution to the column of a), said solution comprising a mixture of free erythropoietin and protein conjugates of erythropoietin and poly (ethylene glycol) wherein each erythropoietin molecule has one or more poly (ethylene glycol) residues,

c) applying a solution having a conductivity of about 21mS/cm to the column and thereby recovering free poly (ethylene glycol) and protein comprising 2 or more poly (ethylene glycol) residues,

d) applying a solution to the column having a conductivity that continuously and linearly increases up to a final value of about 62.5mS/cm and thereby recovering the protein comprising erythropoietin and a single poly (ethylene glycol) residue and free erythropoietin, respectively, wherein the protein comprising erythropoietin and a single poly (ethylene glycol) residue is obtained first.

It has been found that in order to ensure separation of the individual components of the preparation, a solution having a conductivity of about 21mS/cm must first be applied to the chromatographic material.

In one embodiment, the method is a column chromatography method.

In one embodiment, up to 8 column volumes of a solution having a conductivity of about 21mS/cm are applied to the chromatographic material prior to applying the solution comprising the pegylated erythropoietin preparation. In one embodiment, the solution having a conductivity of about 21mS/cm is a solution having a pH of from pH2.5 to pH 3.5. In one embodiment, the solution having a conductivity of about 21mS/cm is a phosphate buffered solution having a pH of from pH2.5 to pH 3.5.

After applying the solution comprising the pegylated erythropoietin preparation, a solution having a conductivity of about 21mS/cm is applied to the column and thereby recovering free poly (ethylene glycol) and the fusion protein (i.e., protein conjugate) comprising 2 or more poly (ethylene glycol) residues from the chromatographic material. In one embodiment, up to 8 column volumes of a solution having a conductivity of about 21mS/cm is applied to the chromatographic material.

After the pegylated erythropoietin has been recovered from the chromatography material, continuous elution using a linear conductivity gradient is started. The conductivity of the mobile phase passing through the chromatographic material is continuously and linearly increased to a conductivity of at least about 62.5 mS/cm. In the linear gradient, monopegylated erythropoietin is first recovered from the column, followed by substantially homogeneous unpegylated erythropoietin. In one embodiment, the conductivity is increased by applying a solution with increasing sodium chloride concentration. In one embodiment, the conductivity is increased by applying a solution having a pH value of pH2.5 to pH 3.5. In one embodiment, the conductivity is increased from a value of about 21mS/cm to a final value of at least 62.5mS/cm, which is achieved within 10 column volumes of the mobile phase volume applied.

In one embodiment, the solution having a conductivity of about 21mS/cm is about 100mM sodium or potassium phosphate buffer solution, having a pH of about pH3.0, and having (i.e., containing) about 120mM sodium chloride.

In one embodiment, the linear gradient is a sodium chloride concentration gradient from about 120mM to about 1000mM sodium chloride in about 100mM sodium or potassium phosphate buffer (pH about pH 3.0).

In one embodiment, a solution comprising a mixture of free erythropoietin and free poly (ethylene glycol) and fusion proteins of erythropoietin and poly (ethylene glycol) (i.e., protein conjugates) wherein each erythropoietin molecule has one or more poly (ethylene glycol) residues is applied to 1ml of chromatographic material in a manner that 1mg/ml to 4mg/ml protein is applied to the chromatographic material.

The term "SP Sephacryl S500HR chromatography material" refers to a cation exchange chromatography material, also known as MacroCap SP (both available from GE Healthcare). In one embodiment, the SP Sephacryl S500HR chromatographic material is a crosslinked copolymer of allyl dextran and N, N-methylene bisacrylamide, which has sulfonic acid as the chromatographic functional group, and is thus a strong cation exchange chromatographic material.

The following examples, sequence listing and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is to be understood that modifications may be made to the steps described without departing from the spirit of the invention.

Description of the sequence listing

SEQ ID NO: 01 amino acid sequence of human erythropoietin.

SEQ ID NO: 02 amino acid sequence of human erythropoietin.

Drawings

Figure 1 elution chromatogram of a purified pegylated erythropoietin preparation using the method reported herein.

Figure 2 SEC analytical chromatograms of peak fractions 1, 2 and 3 of figure 1.

FIG. 3 SEC analytical chromatogram of separated peak fractions 1, 2 and 3, wherein the column has been conditioned with a solution having a higher conductivity.

Figure 4 elution chromatogram of a purified pegylated erythropoietin preparation using a fractionated elution method.

FIG. 5 SEC analytical chromatograms of peak fractions 1, 2 and 3 (regenerated peaks) of FIG. 4.

Figure 6 elution chromatogram of a purified pegylated erythropoietin preparation using the method reported herein, wherein the conductivity of the sample solution was previously adjusted to 20 mS/cm.

Figure 7 SEC analytical chromatograms of peak fractions 1, 2 and 3 of figure 6.

Examples

Materials and methods

Analytical size exclusion chromatography:

resin: TSK3000(Tosohaas)

Column: 300x7.8mm

Flow rate: 0.5ml/min

Elution solution: 200mM potassium phosphate, containing 250mM potassium chloride, adjusted to pH7.0

Wavelength: 220nm

Chromatographic separation of pegylated erythropoietin:

resin: SP Sephacryl S500HR

Bed volume: 2.5ml

Sample loading: mg/ml resin-variables (see examples below)

Flow rate: 0.5ml/min

Solution: a: 100mM potassium phosphate, adjusted to pH3.0

B: 100mM potassium phosphate, 1000mM sodium chloride, adjusted to pH3.0

Loading solution: 88% A and 12% B

Washing solution: 88% A and 12% B

Washing volume: 20ml (8 Column Volumes (CV))

Linear gradient elution solution: 100% B

Linear gradient: to 100% B in 25ml (10 column volumes)

Wavelength: 254nm, 280nm

Example 1

Chromatographic separation of pegylated erythropoietin preparations using SP-Sephacryl chromatography material, conditioned with a solution having a conductivity of about 21mS/cm

Chromatographic separation of pegylated erythropoietin is carried out as described in the materials and methods section.

The elution chromatogram for this method is shown in figure 1. Analytical size exclusion chromatograms of oligo-pegylated erythropoietin peak fraction 1 and mono-pegylated peak fraction 2 and non-pegylated peak fraction 3 are shown in figures 2A-C.

TABLE 2

Example 2

Chromatographic separation of pegylated erythropoietin preparation using SP-Sephacryl chromatography material, conditioned with a solution having a conductivity of about 29mS/cm

As described in the materials and methods section, chromatographic separation of pegylated erythropoietin was performed using the following different parameters:

loading solution: 80% A and 20% B

Washing solution: 80% A and 20% B

Washing volume: 20ml (8 Column Volumes (CV))

Linear gradient elution buffer: 100% B

Linear gradient: up to 50% B in 25ml (10 column volumes)

Analytical size exclusion chromatograms of oligo-pegylated erythropoietin peak fraction 1 and mono-pegylated peak fraction 2 and non-pegylated peak fraction 3 are shown in figures 3A-C.

TABLE 3

Example 3

Regulation using a solution having a conductivity of about 21mS/cm and gradient elution using a solution having a conductivity of 37mS/cm, chromatographic separation of a pegylated erythropoietin preparation using SP-Sephacryl chromatography material

Chromatographic separation of pegylated erythropoietin is carried out as described in the materials and methods section.

The elution chromatogram for this method is shown in figure 4. Analytical size exclusion chromatograms of oligo-pegylated erythropoietin peak fraction 1 and mono-pegylated peak fraction 2 and non-pegylated peak fraction 3 are shown in figures 5A-C. It must be noted that erythropoietin which is not pegylated can only be recovered during column regeneration and cannot be recovered by gradient elution.

TABLE 4

Example 4

Conditioning using a solution having a conductivity of about 21mS/cm and conditioning the sample to a conductivity of 20mS/cm, chromatographic separation of a pegylated erythropoietin preparation using SP-Sephacryl chromatography material

Chromatographic separation of pegylated erythropoietin is carried out as described in the materials and methods section.

The elution chromatogram for this method is shown in fig. 6. Analytical size exclusion chromatograms of oligo-pegylated erythropoietin peak fraction 1 and mono-pegylated peak fraction 2 and non-pegylated peak fraction 3 are shown in figures 7A-C.

TABLE 5

Claims (8)

1. A method for obtaining a protein comprising erythropoietin and a single poly (ethylene glycol) residue, comprising the steps of:

a) applying a solution to a column comprising SP Sephacryl S500HR chromatography material, said solution comprising a mixture of erythropoietin and conjugates of erythropoietin with poly (ethylene glycol) and having one or more poly (ethylene glycol) residues per erythropoietin molecule in said conjugates, said column having applied thereto a solution having a conductivity of 21mS/cm,

b) applying a solution having a conductivity of 21mS/cm to the column, thereby recovering free poly (ethylene glycol) and protein comprising 2 or more poly (ethylene glycol) residues,

c) applying a solution to the column and thereby recovering the protein comprising erythropoietin and a single poly (ethylene glycol) residue and erythropoietin, respectively, wherein the protein comprising erythropoietin and a single poly (ethylene glycol) residue is first recovered, the solution having a conductivity which increases linearly up to a final value of 62.5mS/cm,

wherein the solution having a conductivity of 21mS/cm is a phosphate buffer solution.

2. The method according to claim 1, characterized in that the solution having a conductivity of 21mS/cm is a solution having a pH value of pH2.5 to pH 3.5.

3. The method according to any of the preceding claims, characterized in that the solution comprising erythropoietin and a mixture of conjugates of erythropoietin with poly (ethylene glycol) in which each erythropoietin molecule has one or more poly (ethylene glycol) residues is not adjusted to a conductivity of 21 mS/cm.

4. Method according to any of claims 1-2, characterized in that the solution with linearly increasing conductivity is a solution with linearly increasing sodium chloride concentration.

5. The method according to any of claims 1-2, characterized in that the pH value of the solution with linearly increasing conductivity is pH 2.3 to pH 3.5.

6. The method of any one of claims 1-2, wherein said erythropoietin is human erythropoietin.

7. The method of claim 6, wherein said human erythropoietin has the amino acid sequence of SEQ ID NO: 01 or SEQ ID NO: 02, or a pharmaceutically acceptable salt thereof.

8. The method of any one of claims 1-2, wherein the single poly (ethylene glycol) residue has a molecular weight of 20kDa to 40 kDa.