HK40004614A

HK40004614A - Method for purifying pegylated erythropoietin

Info

Publication number: HK40004614A
Application number: HK19128089.0A
Authority: HK
Inventors: Roberto Falkenstein; Bernhard SPENSBERGER
Original assignee: F. Hoffmann-La Roche Ag
Priority date: 2016-07-15
Filing date: 2017-07-13
Publication date: 2020-04-29

Description

Method for purifying pegylated erythropoietin

Herein is reported a method for the purification of pegylated erythropoietin using a single column method of cation exchange chromatography material.

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 biopharmaceutical active agents for humans, it is particularly necessary 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 (EP 0442724), PEG and erythropoietin (WO 01/02017), chimeric molecules comprising endostatin and immunoglobulins (US 2005/008649), secreted antibody-based fusion proteins (US 2002/147311), albumin-containing fusion polypeptides (US 2005/0100991; human serum albumin US5,876,969), pegylated polypeptides (US 2005/0114037) 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 WO 89/05157, 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 WO 2004/024866 a method for purifying polypeptides by ion exchange chromatography is reported, wherein gradient washing is used to dissociate the polypeptide of interest from one or more contaminants. In EP 0530447, 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 facile purification of mono-pegylated interleukin-1 receptor antagonists. Wang, H.et al, Peptides 26(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.

A method for the purification of pegylated erythropoietin in a SP Sephacryl S500 HR column is reported in WO 2012/035037.

WO 1999/057134 reports protein purification by ion exchange chromatography.

In WO 2009/010270 a process for the purification of monopegylated erythropoietin is reported, which comprises two cation exchange chromatography steps wherein the same type of cation exchange material is used in the cation exchange chromatography step.

Summary of The 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 applying a washing step with a washing solution having an increased pH, it is possible to use cation exchange chromatography materials, for exampleSP-650, a conjugated protein comprising erythropoietin and a single poly (ethylene glycol) residue is obtained in high purity and yield in a single step in an improved and simplified manner.

It has been found that the yield and quality are at least comparable or better with two sequential cation exchange chromatography steps compared to the purification process of a protein conjugate comprising erythropoietin and a single poly (ethylene glycol) residue. One column process allows to obtain the same quality while increasing the robustness of the process. It also reduces manufacturing costs and production time. The final yield can be increased without losing the quality of the monopegylated erythropoietin.

Thus, herein is reported a method for obtaining/purifying/producing a protein comprising erythropoietin and a single poly (ethylene glycol) residue, the method comprising the steps of:

a) applying the solution to a container containingSP-650 as a column of chromatographic material, said solution comprising a mixture of erythropoietin and conjugates of erythropoietin with poly (ethylene glycol) in which each erythropoietin molecule has one or more poly (ethylene glycol) residues, said column having applied thereto a first solution having a pH of about 2.4 to about 2.7,

b) a second solution having an increased pH value (pH of about 2.4 to about 2.7) relative to the first solution is applied,

c) applying a solution having an increased or gradually increased conductivity to the column, thereby recovering a protein comprising erythropoietin and a single poly (ethylene glycol) residue.

As one aspect, herein is reported a method for obtaining/purifying/producing a protein comprising erythropoietin and a single poly (ethylene glycol) residue, the method comprising the steps of:

a) applying a solution to a column comprising a chromatographic material having a methacrylate matrix with sulfopropyl groups as functional groups, the solution comprising a mixture of erythropoietin and conjugates of erythropoietin with poly (ethylene glycol) and erythropoietin in which each erythropoietin molecule has one or more poly (ethylene glycol) residues, the column having applied thereto a first solution having a pH of about 2.4 to about 2.7,

b) a second solution having an increased pH value relative to the first solution (pH of about 2.4 to about 2.7) is applied,

In one embodiment, the method further comprises the step of reapplying the first solution having a pH of about 2.4 to about 2.7 after step b) and before step c). Thus, in one embodiment is reported a method for obtaining/purifying/producing a protein comprising erythropoietin and a single poly (ethylene glycol) residue, the method comprising the steps of:

a) applying a solution to a column comprising a Toyopearl SP-650 chromatographic material, said solution comprising a mixture of erythropoietin and conjugates of erythropoietin with poly (ethylene glycol) and erythropoietin in which each erythropoietin molecule has one or more poly (ethylene glycol) residues, said column having applied thereto a first solution having a pH of about 2.4 to about 2.7,

b) applying a second solution having an increased pH value relative to a first solution having a pH value of about 2.4 to about 2.7,

b1) the first solution having a pH of about 2.4 to about 2.7 is then applied,

In one embodiment, the second solution having an increased pH is a solution having a pH of about 2.7 to about 3.2, preferably a pH of about 2.7 to 3.0.

In one embodiment, the second solution with increased pH is a solution with a constant conductivity value.

In one embodiment, the second solution having an increased pH and the first solution having a pH of about 2.4 to about 2.7 have substantially the same, constant conductivity values.

In one embodiment, the second solution having an increased pH and/or the first solution having a pH of from about 2.4 to about 2.7 has a constant conductivity value of 19mS/cm, preferably from about 17mS/cm to about 19 mS/cm.

In one embodiment, the second solution having an increased pH has a pH of about 2.7 to about 3.2 and has a conductivity value of about 19 mS/cm.

In one embodiment, the second solution having an increased pH is a phosphate buffered solution.

In one embodiment, a solution comprising a mixture of erythropoietin and conjugates of erythropoietin with poly (ethylene glycol) having one or more poly (ethylene glycol) residues per erythropoietin molecule in the conjugate is not adjusted to a conductivity value of about 19 mS/cm.

In one embodiment, the solution with increased or gradually increasing conductivity is a solution with an increasing or gradually increasing concentration of sodium chloride. In one embodiment, the solution with increased or gradually increased conductivity has a pH value of pH 2.3 to pH 3.5.

In one embodiment, a solution with increased or gradually increasing conductivity has a stepwise or linear increase in conductivity.

In one embodiment, the method is for large scale protein production, wherein the chromatography column of step a) has a diameter of at least 30 cm.

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

a1) the first solution having a pH of about 2.4 to about 2.7 is then applied,

b) applying a second solution having an increased pH value relative to a first solution having a pH of about 2.4 to about 2.7,

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 the gradient elution method is on a cation exchange chromatography column, e.g. comprisingOn a column of SP-650, in which cation exchange chromatography is carried outThe SP-650 column has been washed with two wash solutions and then started to recover/elute proteins containing one erythropoietin molecule and one poly (ethylene glycol) residue. Herein, the second wash solution has an increased pH value relative to the first wash solution.

General chromatographic methods and their use are known to the person skilled in the art. See, for example: heftmann, E., (eds.), chromatograpy, 5 th edition, Part A: Fundamentals and technologies, Elsevier science publishing Company, New York (1992); deyl, Z., (ed.), Advanced chromatography and electro migration 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, second 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 wherein 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 apparatus, allowing the chromatographic material to interact with the substances contained in the solution. Depending on conditions such as pH, conductivity, salt concentration, temperature and/or flow rate, some substances in the solution bind to the chromatographic material and can thus be recovered from the chromatographic 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 the applied solution or a buffer solution used for washing the column or for causing 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 the mode of operation of a chromatography step, wherein a solution containing the target substance to be purified is applied to the 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 from 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 is operated 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 consisting of phosphoric acid and/or a salt thereof, or an acetate buffer solution consisting of acetic acid and a salt thereof, or a citrate buffer solution consisting 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. It is understood in the art that the buffer solution is prepared under conditions comparable to those used subsequently. For example, the pH of the buffer solution is adjusted at a temperature comparable to the temperature subsequently used in the intended process. For example, if the buffer solution is used in a chromatography process carried out at 4 ℃, the pH can be adjusted when the buffer solution has a comparable temperature and is, for example, not at 30 ℃. In one embodiment, the second solution having an increased pH is a phosphate buffered solution.

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 2% or 1% variation of 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 terms "step elution", "step elution method" and "step elution method" are used interchangeably in this application and refer to a method wherein e.g. the concentration of the substance causing the elution, i.e. the bound compound is dissolved from the material, is either increased or decreased immediately, i.e. increased directly from one value/level to the next. In this "step elution" one or more conditions, such as pH, ionic strength, salt concentration and/or chromatographic flow rate, are all changed from a first, e.g. starting value, to a second, e.g. final value, i.e. the conditions are changed incrementally, i.e. stepwise, as opposed to linearly. In the "step elution method", a new fraction is collected after each increase in ionic strength. This fraction contains the compounds recovered from the ion exchange material, with a corresponding increase in ionic strength. After each increase, conditions were maintained until the next step in the elution process. In "step elution", one or more conditions are completely changed from a first, e.g., starting value, to a second, e.g., final value, instantaneously. The change may be 10% or more of the concentration of the substance causing elution. That is, in the case where the concentration of the substance causing elution in the first step is 100%, 110% or more in the second step, and 120% or more in the third step. The change may also be 50% or more of the concentration of the substance causing elution. Further, the change may be 120% or more of the concentration of the substance causing elution. "step elution" means that the conditions change incrementally, i.e., stepwise, as opposed to linearly.

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. "ion exchange resin" refers to either 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 resin" refers to: for example, in the case of cation exchange resins, sulfonic acid resins (S) or sulfopropyl resins (SP) or carboxymethyl resins (CM). Depending on the chemistry of the charged groups/substituents, "ion exchange resins" can also be classified as strong or weak ion exchange resins, depending on the strength of the covalently bonded charged substituents. For example, the strong cation exchange resin has a sulfonic acid group, preferably a sulfopropyl group, as a charged substituent, the weak cation exchange resin has a carboxyl group, preferably a carboxymethyl group, as a charged substituent, and the weak anion exchange resin has a diethylaminoethyl group as a charged substituent. In one embodiment, the cation exchange chromatography material is a strong cation exchange chromatography material. In one embodiment, the cation exchange chromatography material isSP-650 chromatographic material. In one embodiment, the cation exchange chromatography material isSP-650M chromatography material.

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" means 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, either resulting from chemical synthesis of the molecule, or as 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 in e.g. EP 0473084, 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 to a poly (ethylene glycol) residue" means the attachment of a poly (ethylene glycol) residue covalently 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 method is also referred to as pegylation and the product thereof is 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., Biomaterials 22(2001) 405-417. 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 Systems 9(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, as described, for example, in WO 00/44785. Fusion can also be carried out on a solid phase according to Lu, Y. et al, Reactive Polymers 22(1994) 221-. According to WO 94/01451, it is also possible to generate non-random 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 polydiethylated erythropoietin (oligomeric pegylated erythropoietin), monopegylated erythropoietin (having a single poly (ethylene glycol residue), non-pegylated erythropoietin, a hydrolysate of an activated PEG ester, and a hydrolysate of the erythropoietin itself.

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

a) applying a solution comprising a mixture of erythropoietin and conjugates of erythropoietin with poly (ethylene glycol) in which each erythropoietin molecule has one or more poly (ethylene glycol) residues to a column comprising a cation exchange chromatography material, the column having applied thereto a first solution having a pH of about 2.4 to about 2.7,

c) applying a solution having a gradually increasing conductivity to the column, thereby separately recovering the protein comprising erythropoietin and a single poly (ethylene glycol) residue, and the erythropoietin, thereby first recovering the protein comprising erythropoietin and a single (ethylene glycol) residue.

In one embodiment, the cation exchange chromatography material is a strong cation exchange chromatography material. In one embodiment, the cation exchange chromatography material isSP-650 chromatographic material. In one embodiment, the cation exchange chromatography material isSP-650M chromatography material.

In one embodiment, the second solution with increased pH is a solution with a constant conductivity value. In one embodiment, the second solution having an increased pH and/or the first solution having a pH of about 2.4 to about 2.7 has a constant conductivity value of about 17mS/cm to about 19 mS/cm. In one embodiment, the second solution having an increased pH has a pH of about 2.7 to about 3.0 and has a conductivity value of about 17mS/cm to about 19 mS/cm.

In one embodiment, the second solution having an increased pH has a pH of about 2.7 to about 3.2 and has a conductivity value of about 17mS/cm to about 19 mS/cm. In one embodiment, the second solution having an increased pH has a pH of about 2.7 to about 3.0 and has a conductivity value of about 17mS/cm to about 19 mS/cm. In a preferred embodiment, the second solution having an increased pH has a pH of about 2.7 to about 2.9 and has a conductivity value of about 17mS/cm to about 19mS/cm, or a pH of about 2.7 to about 3.0 and a conductivity value of about 17mS/cm to about 18 mS/cm.

In one embodiment, the second solution having an increased pH and the first solution having a pH of about 2.4 to about 2.7 have about the same constant conductivity value.

In one embodiment, the cation exchange chromatography material has a matrix of methacrylate with sulfopropyl groups as functional groups. In one embodiment, the cation exchange chromatography material has a particle size of about 65 μm.

The method is particularly suitable for the purification of pegylated recombinant erythropoietin, which is glycosylated, i.e. which has been prepared from mammalian cells, in one embodiment from CHO cells or HEK293 cells or BHK cells or Per.Cells or HeLa cells were prepared and subsequently 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 a 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. for example, 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 red blood cell production 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 (i.e. expression of erythropoietin glycoprotein by endogenous gene activation), see e.g. US5,733,761, US5,641,670, US5,733,746, WO 93/09222, WO 94/12650, WO 95/31560, WO90/11354, WO 91/06667 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 denotes 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 comparable biological activity to the unmodified protein, such as those reported in EP 1064951 or US 6,583,272. 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 uninjected or control individuals. The biological activity of pegylated Erythropoietin obtained and purified according to the methods reported herein can be tested by the method of Bristow, 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

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 of the epsilon amino groups of a lysine residue and/or at the N-terminal amino group. According to Felix, A.M. et al, ACS Symp.Ser.680(Poly (ethylene glycol)) (1997)218-238, selective pegylation at the N-terminal amino acid can be performed. By making N^αCoupling of the PEGylated amino acid derivative to the N-1 terminal amino acid of the peptide chain allows selective N-terminal PEGylation during solid phase synthesis. By making N^εThe coupling of the pegylated lysine derivatives to the growing chain allows the side chain pegylation to be achieved during solid phase synthesis. Combined N-terminal and side chain pegylation is possible either within solid phase synthesis as described above, or by solution phase synthesis by applying 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 a further 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 variety of PEG derivatives suitable for use in the preparation of PEG-protein and PEG-peptide conjugates are available.

Activated PEG derivatives are known in the art and are described, for example, in M Morpurgo, M.et al, J.Bioconjug.chem.7(1996)363-368 (in the case of PEG-vinylsulfone). Both straight-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 and is described, for example, in Hermanson, G.T., Bioconjugate Techniques, Academic Press, San Diego (1996) pp.147-148.

In one embodiment, the PEG species is an activated PEG ester, such as N-hydroxysuccinimidyl propionate or N-hydroxysuccinimidyl butyrate or N-hydroxysuccinimide, such as PEG-NHS (Monfardini, C. et al, Bioconjugate chem.6(1995) 62-69). In one embodiment, PEG is activated by N-hydroxysuccinimide ester

Wherein an alkoxy-PEG-N-hydroxysuccinimide, for example methoxy-PEG-N-hydroxysuccinimide (MW 30000), is used, wherein R and m are as defined above. In one embodiment, the PEG species is N-hydroxysuccinimidyl ester of methoxypoly (ethylene glycol) -butyric acid. The term "alkoxy" refers to an alkyl ether group, wherein the term 'alkyl' refers to a straight or branched chain alkyl group containing a maximum of 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 attached PEG residues. In one embodiment, the pegylated erythropoietin is a mono-pegylated 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 the chromatogram corresponding to the chromatographic method used to obtain the monopegylated erythropoietin.

Herein is reported a process for the purification of pegylated erythropoietin in order to obtain a mono-pegylated erythropoietin in a substantially homogeneous form. It has been found that the purification process can be improved using/utilizing only a single chromatography step, resulting in a simplified and more technically feasible process.

Thus, the present invention provides a method which uses, in a single chromatographic step, a single chromatographic stepSP-650 chromatographic material, obtained/produced/purified mono-pegylated erythropoietin by introducing/applying (in addition to the normal washing step) washing steps using a solution having a pH value which corresponds to the increase of the pH value in the first washing step. It has been found that the removal/reduction of impurities of oligo-pegylated erythropoietin in mono-pegylated erythropoietin can be achieved by using this additionalThe washing step of (2).

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

c) applying a solution having a gradually increasing conductivity to the column, thereby recovering a protein comprising erythropoietin and a single poly (ethylene glycol) residue.

It has been found that the separation of the monopegylated erythropoietin from the by-products/removal of the by-products is improved if after the application of the wash solution with the increased pH a first wash solution with a lower pH is applied. Thus, in one embodiment, the method further comprises the step of reapplying the first solution having a pH of about 2.4 to about 2.7 after step b) and before step c).

It has been found that in the additional/second washing step the second solution should have an increased pH value, wherein this increase reaches a certain magnitude.

In one embodiment, the pH of the second solution having the increased pH value is a solution having a pH of about 2.7 to about 3.1. In a preferred embodiment, the pH of the second solution having the increased pH value is a solution having a pH of about 2.7 to about 3.0. In a preferred embodiment, the pH of the second solution having the increased pH value is a solution having a pH of about 2.7 to about 2.9. In one embodiment, the pH of the second solution having the increased pH value is a solution having a pH of about 2.8 to about 3.0.

In one embodiment, the pH of the second solution with the increased pH value is increased by at most 25% over the first solution. In one embodiment, the pH of the second solution with the increased pH value is increased by up to 20% over the first solution. In one embodiment, the pH of the second solution with the increased pH value is increased by at most 15% over the first solution.

In one embodiment, the pH of the second solution with the increased pH value is increased by 0.5 pH units over the first solution. In one embodiment, the pH of the second solution with the increased pH value is increased by 0.4 pH units over the first solution. In one embodiment, the pH of the second solution with the increased pH value is increased by 0.3 pH units over the first solution. In one embodiment, the pH of the second solution with the increased pH value is increased by 0.2 pH units over the first solution. In one embodiment, the pH of the second solution with the increased pH value is increased by 0.1 pH units over the first solution.

In one embodiment, the pH of the second solution with the increased pH value is increased by 0.1 to 0.5 pH units over the first solution. In one embodiment, the pH of the second solution with the increased pH value is increased by 0.3 to 0.5 pH units over the first solution.

Solutions with increasing pH values should be applied as solutions with constant conductivity, i.e. the conductivity values vary at most +/-10%, preferably at most +/-5%.

In one embodiment, the second solution with increased pH is a solution with a constant conductivity value. In one embodiment, the second solution having an increased pH and/or the first solution having a pH of about 2.4 to about 2.7 has a constant conductivity value of about 17 mS/cm. In one embodiment, the second solution having an increased pH and/or the first solution having a pH of about 2.4 to about 2.7 has a constant conductivity value of about 18 mS/cm. In one embodiment, the second solution having an increased pH and/or the first solution having a pH of about 2.4 to about 2.7 has a constant conductivity value of about 19 mS/cm. In one embodiment, the second solution having an increased pH has a pH of about 2.7 to about 3.0 and has a conductivity value of about 17 mS/cm. In one embodiment, the second solution having an increased pH has a pH of about 2.7 to about 3.0 and has a conductivity value of about 18 mS/cm. In one embodiment, the second solution having an increased pH has a pH of about 2.7 to about 2.9 and has a conductivity value of about 19 mS/cm.

In one embodiment, the second solution having an increased pH has a pH of about 2.7 to about 3.0 and has a conductivity value of about 17mS/cm to about 19 mS/cm. In a preferred embodiment, the second solution having an increased pH has a pH of about 2.7 to about 2.9 and has a conductivity value of about 17mS/cm to about 19 mS/cm.

It is understood in the art that pH and conductivity are related to each other. Thus, if the conductivity also changes, the pH of the wash solution may be different from that described above. For example, as the conductivity is lower, the pH can be increased to a higher degree while achieving comparable purification results.

It has been found that when applying a solution with an increased pH value, the conductivity should remain constant with respect to the first washing solution. In one embodiment, the second solution having an increased pH and the first solution having a pH of about 2.4 to about 2.7 have about the same constant conductivity value.

After recovering the pegylated erythropoietin from the chromatography material, elution of the mono-pegylated erythropoietin is started by increasing the conductivity. The conductivity of the mobile phase through the chromatographic material is linear or stepwise increasing.

In one embodiment, the solution with gradually increasing conductivity has a linear or stepwise increasing conductivity.

In the conductivity gradient, the monopegylated erythropoietin is first recovered from the column, and then the substantially homogeneous non-pegylated erythropoietin is recovered.

In one embodiment, the conductivity is increased by applying a solution with a gradually increasing sodium chloride concentration. In one embodiment, the solution for increasing the conductivity has a pH value of pH 2.5 to pH 3.5.

In one embodiment, a solution comprising a mixture of free erythropoietin and free poly (ethylene glycol) and a fusion protein (i.e., protein conjugate) of erythropoietin with poly (ethylene glycol) wherein each erythropoietin molecule comprises one or more poly (ethylene glycol) residues is applied to a chromatographic material, wherein the protein is applied to 1mL of the chromatographic material at 1mg/mL to 4 mg/mL.

It has been found that the method as reported herein (especially when usedSP-650 as chromatography material) can be used for large scale purification of proteins.

Term "SP-650 chromatographic material "refers to cation exchange chromatographic material (available from Tosohcorporation).SP-650 chromatographic materials have a methacrylate matrix with sulfopropyl groups as functional groups and are therefore strong cation exchange chromatographic materials.SP-650M chromatography material had a particle size of 65 μ M.

In one embodiment, the cation exchange chromatography material has a methacrylate matrix. In one embodiment, the cation exchange chromatography material has sulfopropyl groups as functional groups. In one embodiment, the cation exchange chromatography material has a methacrylate matrix with sulfopropyl groups as functional groups. In one embodiment, the cation exchange chromatography material has a particle size of about 65 μm.

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 will be appreciated that modifications can be made to the method 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.

Description of the drawings

FIG. 1 elution chromatogram of a purified pegylated erythropoietin preparation using the method reported in example 1 (wash at pH 2.5; no additional washing step).

Figure 2 elution chromatogram of a purified pegylated erythropoietin preparation using the method reported in example 3 (wash at pH 2.5; additional wash step at pH 2.8).

Figure 3 an enlarged view (wash at pH 2.5; additional wash step at pH 2.8) of the elution chromatogram (figure 2) of a purified pegylated erythropoietin preparation using the method reported in example 3.

Figure 4 elution chromatogram of a purified pegylated erythropoietin preparation using the method reported in example 2 (wash at pH 3.0; no additional washing step).

Figure 5 elution chromatogram of a purified pegylated erythropoietin preparation using the method reported in example 4 (wash at pH 2.5; additional wash step at pH 3.0).

Example 1

Use ofChromatography of SP-650M chromatography material, pegylated erythropoietin without additional washing step (pH 2.5)

Chromatography of pegylated erythropoietin was performed as follows.

Pegylated erythropoietin chromatography:

resin: SP Toyopearl 650M

Bed volume: 19.6mL

Loading: 1.3mg/mL resin

Flow rate: 1.3 mL/min

Solution: a: 100mM potassium phosphate, 100mM sodium chloride, adjusted to pH 2.5, adjusted to LF ═ 19mS/cm (with 5m sodium chloride)

B: 100mM potassium phosphate, 375mM sodium chloride, adjusted to pH 2.5

Application solution: 100% A

Washing solution: 100% A

Washing volume: 100mL (5 Column Volumes (CV))

Washing solution (washing 2): is free of

Wash volume (wash 2): is free of

Linear gradient elution solution: 100% B

Linear gradient: to 100% B in 196mL (10 column volumes)

Wavelength: 280nm

The elution chromatogram for this method is shown in figure 1.

Example 2

Use ofSP-650M chromatography material, chromatography of pegylated erythropoietin without additional washing step (pH 3.0)

Chromatography of pegylated erythropoietin was performed as follows.

Pegylated erythropoietin chromatography:

resin: SP Toyopearl 650M

Bed volume: 19.6mL

Loading: 1.3mg/mL resin

Flow rate: 1.3 mL/min

Solution: a: 100mM potassium phosphate, 100mM sodium chloride, adjusted to pH 3.0, conductivity value 20.5mS/cm

B: 100mM potassium phosphate, 375mM sodium chloride, adjusted to pH 2.5

Application solution: 100% A

Washing solution: 100% A

Washing volume: 100mL (5 Column Volumes (CV))

Washing solution (washing 2): is free of

Wash volume (wash 2): is free of

Linear gradient elution solution: 100% B

Linear gradient: 196mL (10 column volumes) to 100% B

Wavelength: 280nm

The elution chromatogram for this method is shown in figure 4.

Example 3

Use ofSP-650M chromatography material, chromatography with pegylated erythropoietin using an additional washing step of a solution of pH 2.8 and about 19mS/cm conductivity

Chromatography of pegylated erythropoietin was performed as follows:

resin: SP Toyopearl 650M

Bed volume: 19.6mL

Loading: 1.3mg/mL resin

Flow rate: 1.3 mL/min

B: 100mM potassium phosphate, 375mM sodium chloride, adjusted to pH 2.5

Additional wash (wash 2): 100mM potassium phosphate, 100mM sodium chloride, adjusted to pH 2.8, adjusted to LF ═ 19mS/cm (with water)

Application solution: 100% A

Washing solution: 100% A

Washing volume: 100mL (5 Column Volumes (CV))

Washing solution (washing 2): 100% additional wash (Wash 2)

Wash volume (wash 2): 100mL (5 Column Volumes (CV))

Washing solution (washing 3): 100% A

Wash volume (wash 3): 60mL (3 Column Volumes (CV))

Linear gradient elution solution: 100% B

Linear gradient: 196mL (10 column volumes) to 100% B

Wavelength: 280nm

The elution chromatogram of this method is shown in fig. 2 and the enlarged view is shown in fig. 3.

Example 4

Use ofSP-650M chromatography material, chromatography with pegylated erythropoietin using an additional washing step of a solution of pH 3.0 and about 19mS/cm conductivity

Chromatography of pegylated erythropoietin was performed as follows:

resin: SP Toyopearl 650M

Bed volume: 19.6mL

Loading: 1.3mg/mL resin

Flow rate: 1.3 ml/min

B: 100mM potassium phosphate, 375mM sodium chloride, adjusted to pH 2.5

Additional wash (wash 2): 100mM potassium phosphate, 100mM sodium chloride, adjusted to pH 3.0, adjusted to LF ═ 19mS/cm (with water)

Application solution: 100% A

Washing solution: 100% A

Washing volume: 100mL (5 Column Volumes (CV))

Washing solution (washing 2): 100% additional wash (Wash 2)

Wash volume (wash 2): 100mL (5 Column Volumes (CV))

Washing solution (washing 3): 100% A

Wash volume (wash 3): 60mL (3 Column Volumes (CV))

Linear gradient elution solution: 100% B

Linear gradient: 196mL (10 column volumes) to 100% B

Wavelength: 280nm

The elution chromatogram for this method is shown in figure 5.

Example 5

Use ofSP-650M chromatography material, chromatography of pegylated erythropoietin with additional washing steps using solutions of different pH values (pH 2.8, 2.9 or 3.0) and about 17mS/cm conductivity

Chromatography of pegylated erythropoietin was performed as follows:

resin: SP Toyopearl 650M

Bed volume: 19.2mL

Loading: 0.7mg/mL resin

Flow rate: 150 cm/hour

Solution: a: 100mM potassium phosphate, 100mM sodium chloride, adjusted to pH 2.5, adjusted to LF ═ 17mS/cm (with 5m sodium chloride)

B: 100mM potassium phosphate, 375mM sodium chloride, adjusted to pH 2.5

Additional wash (wash 2): 100mM potassium phosphate, 100mM sodium chloride, adjusted to pH 2.8, 2.9 or 3.0, adjusted to LF ═ 17mS/cm (with water)

Application solution: 100% A

Washing solution: 100% A

Washing volume: 96.2mL (5 Column Volumes (CV))

Washing solution (washing 2): 100% additional wash (Wash 2)

Wash volume (wash 2): 96.2mL (5 Column Volumes (CV))

Washing solution (washing 3): 100% A

Wash volume (wash 3): 57.7mL (3 Column Volumes (CV))

Linear gradient elution solution: 100% B

Linear gradient: 192mL (10 column volumes) to 100% B

Wavelength: 280nm

The results are shown below:

the yield was calculated based on the content of mono-pegylated EPO in the starting material.

Example 6

Use ofSP-650M chromatographic materials with solutions using different pH values (pH 2.8, 2.9 or 3.0) and a conductivity of about 18mS/cmChromatography of pegylated erythropoietin with additional washing step

Chromatography of pegylated erythropoietin was performed as follows:

resin: SP Toyopearl 650M

Bed volume: 19.2mL

Loading: 0.7mg/mL resin

Flow rate: 150cm/h

Solution: a: 100mM potassium phosphate, 100mM sodium chloride, adjusted to pH 2.5, adjusted to LF 18mS/cm (with 5m sodium chloride)

B: 100mM potassium phosphate, 375mM sodium chloride, adjusted to pH 2.5

Additional wash (wash 2): 100mM potassium phosphate, 100mM sodium chloride, adjusted to pH 2.8, 2.9 or 3.0, adjusted to LF 18mS/cm (with water)

Application solution: 100% A

Washing solution: 100% A

Washing volume: 96.2mL (5 Column Volumes (CV))

Washing solution (washing 2): 100% additional wash (Wash 2)

Wash volume (wash 2): 96.2mL (5 Column Volumes (CV))

Washing solution (washing 3): 100% A

Wash volume (wash 3): 57.7mL (3 Column Volumes (CV))

Linear gradient elution solution: 100% B

Linear gradient: 192mL (10 column volumes) to 100% B

Wavelength: 280nm

The results are shown below:

Example 7

Use ofSP-650M chromatography material, chromatography of pegylated erythropoietin with additional washing steps using solutions of different pH values (pH 2.8, 2.9 or 3.0) and about 19mS/cm conductivity

Chromatography of pegylated erythropoietin was performed as follows:

resin: SP Toyopearl 650M

Bed volume: 19.2mL

Loading: 0.7mg/mL resin

Flow rate: 150 cm/hour

B: 100mM potassium phosphate, 375mM sodium chloride, adjusted to pH 2.5

Additional wash (wash 2): 100mM potassium phosphate, 100mM sodium chloride, adjusted to pH 2.8, 2.9 or 3.0, adjusted to LF ═ 19mS/cm (with water)

Application solution: 100% A

Washing solution: 100% A

Washing volume: 96.2mL (5 Column Volumes (CV))

Washing solution (washing 2): 100% additional wash (Wash 2)

Wash volume (wash 2): 96.2mL (5 Column Volumes (CV))

Washing solution (washing 3): 100% A

Wash volume (wash 3): 57.7mL (3 Column Volumes (CV))

Linear gradient elution solution: 100% B

Linear gradient: 196mL (10 column volumes) to 100% B

Wavelength: 280nm

The results are shown below:

the yield was calculated based on the monopegylated EPO content of the starting material.

Claims

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

b) applying a second solution having an increased pH value relative to the first solution,

2. The method of claim 1, characterized in that the method further comprises the step of reapplying the first solution having a pH of about 2.4 to about 2.7 after step b) and before step c).

3. The method according to any of claims 1 or 2, characterized in that the second solution with increased pH value is a solution with a pH of about 2.7 to about 3.0.

4. A method according to any one of claims 1-3, characterized in that the second solution with increased pH value is a solution with constant conductivity value.

5. The method of any one of claims 1-4, characterized in that the second solution having an increased pH value and the first solution having a pH of about 2.4 to about 2.7 have about the same constant conductivity value.

6. The process of any of claims 1-5, characterized in that the second solution having an increased pH value and/or the first solution having a pH of about 2.4 to about 2.7 has a constant conductivity value of about 17mS/cm to about 19 mS/cm.

7. The method of any one of claims 1-6, characterized in that the second solution having an increased pH has a pH of about 2.7 to about 3.0 and has a conductivity value of about 17mS/cm to about 19 mS/cm.

8. The method according to any one of claims 1 to 7, characterized in that a solution comprising erythropoietin and a mixture of conjugates of erythropoietin and poly (ethylene glycol) with one or more poly (ethylene glycol) residues per erythropoietin molecule in the conjugate is not adjusted to a conductivity value of about 19 mS/cm.

9. Method according to any one of claims 1-8, characterized in that the solution with gradually increasing conductivity is a solution with gradually increasing sodium chloride concentration.

10. Method according to any of claims 1-9, characterized in that the solution with gradually increasing conductivity has a linear or stepwise increasing conductivity.

11. The method according to any one of claims 1 to 10, characterized in that the method is used for large scale protein production, wherein the chromatography column of step a) has a diameter of at least 30 cm.

12. Method according to any one of claims 1 to 11, characterized in that the erythropoietin is human erythropoietin.

13. Method according to claim 12, characterized in that human erythropoietin has the amino acid sequence of SEQ ID NO: 01 or SEQ ID NO: 02, or a pharmaceutically acceptable salt thereof.

14. The method of any one of claims 1-13, characterized in that the single poly (ethylene glycol) residue has a molecular weight of 20kDa to 40 kDa.