Purification of pegylated polypeptides

[0001] The present invention belongs to the field of chromatographic separation methods useful for the purification of polypeptides, especially PEGylated erythropoietin.

Background of the invention [0002] Proteins play an important role in the current field of medical techniques. For human use, any therapeutic protein must meet explicit criteria. To ensure the safety of biopharmaceuticals in humans, in particular by-products that accumulate during the manufacturing process should be removed. To meet regulatory requirements, one or more purification steps must follow the manufacturing process. Among other things, purity, efficiency and yield play an important role in determining the appropriate purification process.

[0003] Various methods are well-established and commonly used in purifying proteins, such as protein affinity chromatography of microorganisms (e.g. protein A or protein G affinity chromatography), ion exchange chromatography (e.g. cationic exchange (sulfopropyl or carboxymethyl resins), anion exchange (aminoethyl resins) and mixed ion exchange), thiophilic adsorption (e.g. with betamerkaptoethanol and other SH ligands), hydrophobic interactions or aromatic adsorption chromatography (e.g. with phenyl-sepharose, aza-areophilic or m-aminophenylboronic acid resins), metal chelation affinity chromatography (e.g. with Ni-affinity material (II ) and Cu (II)), size exclusion chromatography and electrophoretic methods (such as gel electrophoresis, capillary electrophoresis) (Vijayalakshmi, MA, Appl. Biochem. Biotech. 75 (1998) 93-102).

[0004] Coupling has been described, for example, for polyethylene glycol (PEG) and interleukin-6 (EP 0 442 724), for PEG and erythropoietin (WO 01/02017), for chimeric molecules containing endostatin and immunoglobulins (US 2005/008649), for proteins fused based secreted antibodies (US 2002/147311), for albumin-containing fusion polypeptides (US 2005/0100991; human serum albumin US 5876969), for PEGylated polypeptides (US 2005/0114037) and for interferon fusion.

[0005] Necina, R. et al. (Biotechnol. Bioeng. 60 (1998) 689-698) described the capture of human monoclonal antibodies directly from cell culture supernatants using ion exchange media showing high charge density. IN WO

89/05157 describes a method of purifying an immunoglobulin product by directly subjecting the cell culture medium to a cation exchange treatment. One-step purification of 2 non-monoclonal IgG antibodies from mouse ascites fluid is described in Danielsson, A. et al., J. Immun. Meth. 115 (1988), 79-88. A method of purifying a polypeptide by ion exchange chromatography is described in WO 2004/024866, where gradient washing was used to separate the polypeptide of interest from one or more impurities. EP 0 530 447 describes a method of purifying IgG monoclonal antibodies by combining three chromatographic steps. Easy purification of the mono-PEGylated interleukin-1 receptor antagonist is described in Yu, G. et al. In Process Biotechnol. 42 (2007) 971-977. Wang et al. (Wang, H. et al., Peptides 26 (2005) 1213-1218) describe the purification of hTFF3 expressed in E. coli using two-step cation exchange chromatography. Yun et al. (Yun, Q. et al., J. Biotechnol. 118 (2005) 67-74) describe the purification of PEGylated rhG-CSF in two successive stages of ion exchange chromatography. WO 2007/039436 and WO 01/087329 describe erythropoietin covalently coupled to polyethylene glycol group (s) and a liquid composition containing erythropoietin protein.

[0006] In Current Protocols in Molecular Biology, chapter 10.10 (by Williams, A. and Frasca, V., John Wiley & Sons, Inc., ISSN printed version: 1934-3639 (1998)) and in Current Protocols in Protein Science, chapter 8.2 (by Williams, A. and Frasca, John Wiley & Sons, Inc., ISSN printed version: 1934-3655 (1999)) describes the planning and conduct of ion exchange chromatography for protein separation.

[0007] In the technical description "Ion Exchange Chromatography: Principles and Methods" (1994) issued by Pharmacia Biotech, the choice of gradient type is described.

[0008] Goding, J. W. he described in Monoclonal Antibodies: Principles and Practice in part

4.2.4 (2nd edition, Adacemic Press, ISBN: 0-12-287021-2 (1986)) that ion exchange chromatography is a useful way to purify immunoglobulins, in particular IgG.

Summary of the Invention The present invention includes a method of purifying mono-PEGylated erythropoietin comprising the steps of providing a solution containing mono-, poly- and non-PEGylated erythropoietin, conducting two consecutive cation exchange chromatography steps, and recovering purified mono-PEGylated erythropoietin in a second cation exchange chromatography step, using in which the same type of cation exchange material is used in both stages of cation exchange chromatography.

[0010] In one embodiment, two subsequent cation exchange chromatography steps are carried out using different elution methods. In another embodiment, the two subsequent steps of cation exchange chromatography include the following steps:

a) introducing an aqueous buffered solution containing a mixture of mono-, poly- and non-PEGylated erythropoietin into the first cation exchange column under conditions suitable for binding this mono-PEGylated erythropoietin to the cation exchange material contained in this first column,

b) recovery of mono-PEGylated erythropoietin from the first cation exchange chromatography column by a step elution method with a gradual increase in the ionic strength of the flowing buffer, the fraction of this mono-PEGylated erythropoietin being increased compared to the mixture introduced in step a),

c) introducing the recovered mono-PEGylated erythropoietin into the second cation exchange chromatography column under conditions suitable for binding this mono-PEGylated erythropoietin to the cation exchange material contained in that second column, the cation exchange material contained in this second column being of the same type as the cation exchange material in first column

d) recovering the purified mono-PEGylated erythropoietin in a substantially homogeneous form from this second cation exchange chromatography column by a continuous elution method with a continuous increase of the ionic strength of the flowing buffer.

[0011] In one embodiment of the method, the cation exchange material is a strong cation exchange material. In a preferred embodiment, the strong cation exchange material is a sulfopropyl cation exchange material. Toyopearl is particularly preferred^® SP 650 M. In another embodiment, the mono-PEGylated erythropoietin is recovered in step d) in a substantially homogeneous form with over 95% purity relative to the surface. In a further embodiment of the method, the gradual increase in ionic strength in step b) of the method is a two-stage increase in ionic strength. Preferably, the mono-PEGylated erythropoietin is recovered in the second step of the stepwise elution method, i.e. after a second increase in ionic strength.

[0012] Another aspect of the present invention is a method of producing mono-PEGylated erythropoietin comprising the following steps:

a) PEGylation of erythropoietin using PEGylating reagent,

b) purification of PEGylated erythropoietin in two successive stages of cation exchange chromatography, with the first and second cation exchange chromatography using the same type of cation exchange material,

c) recovering mono-PEGylated erythropoietin from the second cation exchange chromatography column in a substantially homogeneous form.

Detailed description of the invention [0013] The present invention includes a method of purifying mono-PEGylated erythropoietin comprising two cation exchange chromatography steps, both cation exchange chromatography using the same type of cation exchange material.

[0014] The term "ion exchange material" as used in this application means a stationary high molecular weight matrix carrying covalently bonded substituents used as a stationary phase in ion exchange chromatography. Non-covalent counterions are associated with it for general charge indifference. "Ion exchange material" has the ability to exchange non-covalently associated counterions for similarly charged ions from the surrounding solution. Depending on the charge of its exchanged counterions, "ion exchange resin" is called a cation exchange resin or anion exchange resin. Depending on the nature of the charged group (substituent), "ion exchange resin" is called, for example, in the case of cation exchange resins, a sulfonic acid (S) resin or a sulfopropyl (SP) or carboxymethyl (CM) resin. Depending on the chemical nature of the charged group / substituent, "ion exchange resin" can additionally be classified as a strong or weak ion exchange resin, depending on the strength of the covalently bound charged substituent. For example, strong cation exchange resins contain a sulfonic acid group as a charged substituent, preferably a sulfopropyl group, weak cation exchange resins contain a carboxyl group as a charged substituent, preferably a carboxymethyl group, and weak anion exchange resins contain a diethylaminoethyl group as a charged substituent.

[0015] Various types of ion exchange materials, i.e. stationary phases, are available at _® various names and from many companies, such as Bio-Rex cation exchange materials (e.g. type 70), Chelex^® (e.g. type 100), Macro-Prep^® (e.g. type CM, High S, 25 S), AG^® (e.g. type 50W, MP), all available from BioRad Laboratories, WCX 2 available from Ciphergen, Dowex^® MAC-3 available from Dow chemical company, Mustang C and Mustang S available from Pall Corporation, Cellulose CM (e.g. type 23, 52), hyper-D, partisphere available from Whatman plc., Amberlite^® IRC (e.g. type 76, 747, 748), Amberlite^® GT 73, Toyopearl^® (Eg.

type SP, CM, 650M), all available from Tosoh Bioscience GmbH, CM 1500 and CM 3000 ™ >> available from BioChrom Labs, SP-Sepharose, CM-Sepharose available from GE Healthcare, Poros resins available from PerSeptive Biosystems, Asahipak ES (e.g. type 502C), CXpak P, IEC CM (e.g. type 825, 2825, 5025, LG), IEC SP (e.g. type 420N, 825), IEC QA (e.g. type LG, 825) available from Shoko America Inc. 50W cation exchange resin available from Eichrom Technologies Inc. Preferably the cation exchange material is a strong cation exchange material such as Macro-Prep^® High S or 25S or MacroCap SP or Toyopearl^® SP 650M, or Source S, or SP Sepharose, or POLYCAT A. Examples of anion exchange materials are Dowex^® 1 available from Dow chemical company, AG^®(e.g. type 1, 2, 4), Bio-Rex^® 5, DEAE Bio-Gel 1, Macro-Prep^® DEAE all available from BioRad Laboratories, type 1 anion exchange resin available from Eichrom Technologies Inc., Source Q, ANX Sepharose 4, D EAE Sepharose (e.g. type CL-6B, FF), Q Sepharose, Capto Q, Capto S, all available GE Healthcare, AX-300 available from PerkinElmer, Asahipak ES-502C, AXpak WA (e.g. type 624, G), IEC DEAE all available from Shoko America Inc., Amberlite^® IRA-96, Toyopearl^® DEAE, TSKgel DEAE, all available from Tosoh Bioscience GmbH, Mustang Q available from Pall Corporation. In one embodiment, the cation exchange material is a sulfopropyl cation exchange material.

[0016] The term "cation exchange material of the same type" means that two successive ion exchange chromatography steps are carried out using identical cation exchange material. This means that subsequent steps of cation exchange chromatography are carried out using the first portion of cation exchange material in the first stage of cation exchange chromatography and using a second portion of the same cation exchange material in the second stage of cation exchange chromatography or using the same cation exchange material in both stages of cation exchange chromatography. In one embodiment, the second cation exchange material is a cation exchange material of the same type, but not the same fraction of a cation exchange material as the first cation exchange material.

[0017] The terms "gradual elution" and "gradual elution method", which are used interchangeably in this application, mean a method in which, e.g., the concentration of the substance causing the elution, i.e. detachment of the bound compound from the material, is increased or lowered once, i.e. directly from one value / level to the next value / level. In this "gradual leaching", one or more conditions, for example pH, ionic strength, salt concentration and / or flow in the chromatographic system, all change simultaneously from the first, e.g. initial, value to the second, e.g. final, value, i.e. conditions change incrementally, i.e. gradually, as opposed to a linear change. In the "gradual elution method", a new fraction is collected after each increase in ionic strength. This fraction contains compounds recovered from ion exchange material by correspondingly increasing the ionic strength. After each increase, the conditions are maintained until the next step in the washing method. In "gradual elution," one or more conditions change all simultaneously from the first, e.g., initial, to the second, e.g., final, value. The change in one form is 10% of the concentration of the substance causing leaching or more. This means that in this form the concentration of the substance causing the leaching is 100% in the first stage, 110% or more in the second stage and 120% or more in the third stage. In another embodiment, the change is 50% or more of the concentration of the eluting substance. In another embodiment, the change is 120% or more of the concentration of the eluting substance. "Gradual leaching" means that the conditions are changed incrementally, i.e. gradually, as opposed to a linear change.

[0018] The terms "continuous elution" and "continuous elution method", which are used interchangeably in this application, mean a manner in which, e.g., the concentration of the eluting substance, i.e. the separation of the bound / adsorbed compound from the chromatographic material, increases or decreases continuously, i.e. the concentration varies by successive small steps, each of which has a change of not more than 2%, preferably 1%, of the leaching agent concentration. In this "continuous elution", one or more conditions, for example pH, ionic strength, salt concentration and / or flow in the chromatographic system, can be changed linearly or exponentially or asymptotically. Preferably the change is linear.

[0019] The term "introduction" and its grammatical equivalents as used in this application means a partial step of the purification method in which a solution containing the substance to be purified is brought into contact with the stationary phase. This means that a) the solution is added to the chromatographic equipment in which the stationary phase is located or that b) the stationary phase is added to this solution. In case a) a solution containing an interesting, purified substance passes through the stationary phase allowing interaction between the stationary phase and substances in solution. Depending on the conditions, e.g. pH, conductivity, salt concentration, temperature and / or flow rate, some substances from the solution are bound to the stationary phase and thus removed from the solution. Other substances remain in solution. Substances remaining in solution can be found in the flow-through. The term "flowing fraction" means a solution obtained after passing through a chromatographic device, which can be fed a solution containing the substance or buffer of interest, which is used to wash the column or to induce the elution of one or more substances associated with the stationary phase. In one embodiment, the chromatographic device is a column or cassette. The substance of interest can be recovered from the solution after the purification step by methods known to the person skilled in the art, such as e.g. precipitation, salting out, ultrafiltration, diafiltration, lyophilization, affinity chromatography or reduction of the solvent volume to obtain the substance of interest in a substantially homogeneous form. In the case of b) a stationary phase is added to the solution containing the substance to be purified, e.g. in the form of a solid to allow interaction between the stationary phase and substances in solution. After the interaction, the stationary phase is removed, e.g. by filtration, and the substance of interest is associated with the stationary phase and removed from it in solution or unbound from the stationary phase and remains in solution.

[0020] The term "under conditions suitable for binding" and its grammatical equivalents used in this application means that the substance of interest, e.g. PEGylated erythropoietin, binds to the stationary phase, e.g. ion exchange material, after contact with it. This does not necessarily mean that 100% of the substance of interest binds to the stationary phase, but essentially 100% of the substance of interest binds, i.e. at least 50% of the substance of interest binds, at least 75% of the substance of interest binds, at least 85% of the substance of interest binds, or more than 95% of the substance of interest binds.

[0021] The term "buffered" as used in this application means a solution in which changes in pH due to the addition or release of acidic or basic substances are leveled by the buffering substance. Any buffer substance with such effect can be used. Preferably, pharmaceutically acceptable buffering substances are used, such as e.g. phosphoric acid or its salts, acetic acid or its salts, citric acid or its salts, morpholine, 2- (N-morpholino) ethanesulfonic acid or its salts, histidine or its salts, glycine or its salts or tris (hydroxymethyl) aminomethane (TRIS ) or its salts. In one embodiment, the buffering substance is phosphoric acid or its salts or acetic acid or its salts, or citric acid or its salts, or histidine or its salts. Optionally, the buffered solution may contain additional salt such as, e.g., sodium chloride, sodium sulfate, potassium chloride, potassium sulfate, sodium citrate or potassium citrate.

[0022] General chromatographic methods and their use are known to the person skilled in the art. See, for example, Chromatography, 5th Edition, Part A: Fundamentals and Techniques, Heftmann, E. (ed.), Elsevier Science Publishing Company, New York, (1992); Advanced Chromatographic and Electromigration Methods in Biosciences, Deyl, Z. (ed.), Elsevier Science BV, Amsterdam, the Netherlands, (1998); Chromatography Today, Poole, CF and Poole, S. K., Elsevier Science Publishing Company, New York, (1991); Scopes, Protein Purification: Principles and Practice (1982); Sambrook, J. et al. (ed.), Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; or Current Protocols in Molecular Biology, Ausubel, FM et al. (ed.), John Wiley & Sons, Inc., New York.

[0023] PEGylation of erythropoietin typically results in the formation of a mixture of various compounds, such as poly-PEGylated erythropoietin, mono-PEGylated erythropoietin, non-PEGylated erythropoietin, hydrolysis products of activated PEG ester, for example free PEGylated acid, as well as hydrolysis products of erythropo itself. In order to obtain mono-PEGylated erythropoietin in a substantially homogeneous form, the substances must be separated and the compound of interest must be purified.

[0024] Therefore, an aspect of the present invention is to provide a method for obtaining mono-PEGylated erythropoietin in a substantially homogeneous form comprising the following steps:

a) PEGylation of erythropoietin using activated PEGylating reagent with molecular weight from 20 kDa to 40 kDa,

b) purification of PEGylated erythropoietin obtained in step a) in two subsequent stages of cation exchange chromatography, with the use of the same type of cation exchange material in the first and second stages of cation exchange chromatography,

c) recovering mono-PEGylated erythropoietin from the second cation exchange chromatography column in a substantially homogeneous form.

[0025] This method is particularly useful for purifying PEGylated recombinant polypeptides that are glycosylated, i.e., which have been produced by a mammalian cell, preferably a CHO cell, HEK293 cell, BHK cell, Per.C6 cell^®or HeLa cell and then chemically PEGylated.

[0026] In the first step of the method, the erythropoietin is PEGylated. The polyethylene glycol polymer (PEG) molecules used in the PEGylation reaction have a molecular weight of about 20 kDa to 40 kDa (by the term "molecular weight" as used herein is meant the average molecular weight of PEG because PEG as a polymeric compound is not obtained as a compound of specific molecular weight, but in fact has a molecular weight distribution; the term "about" means that in these PEG preparations some molecules will have a higher molecular weight and some less than the indicated molecular weight, i.e. the term about refers to a molecular weight distribution in which 95% of the PEG particles have a molecular weight in the +/- range 10% of the indicated molecular weight. For example, a molecular weight of 30 kDa means a range from 27 kDa to 33 kDa).

[0027] The term "erythropoietin" refers to a protein with the sequence SEQ ID NO: 1 or SEQ ID NO: 2, or a protein or polypeptide substantially homologous thereto, whose biological properties are associated with stimulation of red blood cell production and stimulation of division and differentiation of determined progenitor cells erythroid cells in the bone marrow. Recombinant erythropoietin can be produced by expression in eukaryotic cells, for example in CHO cells or BHK cells, or HeLa cells using recombinant DNA technology or by activation of an endogenous gene. For example, erythropoietin as a glycoprotein is expressed by activation of an endogenous gene as described in US 5733761, US 5641670, US 5733746, WO 93/09222, WO 94/12650, WO 95/31560, WO 90/11354, WO 91/06667 and WO 91 / 09,955. In one embodiment, the erythropoietin of the invention is based on the human EPO sequence. In another embodiment, human erythropoietin has the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, preferably human erythropoietin has the amino acid sequence set forth in SEQ ID NO: 1. The term "erythropoietin" also means protein variants of 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 exhibit the same biological activity as the unmodified protein, such as described in EP 1 064 951 or US 6583272. The variant may have the amino acid sequence of human erythropoietin containing from 1 to 6 additional glycosylation sites. The specific activity of PEGylated erythropoietin can be determined by various assays known in the art. The biological activity of the purified PEGylated erythropoietin according to this invention is that administration of protein to human patients by injection results in increased production of reticulocytes and red blood cells by bone marrow cells compared to non-injected individuals or control groups. The biological activity of PEGylated erythropoietin obtained and purified in accordance with this invention can be tested by methods according to Pharm. Europa Spec. Issue Erythropoietin BRP Bio 1997 (2).

[0028] The term "PEG" or "PEG group" according to the invention means a residue containing polyethylene glycol as a substantial part. Such PEG may contain further chemical groups that are necessary for the binding reaction, i.e. coupling, which result from the chemical synthesis of the molecule or which constitute a spacer to ensure an adequate distance between the parts of the molecule. These subsequent chemical groups are not used to calculate the molecular weight of the PEG polymer molecule. In addition, such PEG may consist of one or more PEG side chains that are linked together. PEGs with more than one PEG chain are called multi-arm or branched PEGs. Branched PEG can be prepared, for example, by adding polyethylene oxide to various polyols, including glycerol, pentaerythritol and sorbitol. Branched PEG is described, for example, in EP 0 473 084, US 5932462. In one embodiment linear PEG molecules are used as PEG with a molecular weight of 20-35 kDa, and branched PEGs are used as PEG polymers with a molecular weight of over 35 kDa, in particular 40 kDa. In one embodiment, two-arm PEG is used as 40 kDa PEG.

[0029] The term "PEGylation" means the covalent attachment of a polyethylene glycol residue at the N-terminus of the polypeptide and / or at the internal lysine residue. Protein pEGylation is widely known in the art and described in a review by, for example, Veronese, FM, Biomaterials 22 (2001) 405-417. PEG can be coupled using different functional groups and polyethylene glycols with different molecular weights, linear or branched PEGs, as well as various coupling groups (see also Francis,

GE et al., Int. J. Hematol. 68 (1998) 1-18; Delgado, C. et al., Crit. Rev. Ther. Drug Carrier Systems 9 (1992) 249-304). PEGylation of erythropoietin can be carried out in aqueous solution using PEGylating reagents as described, for example, in WO 00/44785, in one embodiment using NHS activated linear or branched PEG molecules with a molecular weight between 5 kDa and 40 kDa. PEGylation can also be carried out on a solid phase according to Lu, Y. et al., Reactive Polymers 22 (1994) 221-229. Non-randomly, the N-terminally PEGylated polypeptide can also be produced according to WO 94/01451. [0030] Such methods produce erythropoietin which is PEGylated at one or more ε-amino groups of lysine residues and / or at the N-terminal amino group. Selective PEGylation at the N-terminal amino acid can be carried out according to Felix, AM et al., ACS Symp. Cheese. 680 (Poly (ethylene glycol)) (1997) 218-238. Selective N-terminal PEGylation can be obtained during solid phase synthesis by N coupling^andPEGylated amino acid derivative with N-1 terminal amino acid of the peptide chain. PEGylation of the side chain can be carried out during solid phase synthesis by coupling to the growing chain of Y-PEGylated lysine derivatives. Combined N-terminus and side chain PEGylation can be performed as described above in solid phase synthesis or in solution phase synthesis using activated PEG reagents for an amino-deprotected peptide.

[0031] Suitable PEG derivatives are activated PEG molecules with an average molecular weight from about 5 to about 40 kDa, in one embodiment from about 20 to about 40 kDa, preferably about 30 kDa to about 35 kDa. The PEG derivative is in one form linear or branched PEG. Many different PEG derivatives suitable for use in the production of PEG-protein and PEG-peptide conjugates can be obtained from Shearwater Polymers (Huntsville, AL, USA;<a href="http://www.nektar.com">www.nektar.com</a>).

[0032] Activated PEG derivatives are known in the art and described, for example, in Morpurgo, M. et al., J. Bioconjug. Chem. 7 (1996) 363-368, for PEG-vinylsulfone. Linear and branched chain PEG types are suitable for producing PEGylated fragments. Examples of reactive PEG reagents are iodoacetylmethoxy-PEG or methoxy-PEG-vinylsulfone (m is preferably an integer from about 450 to about 900, and R is a linear or branched C1- to C6alkyl containing from one to six carbon atoms, such as methyl , ethyl, isopropyl, etc., with R = methyl in one embodiment:

or ψΚ "

[0033] The use of these iodine activated substances is known in the art and described e.g. by Hermanson, GT, in Bioconjugate Techniques, Academic Press, San Diego (1996) pp. 147-148.

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

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or .K ^ Yn ^ YYor

yl ^{H m} .

o using alkoxy-PEG-N-hydroxysuccinimide such as methoxy-PEG-N-hydroxysuccinimide (MW 30,000; Shearwater Polymers, Inc.), where R and m are as defined above. In one embodiment, the type of PEG is methoxy poly (ethylene glycol) butyric acid N-hydroxysuccinimidyl ester. The term "alkoxy" refers to an alkyl ether group, wherein the term "alkyl" means a straight chain or branched chain alkyl group containing up to four carbon atoms, such as methoxy, ethoxy, n-propoxy etc., preferably methoxy.

[0035] The term "substantially homogeneous form" as used in this application means that the PEGylated erythropoietin obtained, contained or used is one containing a defined number of PEG groups attached. In one embodiment, the PEGylated erythropoietin is mono-PEGylated erythropoietin. The preparation may contain unreacted erythropoietin (i.e. no PEG group), poly-PEGylated erythropoietin, as well as polypeptide fragments generated during the PEGylation reaction. The term "substantially homogeneous form" means that the mono-PEGylated erythropoietin formulation contains at least 50% (w / w) of mono-PEGylated erythropoietin, at least 75% mono12 in one form

PEGylated erythropoietin, at least 90% mono-PEGylated erythropoietin or more than 95% mono-PEGylated erythropoietin. The percent values ​​are based on% with respect to the chromatogram surface corresponding to the cation exchange chromatography purification, in which mono-PEGylated erythropoietin is obtained.

[0036] The present invention relates to a method of purifying mono-PEGylated erythropoietin to obtain a substantially homogeneous form of mono-PEGylated erythropoietin. It has surprisingly been found that the combination of two consecutive cation exchange chromatography steps, both steps using the same type of cation exchange material, provides a substantially homogeneous form of mono-PEGylated erythropoietin. Thus, the present invention provides a method of purifying mono-PEGylated erythropoietin comprising the steps of providing a solution containing mono-, poly- and non-PEGylated erythropoietin, performing two consecutive cation exchange chromatography steps, and recovering purified mono-PEGylated erythropoietin in a second cation exchange chromatography step, with both steps cation exchange chromatography uses the same type of cation exchange material. In one embodiment, the recovery of the first cation exchange chromatography step is carried out by a different elution method than the recovery of the second cation exchange chromatography step. In another embodiment, the cation exchange chromatography column is regenerated after the first stage of cation exchange chromatography and after the second stage of cation exchange chromatography.

[0037] The recovery of purified mono-PEGylated erythropoietin in the second cation exchange chromatography step is carried out by eluting the mono-PEGylated erythropoietin from the material of the second cation exchange chromatography. In one embodiment of the method of the invention, the two stages of cation exchange chromatography differ in the elution method used. In one embodiment, the first cation exchange chromatography step is carried out by a step elution method, i.e. the ionic strength of the buffer used is increased gradually, i.e. once from one ionic strength value to the next ionic strength value, preferably by a change of 10% or more. The step elution method is carried out in one embodiment as a three step elution method. In the first stage, mainly poly-PEGylated erythropoietin is eluted from the cation exchange chromatography column. A second increase in ionic strength essentially elutes monoPEGylated erythropoietin with a purity higher than 60% based on the area on the appropriate size exclusion chromatography (% by area). The third increase in ionic strength elutes mainly the remaining unPEGylated erythropoietin from the column.

[0038] The second stage of cation exchange chromatography is carried out in one embodiment as a continuous elution method, i.e. the ionic strength of the buffer is increased continuously, preferably by a change of less than 5%. The eluted fractions containing mono-PEGylated erythropoietin are combined to obtain mono-PEGylated erythropoietin in a substantially homogeneous form, in one form containing less than 0.5% of low molecular weight forms based on the surface on the appropriate chromatogram. The buffer is preferably present at a concentration of 10 mM to 250 mM, in one embodiment from 50 mM to 150 mM, in another embodiment about 100 mM. Thus, in the process of the invention, two further steps of cation exchange chromatography include the following steps:

a) introducing an aqueous buffered solution containing a mixture of mono-, poly- and non-PEGylated erythropoietin and a low molecular weight form into the first cation exchange chromatography column under conditions suitable for binding this mono-PEGylated erythropoietin to the cation exchange material contained in this first column,

b) recovery of mono-PEGylated erythropoietin from the first cation exchange chromatography column by a step elution method with a gradual increase in the ionic strength of the flowing buffer, the relative content of this mono-PEGylated erythropoietin in the recovered solution is increased compared to the mixture introduced in step a),

c) introducing the recovered mono-PEGylated erythropoietin from step b) into the second cation exchange chromatography column under conditions suitable for binding this erythropoietin to the cation exchange material contained in the second column, the cation exchange material contained in this second column being of the same type as the cation exchange material in the first column

d) recovering the purified mono-PEGylated erythropoietin in a substantially homogeneous form from this second cation exchange chromatography column by a continuous elution method with a continuous increase of the ionic strength of the flowing buffer.

[0039] PEGylation of a polypeptide normally does not provide the PEGylation product in a homogeneous form. Furthermore, it is obtained as a mixture of mono-PEGylated, poly-PEGylated and non-PEGylated product. Thus, the solution of PEGylated erythropoietin introduced in step a) of the process is a mixture of mono-, poly- and non-PEGylated erythropoietin and low molecular weight forms or fragments in aqueous buffer. The relative content of various substances is determined by size exclusion chromatography (SE-HPLC). An exemplary chromatogram is shown in Figure 1. The sum of the areas of the correlated peaks, i.e. the area under the peaks, in Figure 1 is the total size of the chromatogram excluding size exclusion.

The fraction for a single peak is given as% relative to the surface, i.e. as the surface fraction relative to the total area of ​​the chromatogram.

[0040] General chromatographic methods, their use and associated terms are known to a person skilled in the art. See, for example, Chromatography, 5th Edition, Part A: Fundamentals and Techniques, Heftmann, E. (ed.), Elsevier Science Publishing Company, New York, (1992) and other related textbooks. During chromatography, the buffer flows through a cation exchange chromatography column. This "flowing buffer" is adapted according to the requirements of the chromatographic method steps. It transports an interesting substance to (input) and from (leaching) chromatographic material.

[0041] In the first stage of cation exchange chromatography, the mixture of mono-PEGylated, poly-PEGylated and non-PEGylated erythropoietin is introduced at a protein concentration of 0.7 to 1.5 mg / ml, preferably about 1 mg / ml, into the first chromatography column cation exchange in an aqueous buffered solution. In one embodiment, the aqueous buffered solution contains about 100 mM potassium phosphate at a pH of about 3.0. The term "about" as used in this application means a range of 10% around the indicated value, i.e. ± 10%. Before and after introduction in one embodiment, the first column is washed with the same buffer solution. For the first step in the stepwise washing method, the buffer changes to a buffer with about 100 mM potassium phosphate, about 90 mM sodium chloride at a pH of about 3.0. This buffer elutes from the cation exchange chromatography column hydrolyzed activated PEG reagent, i.e. suitable PEGylated carbonic acid, unreacted coupling reagent and poly-PEGylated erythropoietin. For the second stage, in a three-step elution method, the buffer changes to a buffer with approximately 100 mM potassium phosphate, approximately 250 mM sodium chloride, pH approximately 3.0. At this stage, mono-PEGylated erythropoietin is recovered from the first cation exchange chromatography column. The collected flowing buffer from this elution step is diluted about 1: 5 (v / v) to 1: 8 (v / v), preferably 1: 5 (v / v), purified water. An exemplary first cation exchange chromatography is shown in Figure 2. In the third step of the three-step elution method, the buffer changes to a buffer with approximately 100 mM potassium phosphate, approximately 750 mM sodium chloride, pH approximately 3.0. At this stage, unPEGylated erythropoietin is recovered from the first cation exchange chromatography column.

[0042] The collected flow buffer from the second stage of the first cation exchange chromatography contains mono-PEGylated erythropoietin in increased relative content, i.e. weight fraction or% relative to the surface (in the chromatogram from the chromatography excluding the size of the collected flow of buffer from the second stage) PEGylated erythropoietin increased compared to the state before the first stage of cation exchange chromatography. In one embodiment, the relative content of monoPEGylated erythropoietin is at least 60% by area. In another embodiment, the relative content of mono-PEGylated erythropoietin is at least 80% by area.

[0043] To further purify the mono-PEGylated erythropoietin, a second cation exchange chromatography step is carried out. In the second cation exchange chromatography, the collected and diluted buffer flowing from the second elution step adjusted to a potassium phosphate concentration of about 100 mM and a pH of about pH 3.0 is introduced into a second cation exchange chromatography column containing cation exchange material of the same type as the first cation exchange chromatography column. In one embodiment, the second cation exchange column and the cation exchange material contained therein are the same as in the first cation exchange chromatography step. Mono-PEGylated erythropoietin is recovered from the second cation exchange chromatography column by applying a linear gradient starting from a potassium phosphate buffer at a concentration of about 100 mM with about 50 mM sodium chloride at pH about 3.0 and ending with a potassium phosphate buffer at a concentration of about 100 mM with about 500 mM sodium chloride at a pH of about 3.0. The change in sodium chloride concentration is linear over ten column volumes. The flowing buffer is fractionated and each fraction is diluted with 1 M potassium hydrogen phosphate to increase the pH to about pH 6 to 8. An exemplary chromatogram is shown in Figure 3.

[0044] After the second cation exchange chromatography step, mono-PEGylated erythropoietin is obtained in a substantially homogeneous form, in one form at least 95% pure by area.

[0045] The skilled person is familiar with ion exchange chromatography technology. In the recovery step of the polypeptide bound to the cation exchange material, the ionic strength, i.e. conductivity, of the buffer / solution flowing through the ion exchange column is increased. This is achieved by increasing the concentration of buffering salt or by adding other salts to the buffer solution, so-called elution salts. Depending on the elution method, the buffer / salt concentration is increased once (step elution method) or continuously (continuous elution method) by partially adding a concentrated buffer solution or elution salt. Preferred elution salts are sodium citrate, sodium chloride, sodium sulfate, sodium phosphate, potassium chloride, potassium sulfate, potassium phosphate or other salts of citric acid or phosphoric acid or any mixture of these ingredients. In one embodiment, the elution salt is sodium citrate, sodium chloride, potassium chloride or mixtures thereof.

[0046] In one embodiment of the present method, the cation exchange material is a strong cation exchange material, such as preferably Toyopearl^® SP 650 M. The concentration of the eluting salt is in one form in the range from 5 mM to 500 mM, preferably in the range from 5 mM to 400 mM, and more preferably in the range from 5 mM to 250 mM. In another embodiment of the invention, the leaching salt is simultaneously used as a buffering substance, such as, for example, citric acid or its salts or phosphoric acid or its salts.

[0047] Mono-PEGylated erythropoietin can be used in injectable pharmaceutical compositions with a pharmaceutically acceptable carrier or vehicle by methods known in the art. For example, suitable compositions are described in WO 97/09996, WO 97/40850, WO 98/58660 and WO 99/07401. Preferred pharmaceutically acceptable carriers for the formulation of products of the invention include human serum albumin, human plasma proteins, etc. The compounds of the present invention can be formulated in a buffer based on 10 mM sodium / potassium phosphate at pH 7 containing a tonic agent, e.g. 132 mM sodium chloride. Optionally, the pharmaceutical composition may contain a preservative. The pharmaceutical composition may contain various amounts of mono-PEGylated erythropoietin, for example 101,000 µg / ml, e.g. 50 μg or 400 μg.

[0048] Administration of glycoprotein products in the form of erythropoietin according to the present invention results in the formation of red blood cells in humans. Thus, administration of a glycoprotein product in the form of mono-PEGylated erythropoietin supplements this protein, erythropoietin, which is important in the production of red blood cells. Pharmaceutical compositions containing glycoprotein products in the form of mono-PEGylated erythropoietin can be formulated in a concentration effective for administration by various routes to a human patient suffering from a blood disorder characterized by low or abnormal production of red blood cells, either alone or as part of a condition or disease. Pharmaceutical compositions may be administered by injection, such as subcutaneous or intravenous injections. Average amounts of mono-PEGylated erythropoietin glycoprotein may vary. The exact amount of conjugate is a matter of preference depending on factors such as the type of condition being treated, the condition of the patient being treated, as well as the ingredients of the composition. For example, 0.01 to 10 μg per kg body weight, preferably 0.1 to 1 μg per kg body weight, e.g. once a week, may be administered.

[0049] The following examples, sequence listing and figures are provided to facilitate understanding of the present invention, the true scope of which is set out in the appended claims. It should be understood that modifications may be made to the procedures set forth without departing from the spirit of the invention.

Description of the figures [0050]

Figure 1 SE-HPLC mixtures of differently PEGylated erythropoietins containing a correlation of peaks and substances.

Figure 2 Example of a chromatogram for a stepwise elution method.

Figure 3 Example of a chromatogram for a continuous elution method.

Materials and methods

SE-HPLC [0051] SE-HPLC separates proteins by their apparent molecular weight. Thus, this method is able to detect the presence of mono-PEGylated erythropoietin, low molecular weight forms and fragments, poly-PEGylated forms and larger erythropoietin aggregates. The HPLC device is equipped with a detector for 220 nm and a Superose 6 HR column (dimensions 10 x 300 mm, Pharmacia Biotech, catalog number: 170537-01) or a Superose 6 10/300 GL column (Pharmacia Biotech, catalog number: 17 -5172-01). The column is isocratic at room temperature using a flow rate of about 0.4 ml / min. The mobile phase buffer is a 50 mM potassium phosphate buffer with 300 mM sodium chloride, pH 6.8. Depending on the HPLC system used, the method can be carried out with a sample volume of 100 μl or 500 gl. Samples are diluted with the mobile phase buffer to a protein concentration of about 0.5 mg / ml (100 μ ^ aliquot or 0.1 mg / ml (500 μl aliquot. Samples with less than 0.1 mg / ml protein can be used undiluted) Eluted proteins are detected at a detector wavelength of 220 nm.

Example 1

Fermentation and purification of erythropoietin [0052] Erythropoietin can be prepared, e.g., according to WO 01/87329 and purified as described in WO 96/135718.

Example 2

PEGylation of erythropoietin with bifunctional reagents

a) Activation of erythropoietin [0053] The specified amounts of reagent containing blocked thiol, SATA (succinimidyl acetylthioacetate) or SATP (succinimidyl acetylthiopropionate) (dissolved in DMSO at 10 mg / ml) were added to the benzyl protected erythropoietin solution here to 1 ml 5 mg / ml protein in a buffer based on 10 mM potassium phosphate supplemented with 50 mM sodium chloride at pH 7.3. The reaction mixture was stirred for about 30 minutes (at 25 ° C) and the reaction was stopped by adding a 1 M lysine solution to a final concentration of 10 mM. Excess amounts of SATA and SATP were removed by dialysis against a buffer based on 10 mM potassium phosphate containing 50 mM sodium chloride and 2 mM EDTA at pH 6.2. The acetyl protecting group was removed with hydroxylamine.

b) PEGylation of activated erythropoietin [0054] In a solution containing 95 mg of activated erythropoietin (4.5 mg / ml in a buffer based on 10 mM potassium phosphate with 50 mM sodium chloride and 2 mM EDTA, pH 6.2), 380 mg of methoxy was dissolved. PEG-maleimide (MW 30,000; Shearwater Polymers, Inc., Huntsville (Alabama, USA)). The resulting molar ratio between activated erythropoietin and methoxy-PEG-maleimide in solution was 1: 2 to 1: 4. By adding 1 M aqueous hydroxylamine solution to a final concentration of 30 mM (pH 6.2) to the above solution, the covalently blocked thiol groups of activated erythropoietin were unblocked. The resulting activated erythropoietin contained free thiol (-SH) groups in the reaction mixture. Immediately after unblocking the thiol groups, a coupling reaction between activated erythropoietin now containing free thiol groups (-SH) and methoxy-PEG-maleimide was carried out for 90 minutes (under stirring at 25 ° C). The coupling reaction was stopped by adding 0.2 M aqueous cysteine ​​solution to the reaction mixture to a final concentration of 2 mM. After 30 minutes, excess free thiol groups of activated erythropoietin that did not react with methoxy-PEG-maleimide were blocked by the addition of a 0.5 M solution of N-methylmaleimide in DMSO to achieve a final concentration of 5 mM. After 30 minutes it was possible to purify the resulting reaction mixture now containing PEGylated erythropoietin.

Example 3

Purification of mono-PEGylated erythropoietin

a) First chromatography on Toyopearl 650 M [0055] The first product chromatography was performed on a sulfopropyl (SP) column packed with Toyopearl 650M SP. The column was operated at room temperature. The maximum load on the first column was defined as 1.5 g protein per liter column volume (CV). The column was equilibrated with 100 mM potassium phosphate buffer at pH 2.9 to 3.1 (SP-A buffer). After the loading step, the column was washed and eluted with a series of potassium phosphate buffers containing increasing amounts of NaCl. Hydrolyzed PEG reagent and poly-PEGylated forms were removed in the flowing fraction and the subsequent washing step respectively with SP-A buffer and 100 mM potassium phosphate buffer, pH 2.9 to 3.1, containing 90 mM sodium chloride (buffer SP-B ).

[0056] Mono-PEGylated erythropoietin was eluted by the use of a buffer based on 100 mM potassium phosphate, pH 2.9 to 3.1, containing 250 mM sodium chloride (buffer SP-C), collected in a vessel and directly diluted with purified water 1: 5 . This collected elution fraction was named as "pool and eluted from SP".

[0057] The column was then washed with 100 mM potassium phosphate buffer, pH 2.9 to 3.1 containing 750 mM sodium chloride (SP-D buffer) to remove unreacted erythropoietin and regenerate the column.

b) Second chromatography on SP Toyopearl 650 M [0058] The second column was operated at room temperature. After equilibration with SP-A buffer, pool I washed with SP was added to the column, and then the column was washed with SP-A buffer. Mono-PEGylated erythropoietin was eluted by applying a linear gradient from 50 to 500 mM sodium chloride over ten column volumes in a buffer based on 100 mM potassium phosphate at pH 2.9 to 3.1. The product peak was fractionated into up to 8 individual fractions and each fraction was directly diluted with 1 M potassium hydrogen phosphate to increase the pH to 6 to 8.

[0059] After the elution of mono-PEGylated erythropoietin is complete, the gradient can be increased, leading to immediate column washing with 100 mM potassium phosphate pH 2.9 to 3.1 containing 500 mM sodium chloride.

c) Regeneration of SP Toyopearl 650 M columns [0060] The resins from both columns were regenerated in a seven-step sequence. The columns were washed with purified water followed by 0.5 M sodium hydroxide solution. The alkaline solution was displaced with purified water and then with acid (0.5 M sodium dihydrogen phosphate,

1 M phosphoric acid). After the next step with purified water, pyrogens were removed from the columns using 0.5 M sodium hydroxide for&gt; 4 hours. After regeneration with base, the columns were again washed with purified water. List of parameters for columns see Table 1 and Table 2.

Table 1: Parameters of the first chromatographic column

<td>Stage</td><td>Buffer solution</td><td>volume column</td><td>Intensity flow [L / min]</td>

<td>balancing</td><td>100 mmol / l potassium phosphate, pH 2.9 -3.1 (buffer SP-A)</td><td> &gt; 6</td><td> 1,6 - 2,1</td>

<td>Entering a column</td><td>reaction mixture, diluted SP-A (1: 5)</td><td>nd</td><td> 1,6 - 2,1</td>

<td>Stage</td><td>Buffer solution</td><td>volume column</td><td>Intensity flow [L / min]</td>

<td>SP-A washing</td><td>100 mmol / L potassium phosphate, pH 2.9 -3.1 (buffer SP-A)</td><td> 2</td><td> 1,6 - 2,1</td>

<td>SP-B washing</td><td>100 mmol / L potassium phosphate, pH 2.9 -3.1, 90 mmol / l NaCl (buffer SP-B)</td><td> 2-3</td><td> 1,6 - 2,1</td>

<td>SP-C leaching</td><td>100 mmol / L potassium phosphate, pH 2.9 -3.1, 250 mmol / l NaCl (buffer SP-C)</td><td> 2-3</td><td> 1,6 - 2,1</td>

<td>SP-D washing</td><td>100 mmol / l potassium phosphate, pH 2.9 -3.1, 750 mmol / l NaCl (buffer SP-D)</td><td> 2-3</td><td> 1,6 - 2,1</td>

<td>Rinsing</td><td>PW III</td><td> &gt; 2</td><td> 1,6 - 2,1</td>

<td>Regeneration of the column with the principle of</td><td>0.5 mol / L NaOH</td><td> &gt; 2</td><td> 1,6 - 2,1</td>

<td>Rinsing</td><td>PW III</td><td> &gt; 2</td><td> 1,6 - 2,1</td>

<td>Acid regeneration of the column</td><td>1 mol / l phosphoric acid, 0.5 mol / l potassium dihydrogen phosphate</td><td> &gt; 3</td><td> 1,6 - 2,1</td>

<td>Rinsing</td><td>PW III</td><td> &gt; 2</td><td> 1,6 - 2,1</td>

<td>Regeneration of the column with principle II</td><td>0.5 mol / L NaOH</td><td> &gt; 3</td><td>nd</td>

<td>Rinsing</td><td>PW III</td><td> &gt; 2</td><td> 1,6-2,1</td>

N / A: Not applicable

Table 2: Parameters of the second column chromatography

<td>Stage</td><td>Buffer solution</td><td>volume column</td><td>Intensity flow [L / min]</td>

<td>balancing</td><td>100 mmol / l potassium phosphate, pH 2.9-3.1 (buffer SP-A)</td><td> &gt; 6</td><td> 1,6-2,1</td>

<td>Entering into the column</td><td>pool I washed out of SP, diluted PW III (1: 5)</td><td>nd</td><td> 1,6-2,1</td>

<td>Wash SPA</td><td>100 mmol / l potassium phosphate, pH 2.9-3.1, (buffer SP-A)</td><td> 2-3</td><td> 1,6-2,1</td>

<td>Gradient and washout</td><td>Gradient 50-500 mmol / L NaCl over 10 CV between SP-A buffer and 100 mmol / l potassium phosphate, 500 mmol / l NaCl pH 2.9 - 3.1 (buffer SP-E)</td><td> 10</td><td> 1,6-2,1</td>

<td>Rinsing</td><td>PW III</td><td> &gt; 2</td><td> 1,6-2,1</td>

<td>Regeneration of the column with the principle of</td><td>0.5 mol / L NaOH</td><td> &gt; 2</td><td> 1,6-2,1</td>

<td>Stage</td><td>Buffer solution</td><td>volume column</td><td>Intensity flow [L / min]</td>

<td>Rinsing</td><td>PW III</td><td> &gt; 2</td><td> 1,6-2,1</td>

<td>Acid regeneration of the column</td><td>1 mol / l phosphoric acid, 0.5 mol / l potassium dihydrogen phosphate</td><td> &gt; 3</td><td> 1,6-2,1</td>

<td>Rinsing</td><td>PW III</td><td> &gt; 2</td><td> 1,6-2,1</td>

<td>Regeneration of the column with principle II</td><td>0.5 mol / L NaOH</td><td> &gt; 3</td><td>nd</td>

<td>Rinsing</td><td>PW III</td><td> &gt; 2</td><td> 1,6-2,1</td>

n.d: not applicable

LIST OF SEQUENCES [0061] &lt;110&gt; F. Hoffmann-La Roche AG &lt;120&gt; Purification of PEGylated polypeptides &lt;130&gt; 24380 FT &lt;150&gt; EP 07013959.7 &lt;151&gt; 2007-07-17 &lt;160&gt; 2 &lt;170&gt; Patent In version 3.2 &lt; 210&gt; 1 &lt;211&gt; 165 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 1

Ala Pro Pro Arg Leu Ile Cys Asp Cheese Arg Val Leu Glu Arg Tyr Leu 15 10 15

Leu Glu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His 20 25 30

Cys Ser Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe 35 40 45

Tyr Ala Trp Lys Arg Met Glu Val Gly Gin Gin Ala Val Glu Val Trp 50 55 60

Gin Gly Leu Ala Leu Leu Cheese Glu Ala Val Leu Arg Gly Gin Ala Leu 65 70 75 80

Leu Val Asn Cheese Ser Gin Pro Trp Glu Pro Leu Gin Leu His Val Asp 85 90 95

Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu 100 105 110

Gly Ala Gin Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala 115 120 125

Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val 130 135 140

Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala 145 150 155 160

Cys Arg Thr Gly Asp 165 &lt;210&gt; 2 &lt;211&gt; 166 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 2

<td>ala 1</td><td>Pro</td><td>Pro</td><td>Arg</td><td>Leu 5</td><td>How much</td><td>Cys</td><td>asp</td><td>Cheese</td><td>Arg 10</td><td>val</td><td>Leu</td><td>Glu</td><td>Arg</td><td>Tyr 15</td><td>Leu</td>

<td>Leu</td><td>Glu</td><td>ala</td><td>lys twenty</td><td>Glu</td><td>ala</td><td>Glu</td><td>own</td><td>How much 25</td><td>Thr</td><td>Thr</td><td>Gly</td><td>Cys</td><td>ala thirty</td><td>Glu</td><td>His</td>

<td>Cys</td><td>Cheese</td><td>Leu 35</td><td>own</td><td>Glu</td><td>own</td><td>How much</td><td>Thr 40</td><td>val</td><td>Pro</td><td>asp</td><td>Thr</td><td>lys 45</td><td>val</td><td>own</td><td>phe</td>

<td>Tyr</td><td>ala 50</td><td>Trp</td><td>lys</td><td>Arg</td><td>Underworld</td><td>Glu 55</td><td>val</td><td>Gly</td><td>Gin</td><td>Gin</td><td>ala 60</td><td>val</td><td>Glu</td><td>val</td><td>Trp</td>

<td>Gin 65</td><td>Gly</td><td>Leu</td><td>ala</td><td>Leu</td><td>Leu 70</td><td>Cheese</td><td>Glu</td><td>ala</td><td>val</td><td>Leu 75</td><td>Arg</td><td>Gly</td><td>Gin</td><td>ala</td><td>Leu 80</td>

<td>Leu</td><td>val</td><td>own</td><td>Cheese</td><td>Cheese 85</td><td>Gin</td><td>Pro</td><td>Trp</td><td>Glu</td><td>Pro 90</td><td>Leu</td><td>Gin</td><td>Leu</td><td>His</td><td>val 95</td><td>asp</td>

<td>lys</td><td>ala</td><td>val</td><td>Cheese 100</td><td>Gly</td><td>Leu</td><td>Arg</td><td>Cheese</td><td>Leu 105</td><td>Thr</td><td>Thr</td><td>Leu</td><td>Leu</td><td>Arg 110</td><td>ala</td><td>Leu</td>

<td>Gly</td><td>ala</td><td>Gin 115</td><td>lys</td><td>Glu</td><td>ala</td><td>How much</td><td>Cheese 120</td><td>Pro</td><td>Pro</td><td>asp</td><td>ala</td><td>ala 125</td><td>Cheese</td><td>ala</td><td>ala</td>

<td>Pro</td><td>Leu 130</td><td>Arg</td><td>Thr</td><td>How much</td><td>Thr</td><td>ala 135</td><td>asp</td><td>Thr</td><td>phe</td><td>Arg</td><td>lys 140</td><td>Leu</td><td>phe</td><td>Arg</td><td>val</td>

<td>Tyr</td><td>Cheese</td><td>own</td><td>phe</td><td>Leu</td><td>Arg</td><td>Gly</td><td>lys</td><td>Leu</td><td>lys</td><td>Leu</td><td>Tyr</td><td>Thr</td><td>Gly</td><td>Glu</td><td>ala</td>

145 150 155 160

Cys Arg Thr Gly Asp Arg 165