Purification of pegylated polypeptides

FIELD: chemistry.

SUBSTANCE: present invention relates to a method of purifying mono-pegylated erythropoietin using two cation-exchange chromatography steps, where the same type of cationite is used at both cation-exchange chromatography steps.

EFFECT: method of producing mono-pegylated erythropoietin in a substantially homogeneous form.

17 cl, 3 dwg, 2 tbl, 3 ex

 

The present invention relates to the field of methods of chromatographic separation, applicable for purification of polypeptides, in particular pegylated erythropoietin.

Prerequisites to the creation of inventions

Proteins play an important role in modern medical Arsenal. For use on people every therapeutic protein must meet certain criteria. In order to ensure the safety of biopharmaceutical products for people, it is important to take care to remove by-products generated during the manufacturing process. To meet the regulatory requirements following the manufacturing process must follow one or several stages of purification. Among other parameters important role in the selection of a suitable method of purification of play cleanliness, productivity and output.

Universally recognized and widely used by a variety of methods protein purification, such as affinity chromatography with microbial proteins (for example, affinity chromatography with a protein or G-protein), ion-exchange chromatography (e.g. cation exchange (sulfopropyl or carboxymethyl resins), anion exchange (iminoethylene resin) and mixed mode), teofilina adsorption chromatography (e.g. with beta-mercaptoethanol and other SH ligands), hydrophobic chromatography or adsorption chromatography is raffia with aromatic groups (for example, with phenyltetrazol, Aza-uranopilite resins, or m-aminophenylarsonic acid), metallokhelaty affinity chromatography (for example, a carrier with an affinity for Ni(II)- and Cu(II)), size-exclusion chromatography and electrophoretic methods (such as gel electrophoresis, capillary electrophoresis) (Vijayalakshmi, M.A., Appl. Biochem. Biotech. 75 (1998) 93-102). It was reported conjugation, for example, polyethylene glycol (PEG) and interleukin-6 (European patent EP 0442724), PEG and erythropoietin (international patent application WO 01/02017)containing endostatin chimeric molecules of immunoglobulins (application for U.S. patent US 2005/008649), hybrid proteins on the basis of secreted antibodies (application for U.S. patent US 2002/147311)containing albumin hybrid polypeptides (application for U.S. patent US 2005/0100991; human serum albumin - US patent US 5876969), pegylated polypeptides (application for U.S. patent US 2005/0114037) and hybrid interferons.

Necina, R. and others (Biotechnol. Bioeng. 60 (1998) 689-698) reported the capture of human monoclonal antibodies directly from the supernatant fluid cell cultures using ion-exchange environments, characterized by high charge density. In the international patent application WO 89/05157 reported method of purification of the obtained antibodies by direct cation-exchange processing of cell culture medium. Adnotation what I monoclonal antibodies purification of IgG (immunoglobulin G) from peritoneal exudates of mice described in Danielsson, A., and others, J. Immun. Meth. 115 (1988), 79-88 [in Russian]. The method of purification of the polypeptide using ion-exchange chromatography is described in the international patent application WO 2004/024866 in which to separate the desired polypeptide from one or more pollutants apply a gradient elution. In the European patent EP 0530447 described method of purification of monoclonal IgG antibodies using a combination of three chromatographic steps. Convenient way to clean monopegylated receptor antagonist interleukin-1 described in Yu, G., and others, in the Process, Biotechnol. 42 (2007) 971-977. Wang and others (Wang, H., and others, Peptides 26 (2005) 1213-1218) reported the purification of a protein hTFF3 expressed E. coli E. coli, using two-step cation-exchange chromatography. Yun and others (Yun, Q., and others, J. Biotechnol. 118 (2005) 67-74) reported the purification of pegylated recombinant human granulocyte colony-stimulating factor human rhG-CSF using two sequential ion-exchange chromatographic steps. In the international patent applications WO 2007/039436 and WO 01/087329 described erythropoietin associated covalently with polietilenglikoli group(or groups), and liquid pharmaceutical composition comprising eritropoetinov protein.

Brief description of the invention

The object of the present invention is a method of cleaning monopegylated erythropoietin, which includes stages of a solution of sod is rasego mono-, poly - and naegeliana erythropoietin, the implementation of two consecutive cation exchange chromatography steps and highlight cleaned monopegylated erythropoietin on the second cation exchange chromatography stage, and for both cation-exchange chromatographic stages use the same type of cation exchange resin.

In one of the embodiments of this method, two consecutive cation exchange chromatography stage is performed with the use of different methods of elution. In another embodiment, the present method two consecutive cation exchange chromatography stage include the following steps;

an introduction to a buffered aqueous solution containing a mixture of mono-, poly - and naegeliana erythropoietin, the first cation exchange chromatography column under conditions suitable for binding mentioned monopegylated erythropoietin contained in one column of cation exchange resin,

b) isolation monopegylated erythropoietin from the first cation exchange chromatography column by using the method of stepwise elution with a stepwise increase ionic strength eluting buffer solution, resulting in the content of the above monopegylated erythropoietin increases in comparison with the mixture, sadas savannas in step a),

C) the introduction of a dedicated monopegylated erythropoietin second cation exchange chromatography column under conditions suitable for binding mentioned monopegylated erythropoietin contained in such second column of cation exchange resin, where contained in said second column of cation exchange resin refers to the same type as the cation exchange resin in the first column

g) isolation of purified monopegylated erythropoietin mentioned second cation exchange chromatography column in a form which is substantially homogeneous, using the method of continuous elution with a continuous increase in ionic strength eluting buffer solution.

In one variation of this method, the cation exchange resin is a strong cation exchanger. In a preferred embodiment of the present invention is a strong cation exchanger is sulfopropyl cation exchange resin. Especially preferred is Toyopearl® SP 650 M. In another embodiment, monopegylated secrete erythropoietin in step d) in a form which is substantially homogeneous, with a purity of more than 95% of the chromatographic peak area. In another embodiment, this method stepwise increase of the ionic strength on the step b) of the method is the increase of ionic strength in two stages. Preferably monopegylated the config secrete erythropoietin in the second stage of the method of stepwise elution, i.e. after the second increase in ionic strength.

In another embodiment of the present invention, its object is a method for monopegylated erythropoietin, comprising the following stages:

and tahilramani erythropoietin using philonous agent,

b) purification of pegylated erythropoietin using two consecutive cation exchange chromatography steps, where the first and second cation exchange chromatographic stages involved the same type of cation exchange resin,

in the selection monopegylated erythropoietin from the second cation exchange chromatography column in a form which is substantially homogeneous.

Detailed description of the invention

The object of the present invention is a method of cleaning monopegylated erythropoietin, including two cation-exchange chromatographic step, where both cation-exchange chromatographic stages uses a cation exchange resin of the same type.

The term "ion-exchange substance (ion exchanger)as used in this application means a fixed matrix of high molecular weight, carrier covalently linked charged substituents used as stationary phase in ion-exchange chromatography. For the overall electroneutrality with her ecovalence associated counterions. "ionoobmennoe substance" is characterized by the ability to exchange their ecovalence associated counterions for similarly charged ions from the surrounding solution. Depending on the charge exchanged their counterions "ion exchange resin" refers to a cation exchange resin or anion exchange resin. Depending on the nature of the charged group (Deputy) "ion exchange resin" refers, in the case of, for example, cation exchange resins, to sulfoxylates resin (S), or sulfopropyl resin (SP), or carboxymethyl resin (CM). Depending on the chemical nature of the charged group/Deputy "ion exchange resin" may be further classified as strong or weak ion-exchange resin, which depends on the strength of covalently bound charged substituent. For example, strong cation exchange resins contain sulfonylurea group, preferably sulfopropyl group, as a charged substituent, and weak cation-exchange resin containing carboxyl group, preferably carboxymethyl group, as a charged substituent, weak anion exchange resins contain diethylaminoethyl group as a charged substituent.

Various types of ion exchange materials, i.e. stationary phases available under different names from many companies, including, for example, the cation exchange resin Bio-Rex® (e.g. type 70), Chelex® (for example, type 100), Macro-Prep® (for example, type CM, High S, 25 S), AG® (e.g. type 50W, Mr), provided by the company BioRad Laboratories, WCX 2 from the company is Ciphergen, Dowex® MAC-3 from Dow Chemical Company, Mustang and Mustang's from Pall Corporation, cellulose CM (for example, type 23, 52), hyper-D, partisphere from Whatman plc., Amberlite® IRC (for example, type 76, 747, 748), Amberlite® GT 73, Toyopearl® (e.g., type SP, CM, 650M), provided by the company Tosoh Bioscience GmbH, CM 1500 and 3000 CM from BioChrom Labs, SP-sepharoseTMCM-sepharoseTMfrom GE Healthcare, Poros resin from PerSeptive Biosystems, Asahipak ES (for example, type S), CXpak P, IEC CM (for example, type 825, 2825, 5025, LG), IEC SP (for example, type 420N, 825), IEC QA (for example, type LG, 825), provided by the company Shoko America Inc., cation-exchange resin 50W from Eichrom Technologies Inc. Preferably the cation exchange resin is a strong cation exchanger, 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 resins are Dowex® 1 from Dow Chemical Company, AG® (e.g. type 1, 2, 4), Bio-Rex® 5, DEAE (diethylaminoethyl) Bio-Gel 1, Macro-Prep® DEAE provided by the campaign BioRad Laboratories, anion-exchange resin type 1 from Eichrom Technologies Inc., Source Q, ANX sepharose 4, DEAE-sepharose (for example, type CL-6B, FF), Q sepharose, Capto Q, Capto S, provided by GE Healthcare, AX-300 from PerkinElmer, Asahipak ES-502C, AXpak WA (for example, type 624, G), IEC, DEAE provided by the campaign Shoko America Inc., Amberlite® IRA-96, Toyopearl® DEAE, TSKgel DEAE provided by the campaign Tosoh Bioscience GmbH, Mustang Q from Pall Corporation. In one embodiment, the implementation of this is th invention, the cation exchanger is sulfopropyl cation exchange resin.

The expression "the same type of cation exchange resin" refers to two consecutive ion-exchange chromatographic stages, carried out with the use of identical cation. This means that sequential cation exchange chromatographic stages are carried out either by using the first portion of the cation to the first cation exchange chromatographic stage and the second portion of the same cation exchange resin for the second cation exchange chromatography stage, or using the same cation for both cation-exchange chromatographic steps. In one of the embodiments of the present invention, the second cation is the same type of cation exchange resin, the first cation exchange resin, but not the same its serving.

The terms "step elution" and "method of stepwise elution", as used in this application interchangeably, indicate the way in which, for example, the concentration of the substance causing elution, i.e. the dissolution of the associated connection of the substance carrier, abruptly increase or decrease, i.e. directly from one value/level to another value/level. When this step elution" one or more conditions, such as pH, ionic strength, salt concentration and/or rate of elution, stepwise change from the first, for example source is th, value to the second, for example, end, size, i.e. conditions change discretely, i.e. speed, in contrast to linear changes. In method step elution" collect new faction after each increase in ionic strength. This fraction contains compounds selected from ion-exchange substances with a corresponding increase in ionic strength. After each of improving the conditions to support the next stage of this method of elution. When "step elution" one or more conditions together abruptly change from the first, for example, the original value before the second, for example finite value. In one of the embodiments of the present invention, the magnitude of the change is 10% or more of the concentration causing elution of the substance. This means that in this embodiment of the present invention, the concentration causing elution of the substance is 100% at the first stage, 110% or more on the second level and 120% or more of the third stage. In another embodiment of the present invention, the magnitude of the change is 50% or greater concentration causing elution of the substance. Even in one of the embodiments of the present invention, the magnitude of the change is 120 percent or more of the concentration causing elution of substances the. The term "stepwise elution" means that conditions change discretely, i.e. speed, in contrast to linear changes.

The terms "continuous elution" and "method of continuous elution", as used in this application interchangeably, mean a way in which, for example, the concentration of the substance causing elution, i.e. the dissolution of the bound/adsorbed compounds of chromatographic media, increase or decrease continuously, i.e. the concentration change of successive small steps, each of which occurs not more than two percent, preferably one percent, the change in the concentration causing elution of the substance. In this "continuous elution" one or more conditions, such as pH, ionic strength, salt concentration and/or rate of elution may be changed linearly or exponentially or asymptotically. Preferably the change is linear.

The term "introduction" and its grammatical equivalents, as used in this application means the substage, MIS cleaning method, which contains the desired substance solution is injected into contact with the stationary phase. This means that a) the solution is injected in the chromatographic device, which is the stationary phase, or b) the stationary phase is added to the solution is. In the case of (a) a solution containing subjected to the purification of the desired substance passes through the stationary phase, providing interaction between the stationary phase and the substances in solution. Depending on conditions, such as pH, conductivity, salt concentration, temperature and/or rate of elution, some substances from the solution contacted with the stationary phase and thus removed from solution. Other substances remain in solution. Remaining in solution substances can be detected in the flow-through fraction. The term "flow-through fraction" means the solution obtained after passing through the chromatographic device, which can be introduced as a solution containing the desired substance, and the buffer solution used to wash the column and elution of one or more related stationary phase substances. In one of the embodiments of the present invention the chromatographic device is a column or cartridge. The desired substance can be separated from the solution after purification stages with well-known expert in the appropriate field methods, such as, for example, precipitation, salting out, ultrafiltration, diafiltration, lyophilization, affinity chromatography, or the reduction of the volume of the solvent, to obtain the desired substance in the form aplause which are substantially homogeneous. In case b) to the solution containing subjected to the purification of the desired substance, type of stationary phase, such as solid, providing interaction between the stationary phase and the substances in solution. After interaction of the stationary phase is removed, for example by filtration, and the desired substance or is connected with the stationary phase and is removed along with it from the solution, or is not associated with the stationary phase and remains in solution.

The expression "suitable for binding conditions" and its grammatical equivalents, as used in this application means that the desired substance, such as pegylated erythropoietin is associated with the stationary phase when introduced into interaction with it, such as with ion exchange substance. This does not necessarily mean that all connect 100% of the desired substance, but 100% of the required substances are associated in a significant amount, i.e. with stationary phase is associated with at least 50% of the desired substance is associated with at least 75% of the desired substance is associated with at least 85% of the desired substance or binds more than 95% of the desired substance.

The term "buffered"as used in this application means a solution in which the pH changes due to the addition or release of acidic or basic substances are aligned with the buffer prophetic the STV. Can be used any buffering agent, leading to such effect. Preferably use pharmaceutically acceptable buffering agents, such as, for example, phosphoric acid or a salt thereof, acetic acid or a salt thereof, citric acid or its salts, morpholine, 2-(N-morpholino)econsultation or a salt thereof, histidine or salts thereof, glycine or its salt or Tris(hydroxymethyl)aminomethan (Tris) or its salt. In one of the embodiments of the present invention as a buffer substance use phosphoric acid or a salt thereof, or acetic acid or a salt thereof, or citric acid or a salt thereof, or a histidine or salts thereof. The buffer solution may optionally contain additional salt such as sodium chloride, sodium sulfate, potassium chloride, potassium sulfate, sodium citrate or potassium citrate.

Common methods of chromatography and their application well-known specialist in the relevant field. See, for example, Chromatography (Chromatography), 5th edition, part a: Fundamentals and Techniques (Principles and methods), Ed. by Heftmann, E., Elsevier Science Publishing Company, New York, (1992); Advanced Chromatographic and Electromigration Methods in Biosciences (Advanced chromatographic and electromigration methods in the biological Sciences), edited by Deyl, Z., Elsevier Science BV, Amsterdam, The Netherlands, (1998); Chromatography Today (Chromatography today), Poole, c.f.nielsen (Denmark), and Poole, S.K., Elsevier Science Publishing Company, New York, (1991); Scopes, Protein Purificatin: Principles and Practice (protein Purification: principles and practice (1982); Ed. by Sambrook, J., and others, Molecular Cloning: A Laboratory Manual (Molecular cloning: a laboratory manual), 2nd edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; or Current Protocols in Molecular Biology (Modern methods in molecular biology), edited by Ausubel, F.., etc., John Wiley & Sons, Inc., New York.

Tahilramani erythropoietin leads, as a rule, mixtures of different compounds, such as paliperidone erythropoietin, monopegylated erythropoietin, naegeliana erythropoietin products of the hydrolysis of ester activated erythropoietin, for example pegylated acid in free form, as well as the products of hydrolysis of the erythropoietin. With the aim of obtaining monopegylated erythropoietin in a form which is substantially homogeneous, these compounds must be separated, and the desired connection must be cleared.

In this regard, an object of the present invention is a method for monopegylated erythropoietin in a form which is substantially homogeneous, comprising the following stages:

and tahilramani erythropoietin with activated philonous reagent with a molecular mass of 20 kDa to 40 kDa,

b) purification obtained in stage a) pegylated erythropoietin using two consecutive cation exchange chromatography steps, where the first and second to ionoobmennyh chromatographic stages involved in the cation exchange resin of the same type,

in the selection monopegylated erythropoietin from the second cation exchange chromatography column in a form which is substantially homogeneous.

This method is especially useful for the purification of pegylated recombinant polypeptide which is glycosylated, i.e. produced in mammalian cells, preferably in a cell Chinese hamster ovary (Cho), human embryonic cell of the kidney (HEK293)cell kidney baby hamster (BHK)cell line Regs® or cancer cells HeLa, and subsequently chemically pegylated.

In the first stage of the method erythropoietin philonous. Polymer molecules of polyethylene glycol (PEG)used in the reaction of tahilramani, are characterized by a molecular mass of approximately 20 kDa to 40 kDa (the term "molecular weight"as used here should be understood in the average molecular weight of the PEG because PEG, which is a polymer compound cannot be obtained with a certain molecular weight, in fact it is characterized by molecular weight distribution. The term "about" indicates that samples of PEG, some molecules will have more and some less weight compared with the specified molecular weight, i.e. the term "approximately" refers to the molecular weight divided the Yu, in which 95% of the PEG molecules are characterized by a molecular weight within +/- 10% of specified molecular weight. For example, a molecular weight of 30 kDa indicates the range of 27 to 33 kDa kDa).

The term "erythropoietin" refers to a protein characterized by the sequence number 1 or number 2 from the following description of the sequence, or to a significant extent it is homologous to a protein or polypeptide biological properties which belong to stimulate production of red blood cells and to stimulate the division and differentiation of commiteeman erythroid precursor cells in the bone marrow. Recombinant erythropoietin can be obtained by expression in eukaryotic cells, such as cells of Cho, or cells KSS, or HeLa cells, using techniques based on recombinant DNA, or by endogenous gene activation. For example, eritropoetinov glycoprotein is expressed by endogenous gene activation, as reported in US Patents US 5733761, US 5641670, US 5733746 and international patent applications WO 93/09222, WO 94/12650, WO 95/31560, WO 90/11354, WO 91/06667 and WO 91/09955. In one of the embodiments of the present invention erythropoietin according to the present invention is based on the sequence of human erythropoietin (EPO). In another embodiment, implementation of the program of the present invention human erythropoietin is characterized by the amino acid sequence, presented in the description below, under number 1 or number 2, preferably human erythropoietin is characterized by the amino acid sequence presented in the description below, under number 1. The term "erythropoietin" also applies to variants of the protein according to sequence number 1 or number 2, in which one or more amino acid residues are replaced, deleted or inserted, and which have the same biological activity as unmodified protein, as, for example, described in European patent EP 1064951 or in U.S. patent US 6583272. Option may have the amino acid sequence of human erythropoietin containing from one to six additional sites for glycosylation. The specific activity of the pegylated erythropoietin may be determined by various assays known in the relevant field. Biological activity of the purified pegylated erythropoietin of the present invention is that the introduction of the protein by injecting patients-people leads to increased production by the bone marrow cells of reticulocytes and red blood cells compared to unexposed injection or control groups of patients. Biological activity pagalilauan what about erythropoietin, obtained and purified in accordance with the present invention, can be investigated using methods according to Pharm. Europa Spec. Issue Erythropoietin BRP Bio 1997(2).

The term "PEG" or "PEG-group" according to the present invention means a residue containing polyethylene glycol as its main part. This PEG can contain other chemical groups necessary for the coupling reaction, i.e. conjugation, resulting from the chemical synthesis of molecules or a spacer for optimal distances between parts of the molecule. These additional chemical groups are not taken into account when calculating the molecular weight of the polymer molecules of PEG. In addition, the PEG may include one or more side chains of PEG connected with each other. Options PEG with more than one chain of PEG-called multi-beam or branched PEG. Branched PEG can be obtained, for example, by adding polyethylene oxide to various polyols, including glycerol, pentaerythritol and sorbitol. Branched PEG is described, for example, in European patent EP 0473084 and U.S. Patent US 5932462. In one of the embodiments of the present invention as PEG with molecular weight of 20-35 kDa used linear PEG molecules, and polymers of PEG with a molecular mass of 35 kDa, in particular 40 kDa, COI is lsout branched variants PEG. In one of the embodiments of the present invention as a PEG with a mass of 40 kDa using PEG with two chains.

The term "tahilramani" means the covalent binding of polietilenglikoli residue at the N end of the polypeptide and/or internal lysine residue. Tahilramani proteins is well known in the relevant field, an overview of it is given, for example, Veronese, F.M., Biomaterials 22 (2001) 405-417. PEG can be attached using a variety of functional groups and polyethylene glycols of various molecular weights, linear and branched variants PEG, as well as various connecting groups (see also Francis, G.E., and others, Int. J. Hematol. 68 (1998) 1-18; Delgado, C., and others, Crit. Rev. Ther. Drug Carrier Systems 9 (1992) 249-304). Tahilramani erythropoietin can be carried out in the same solution using philonous reagents, as described, for example, in international patent application WO 00/44785, in one of the embodiments using the activated N-hydroxysuccinimide (NGS) linear or branched PEG molecules with a molecular weight between 5 kDa and 40 kDa. Tahilramani can also be carried out in the solid phase according to Lu, Y., and others, Reactive Polymers 22 (1994) 221-229. Often pegylated at the N end of the proteins can also be obtained according to the international patent application WO 94/01451.

Such methods lead to erythropoietin, spotted the new one or more ε-amino lysine residues and/or N-terminal amino group. Selective tahilramani N-terminal amino acid can be carried out according to Felix, A.M., and others, ACS Symp. Ser. 680 (polyethylene glycol) (1997) 218-238. Selective tahilramani N-end can be reached during solid-phase synthesis through a combination of Nαpegylated amino acid derivative with N-1 terminal amino acid of the peptide chain. Tahilramani in the form of a side chain can be carried out during solid-phase synthesis through a combination of Nεpegylated derivatives of lysine to the growing chain. The combined tahilramani N-end and with the formation of the side chain is feasible or as described above during solid-phase synthesis, or by synthesis in solution, subjecting the peptide is removed from the protection of the amino group to the action of activated philonous reagents.

Suitable PEG derivatives are activated molecules of PEG with an average molecular weight of from about 5 to about 40 kDa, and in one of the embodiments of the present invention from about 20 to about 40 kDa, preferably from about 30 kDa to about 35 kDa. In one of the embodiments of the present invention pegylated derivative is a linear or branched PEG. A large variety of PEG derivatives that are suitable for application in which I am receiving PEG-protein and PEG-peptide conjugates, can be obtained from the company (Huntsville, AL, U.S.A.; www.nektar.com).

Activated PEG derivatives known in the relevant field and are described, for example, Morpurgo, M., and others, J. Bioconjug. Chem. 7 (1996) 363-368, for the case of PEG-vinylsulfonic. Linear and branched types of PEG suitable for receiving pegylated fragments. Examples of the reactive PEG reagents are iodoacetate-PEG-or methoxy-PEG-vinylsulfonic (preferably m is an integer from about 450 to about 900, a R is a (C1-C6)alkyl, linear or branched and contains from one to six carbon atoms, such as methyl, ethyl, isopropyl, etc. In accordance with this, in one of the embodiments of the present invention R is the stands):

or

The application of these activated iodine substances known in the relevant field and are described, for example, Hermanson, G. T. in Bioconjugate Techniques (Methods bioconjugates), Academic Press, San Diego (1996), str-148.

In one of the embodiments of the present invention, the PEG is activated complex ether of PEG, for example N-hydroxysuccinimide, or N-hydroxysuccinimidyl, or N-hydroxysuccinimidyl, such as PEG-NRS (Monfardini, C., and others, Bioconjugate Chem. 6 (1995) 62-69). In one variant of implementation n the present invention is an ester of N-hydroxysuccinimide is

or

using alkoxy-PEG-N-hydroxysuccinimide, such as methoxy-PEG-N-hydroxysuccinimide (MW 30000; Shearwater Polymers, Inc.), where R and m meet the above definition. In one of the embodiments of the present invention, the PEG is a complex N-hydroxysuccinimidyl ether methoxypolyethyleneglycol acid. The term "alkoxygroup" refers to the group of simple Olkiluoto ether, where the term "alkyl" means an unbranched or branched alkyl group containing not more than four carbon atoms, such as methoxy group, ethoxypropan, n-propoxylate and the like, preferably a methoxy group.

The expression "substantially homogeneous form"as used in this application means that the obtained contained or used pegylated erythropoietin contains a certain number of attached PEG groups. In one of the embodiments of the present invention pegylated erythropoietin is monopegylated with erythropoietin. The product may contain unreacted (i.e., not bearing group PEG) erythropoietin, paliperidone erythropoietin, as well as fragments of the polypeptide, formed during the reaction of tahilramani. The term "substantial flat surface is homogeneous form" means, what drug monopegylated erythropoietin contains in one of the embodiments of the present invention at least 50% (wt./mass.) monopegylated erythropoietin, at least 75% monopegylated erythropoietin, at least 90% monopegylated erythropoietin or more than 95% monopegylated erythropoietin. Percentage based on the percentage area of the chromatogram, corresponding to the results of the cation-exchange chromatographic purification, after which get monopegylated erythropoietin.

The object of the present invention is a method of cleaning monopegylated erythropoietin, with the aim of obtaining monopegylated erythropoietin in a form which is substantially homogeneous. It was unexpectedly discovered that the combination of two consecutive cation exchange chromatography steps, using the same type of cation, leads to monopegylated erythropoietin in a form which is substantially homogeneous. Thus, an object of the present invention is a method of cleaning monopegylated erythropoietin, which includes stages of a solution containing mono-, poly - and naegeliana erythropoietin, the implementation of two consecutive cation exchange chromatography steps and highlight cleaned monopegylated eritropoyetina on the second cation exchange chromatography stage, moreover, on both cation-exchange chromatographic stages use the same type of cation exchange resin. In one of the embodiments of the present invention the selection of the first cation exchange chromatographic stage is carried out using another method of elution than the allocation for the second cation exchange chromatography stage. In another embodiment, the present invention cation-exchange chromatographic column regenerate after the first cation exchange chromatographic stage and after the second cation exchange chromatography stage.

The selection is cleared monopegylated erythropoietin on the second cation exchange chromatographic stage is implemented by elution monopegylated erythropoietin involved in the second chromatographic stage of the cation. In one embodiment, the process according to the present invention two cation-exchange chromatographic stage differ sodastream method of elution. In this embodiment, the first cation exchange chromatographic stage is carried out using the method of stepwise elution, i.e. the ionic strength of the buffer used to increase speed, i.e. abruptly from one value of the ionic strength to the next value of the ionic strength, preferred is entrusted, by a change of 10% or more. In one of the embodiments of the present invention, the method of stepwise elution is carried out in the form of a three-step elution. The first stage of the cation-exchange chromatographic column mainly eluted paliperidone erythropoietin. The second increase in ionic strength mainly eluted monopegylated erythropoietin with a purity of more than 60% based on the square of the corresponding exclusion chromatogram (% area). When the third increase in ionic strength mainly from the column eluted remaining naegeliana erythropoietin.

The second cation exchange chromatographic stage is carried out in one of the embodiments of the present invention using the method of continuous elution, i.e. the ionic strength of the buffer increases continuously, preferably, by a change to a less than 5%. Eluruume fractions containing monopegylated erythropoietin, together with the purpose of obtaining monopegylated erythropoietin in a form which is substantially homogeneous, and contains, in one of the embodiments of the present invention, less than 0.5% of forms with low molecular weight according to the peak areas on the corresponding chromatogram. The buffer is preferably present in a concentration of from 10 mm to 20 mm, and in one of the embodiments of the present invention is from 50 mm to 150 mm, in another embodiment, the present invention is approximately 100 mm. Thus, in the method according to the present invention, two consecutive cation exchange chromatography stage represent the following sequence of steps:

an introduction to a buffered aqueous solution containing a mixture of mono-, poly - and naegeliana erythropoietin, and forms low molecular weight, in the first cation exchange chromatography column under conditions suitable for binding mentioned monopegylated erythropoietin with cation exchange resin contained in the specified first column

b) isolation monopegylated erythropoietin from the first cation exchange chromatography column by using the method of stepwise elution with a stepwise increase of the ionic strength of the running buffer, where the relative content monopegylated erythropoietin in the selected solution is improved in comparison with the mixture, put on stage a),

C) the introduction of a dedicated monopegylated erythropoietin from stage b) to a second cation exchange chromatography column under conditions suitable for binding mentioned monopegylated erythropoietin with cation exchange resin contained in the specified vtoro the column, moreover, the cation exchange resin contained in such second column, of the same type as the cation exchange resin in the first column

g) isolation of purified monopegylated erythropoietin in a form which is substantially homogeneous, mentioned the second cation exchange chromatography column by using the method of continuous elution with a continuous increase of the ionic strength of the running buffer.

Tahilramani polypeptide does not, as a rule, to the product of tahilramani in homogeneous form. Moreover, it is obtained in the form of a mixture monopegylated, paliperidone and naegeliana product. Therefore, the solution of pegylated erythropoietin, administered at the stage a) of the method is a mixture of mono-, poly - and naegeliana erythropoietin, as well as forms or fragments of low molecular weight in an aqueous buffer solution. The relative content of various substances was determined by exclusion high-performance liquid chromatography (AWASH). An example chromatogram is shown in figure 1. The sum of the areas assigned peaks, i.e. the area under the peaks, figure 1 is a General size exclusion chromatogram. The share of individual peak is given as a percentage of the square, i.e. the relative share of area from the total area of the chromatogram.

Common chromatographic methods, their application and putting what I said to him the terms well-known specialist in the relevant field. See, for example, Chromatography (Chromatography), 5th edition, Part A: Fundamentals and Techniques (part a: Fundamentals and methods, Ed. by Heftmann, E., Elsevier Science Publishing Company, New York, (1992) and other related books. In continuation of chromatographically through the cation-exchange chromatographic column runs the buffer. This "running buffer" shall be chosen in accordance with the requirements of the stages of chromatographic procedures. He transfers the desired substance to a chromatographic stationary phase (introduction) and out (elution).

At first cation exchange chromatographic phase mixture monopegylated, paliperidone and naegeliana erythropoietin is administered at a concentration of protein from 0.7 to 1.5 mg/ml, preferably about 1 mg/ml, in the first cation exchange chromatography column in buffered aqueous solution. In one of the embodiments of the present invention buffered aqueous solution contains about 100 mm potassium phosphate at pH of 3.0. The term "about" ("approximately"), as used in this application means a ten percent range around the specified value, i.e. ±10%. In one of the embodiments of the present invention the first column is washed before and after the introduction of the same buffer solution. For the first stage in the method of stepwise elution buffer is replaced with a buffer containing approximately the 100 mm potassium phosphate and about 90 mm sodium chloride at pH of 3.0. Under the action of this buffer from the cation exchange chromatography column suiryudan hydrolyzed activated PEG reagent, i.e. the corresponding pegylated carboxylic acid, unreacted agent for combination and paliperidone erythropoietin. For the second stage in the method of the three-step elution buffer solution is replaced by a buffer containing 100 mm potassium phosphate and about 250 mm sodium chloride at pH of 3.0. At this stage from the first cation exchange chromatography column allocate monopegylated erythropoietin. The collected flow-through buffer this stage elution was diluted in a ratio of approximately 1:5 (vol./about.) to 1:8 (vol./vol.), preferably 1:5 (vol./vol.), purified water. An example of the first cation exchange chromatographic stage is represented in figure 2. For the third step in the three-way elution buffer is replaced with a buffer containing 100 mm potassium phosphate and about 750 mm sodium chloride at pH of 3.0. At this stage, from the first cation exchange chromatography column allocate naegeliana erythropoietin.

The collected flow-through buffer from the second stage of the first cation exchange chromatographic stage contains monopegylated erythropoietin with increased relative content, i.e. the mass fraction or percentage of the square (x is autogramme exclusion chromatography of the collected flow-through buffer from the second stage) monopegylated erythropoietin increased in comparison with the state, before the first cation exchange chromatography stage. In one of the embodiments of the present invention relative content monopegylated erythropoietin is not less than 60% by area. In another embodiment, the present invention relative content monopegylated erythropoietin is not less than 80% by area.

For further purification monopegylated erythropoietin carry out the second cation exchange chromatography stage. On the second cation exchange chromatography stage, the collected and diluted with running buffer from the second stage of the elution lead to the potassium phosphate concentration of about 100 mm and a pH of about 3.0, and enter it in the second cation exchange chromatography column containing a cation exchange resin of the same type as the first cation exchange chromatography column. In one of the embodiments of the present invention, the second cation-exchange column and its cation are the same as on the first cation exchange chromatography stage. Monopegylated erythropoietin isolated from the second cation exchange chromatography column by application of a linear gradient, starting from potassium phosphate buffer with a concentration of about 100 mm, containing about 50 mm of sodium chloride p and pH of 3.0, and ending potassium phosphate buffer with a concentration of about 100 mm, containing 500 mm sodium chloride at pH of 3.0. The concentration of sodium chloride varies linearly in the continuation of the transmission volume of eluent is equal to ten volumes of the column. Running buffer is separated into fractions and diluted per faction 1 M solution of monohydrogenphosphate potassium to increase the pH to about 6-8. An example chromatogram is presented in figure 3.

After the second cation exchange chromatographic stage monopegylated erythropoietin get in shape, which is substantially homogeneous, and in one of the embodiments of the present invention with a purity of at least 95% by area.

Specialist in the relevant field should be familiar with the methodology ion-exchange chromatography. At the stage of selection of the polypeptide that is associated with the cation, ionic strength, i.e. conductivity, buffer/solution flowing through the ion exchange column, increase. This can be done either by increasing the concentration of the buffer salt, or by adding other salts, so-called eluting salts to buffer the solution. Depending on the method of elution concentration of the buffer/salt increases abruptly (method stepwise elution) or continuously (continuous way, elwi the Finance) by adding parts of concentrated buffer or solution eluting salt. Preferred eluting salts are sodium citrate, sodium chloride, sodium sulfate, sodium phosphate, potassium chloride, potassium sulfate, potassium phosphate or other salt of citric acid or phosphoric acid, or any mixture of these components. In one of the embodiments of the present invention eluting salt is sodium citrate, sodium chloride, potassium chloride or mixtures thereof.

In one of the embodiments of the present invention the cation exchange resin is a strong cation exchanger, such as, preferably, Toyopearl® SP 650 M Concentration causing elution of the salt is in one of the embodiments of the present invention, in the range from 5 mm to 500 mm, preferably in the range from 5 mm to 400 mm, more preferably in the range from 5 mm to 250 mm. In another embodiment, the present invention causes the elution salt is used at the same time and as a buffer substance, such as, for example, citric acid or its salts or phosphoric acid or a salt thereof.

Monopegylated erythropoietin can be used in suitable for injection pharmaceutical compositions with a pharmaceutically acceptable carrier or substrate using known in the relevant field methods. For example, suitable compositions are described in international patent applications WO 97/09996, WO 97/4050, WO 98/58660 and WO 99/07401. Among the preferred pharmaceutically acceptable carriers for the manufacture of drug products of the present invention can be mentioned the human serum albumin, proteins of human blood plasma and the like, Compounds of the present invention can be introduced into the product in 10 mm potassium/sodium phosphate buffer at pH 7 containing means for maintaining toychest, for example 132 mm sodium chloride. The pharmaceutical composition may optionally contain a preservative. The pharmaceutical composition may contain different amounts of monopegylated erythropoietin, for example, 10-1000 μg/ml, 50 µg or 400 µg.

Introduction eritropoetinov glycoprotein products according to the present invention leads to the production people of red blood cells. Thus, the introduction monopegylated eritropoetinovmi glikoproteinov product replenishes the amount of that eritropoetinovmi protein, which is essential for production of red blood cells. Pharmaceutical compositions containing monopegylated eritropoetinov glycoprotein products can be used in drugs with the same action that would be effective with the introduction of a variety of ways to the patient-the person suffering from a hematological disorder characterized by low the or impaired production of red blood cells, as an end in itself and as a component of the condition or disease. The pharmaceutical compositions may be introduced by injection, such as subcutaneous or intravenous injection. The average number monopegylated eritropoetinovmi glikoproteinov product may vary. The exact amount of conjugate is a matter of preference, depending on such factors as the exact type being treated condition, the condition being treated patient, as well as other components of the composition. For example, can be entered, for example, once a week from 0.01 to 10 μg per kilogram of body weight, preferably from 0.1 to 1 microgram per kilogram of body weight.

The following examples describe sequences and figures are given as supporting material for understanding the present invention, the true scope of which is set forth in the attached claims. It should be understood that the following methodologies can be introduced modifications without departing from the spirit of the invention.

Description of figures

Figure 1. AWASH mixture in various ways pegylated derivatives of erythropoietin, including the assignment of the peaks to substances.

Figure 2. An example chromatogram of the method of stepwise elution.

Figure 3. An example chromatogram of the method of continuous elution.

The materials and Mody

AWASH

AWASH separates proteins according to their average molecular weight. Thus, this method is able to detect the presence monopegylated erythropoietin, forms and fragments with low molecular weight, paliperidone forms and higher units of erythropoietin. Installation HPLC equipped with a detector wave length of 220 nm and column Superose 6 HR (dimensions 10×300 mm, Pharmacia Biotech, catalog number: 17-0537-01) or column Superose 6 10/300 GL (Pharmacia Biotech, catalog number: 17-5172-01). Column works in isocratic conditions at room temperature at flow rate of elution of about 0.4 ml/min Buffer mobile phase is 50 mm sodium phosphate buffer containing 300 mm sodium chloride at pH 6.8. Depending on the HPLC system study this method can be performed if the volume of the injected sample 100 ál or 500 ál. Samples diluted with buffer mobile phase to a protein concentration of about 0.5 mg/ml (load 100 ál) or 0.1 mg/ml (load 500 ál). Samples with a protein concentration less than 0.1 mg/ml can be used in undiluted form. Eluruume proteins are logged when the wavelength detector 220 nm.

Example 1

Fermentation and purification of erythropoietin

Erythropoietin can be obtained according to, for example, international patent application WO 01/87329 and purified as OPI the ANO in the international patent application WO 96/135718.

Example 2

Tahilramani erythropoietin bifunctional reagents

a) Activation of erythropoietin

The specified amount of the reagent containing blocked Tilney group, SATA (Succinimidyl-S-acetylthiourea) or SATP (Succinimidyl-3(acetylthio)propionate), (dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mg/ml), added to a solution of benzyl-protected erythropoietin to 1 ml of protein solution concentration of 5 mg/ml in 10 mm potassium phosphate buffer with the addition of 50 mm sodium chloride at pH of 7.3. The reaction mixture is stirred for about 30 minutes (at 25°C) and stop the reaction by adding 1 M solution of lysine to a final concentration of 10 mm. The excess SATA and SATP removed by dialysis against 10 mm potassium phosphate buffer containing 50 mm sodium chloride and 2 mm ethylenediaminetetraacetic acid (adtc) at pH of 6.2. Acetyl protective group is removed using hydroxylamine.

b) Tahilramani activated erythropoietin

380 mg of methoxy-PEG-maleimide (molecular weight 30,000; Shearwater Polymers, Inc., Huntsville (Alabama, USA)is dissolved in a solution containing 95 mg of activated erythropoietin (4.5 mg/ml in 10 mm potassium phosphate buffer containing 50 mm sodium chloride and 2 mm add, pH of 6.2). Thus obtained the molar ratio between the activated erythropoietin and methoxy-PEG-Mal is the imide in the solution is from 1:2 to 1:4. By adding to the above solution of 1 M aqueous solution of hydroxylamine to a final concentration of 30 mm (pH of 6.2) leads to the release of covalently linked blocked tylnej groups in the activated erythropoietin. Thus obtained in the reaction mixture solution activated erythropoietin contains free tirinya group (-SH). After the release of tylnej groups immediately carry out the reaction mix between activated erythropoietin, containing now available tirinya group (-SH), and methoxy-PEG-maleimide for 90 minutes with stirring, at 25°C). The reaction mix is stopped by adding to the reaction mixture of 0.2 M aqueous solution of cysteine to a final concentration of 2 mm. After 30 minutes the excess of free tylnej groups reacted with methoxy-PEG-maleimide activated erythropoietin block by adding 0.5 M solution of N-methylmaleimide in DMSO to achieve a final concentration of 5 mm. After 30 minutes, the thus obtained reaction mixture, containing now pegylated erythropoietin, may be subjected to cleaning.

Example 3

Cleaning monopegylated erythropoietin

a) the First chromatographic stage on SP Toyopearl 650 M

First chromatographic stage of achadirect carry on sulfopropyl (SP) column, filled JV Toyopearl 650 M Column operates at room temperature. The maximum capacity of the first column is defined as 1.5 grams of protein per liter of column volume. The column is brought into equilibrium with 100 mm potassium phosphate buffer with a pH from 2.9 to 3.1 (buffer SP-A). After the boot stage column is washed and carry out elution with sequences potassium phosphate buffers containing increasing amounts of NaCl. Hydrolyzed reagent PEG and paliperidone forms are removed in the flow-through fraction, and in the next step of washing buffer SP-a and 100 mm potassium-phosphate buffer, pH 2.9 to 3.1, containing 90 mm sodium chloride (buffer SP-B), respectively.

Monopegylated erythropoietin elute with 100 mm potassium phosphate buffer, pH 2.9 to 3.1, containing 250 mm sodium chloride (buffer NP-In), collected in a vessel and directly diluted with purified water at a ratio of 1:5. This collected eluate is called "SP ouatim pool I".

After that, the column was washed with 100 mm potassium phosphate buffer, pH 2.9 to 3.1, containing 750 nm sodium chloride (buffer SP-D), in order to remove unreacted erythropoietin and regeneration of the column.

b) a Second chromatographic stage on JV Toyopearl 650 M

The second column operates at room temperature. After it is brought into equilibrium with the buffer SP-a in the Alonso enter JV blatny pool I, then the column was washed with buffer SP-A. Monopegylated erythropoietin elute under the action of a linear gradient with a slope of approximately from 50 to 500 mm sodium chloride at ten column volumes, buffered with 100 mm potassium phosphate buffer at pH from 2.9 to 3.1. Peak product separated up to eight and directly diluted per faction 1 M solution of monohydrogenphosphate potassium to increase the pH to 6-8.

After elution monopegylated erythropoietin slope gradient can be increased, which leads to immediate washing of the column with 100 mm potassium phosphate buffer with a pH from 2.9 to 3.1 containing 500 mm sodium chloride.

in the Regeneration column SP Toyopearl 650 M

The resin from both columns regenerate using simistatin sequence. The column was washed with purified water, and then 0.5 M solution of sodium hydroxide. The alkaline solution displace purified water, and then conduct acid washing (0.5 M sodium dihydrophosphate, 1 M phosphoric acid). After another stage with purified water column depyrogenation 0.5 M solution of sodium hydroxide for ≥4 hours. After alkaline regeneration of the column is again washed with purified water. Summary of column parameters given in table 1 and table 2.

Table 1
The settings column on the first chromatographic stage
StageBuffer solutionVolume columnThe volumetric rate [l/min]
Bringing balance100 mmol/l potassium phosphate, pH 2,9-3,1 (Buffer SP-A)≥61,6-2,1
Download columnDiluted SP-And the reaction mixture (1:5)the concentration is1,6-2,1
Lavage SP-A100 mmol/l potassium phosphate, pH 2,9-3,1 (Buffer SP-A)21,6-2,1
Lavage SP-B100 mmol/l potassium phosphate, pH 2,9-3,1, 90 mmol/l NaCl (Buffer SP-B)2-31,6-2,1
Elution SP-In100 mmol/l potassium phosphate, pH 2,9-3,1, 250 mmol/l NaCl (Buffer NP-In)2-31,6-2,1
Lavage SP-G100 mmol/l potassium phosphate, pH 2,9-31, 750 mmol/l NaCl (Buffer SP-D)2-31,6-2,1
FlushingPurified water III≥21,6-2,1
Alkaline regeneration column I0.5 mol/l NaOH≥21,6-2,1
FlushingPurified water III≥21,6-2,1
Acid regeneration column1 mol/l of phosphoric acid, 0.5 mol/l of sodium dihydrophosphate≥31,6-2,1
FlushingPurified water III≥21,6-2,1
Alkaline regeneration column II0.5 mol/l NaOH≥3the concentration is
FlushingPurified water III≥21,6-2,1

Table 2
The settings column on the second chromatographic stage
StageBuffer solutionVolume columnThe volumetric rate [l/min]
Bringing balance100 mmol/l potassium phosphate, pH 2,9-3,1 (Buffer SP-A)≥61,6-2,1
Download columnJV blatny pool I, diluted with purified water III (1:5)the concentration is1,6-2,1
Lavage SP-A100 mmol/l potassium phosphate, pH 2,9-3,1 (Buffer SP-A)2-31,6-2,1
The gradient elutionGradient of 50-500 mmol/l NaCl ten column volumes from buffer SP And 100 mmol/l potassium phosphate and 500 mmol/l NaCl, pH 2,9-3,1 (buffer SP-D)101,6-2,1
FlushingPurified water III≥21,6-2,1
Alkaline regeneration column I0.5 mol/l NaOH ≥21,6-2,1
FlushingPurified water III≥21,6-2,1
Acid regeneration column1 mol/l of phosphoric acid, 0.5 mol/l of sodium dihydrophosphate≥31,6-2,1
FlushingPurified water III≥21,6-2,1
Alkaline regeneration column II0.5 mol/l NaOH≥3the concentration is
FlushingPurified water III≥21,6-2,1
concentration: not defined

1. Cleaning method monopegylated erythropoietin, which includes stages of a solution containing mono-, poly - and naegeliana erythropoietin, the implementation of two consecutive cation exchange chromatography steps and highlight cleaned monopegylated erythropoietin on the second cation exchange chromatography stage, characterized in that on both cation x is autographically stages use the same type of cation exchange resin, and the fact that two consecutive cation exchange chromatography stage is performed with the use of different methods of elution, whereby two consecutive cation exchange chromatography stage include the following steps:
an introduction to a buffered aqueous solution containing a mixture of mono-, poly - and naegeliana erythropoietin, and forms low molecular weight, in the first cation exchange chromatography column under conditions suitable for binding mentioned monopegylated erythropoietin with cation exchange resin contained in the specified first column
b) isolation monopegylated erythropoietin from the first cation exchange chromatography column by using the method of stepwise elution with a stepwise increase of the ionic strength of the running buffer, where the relative content monopegylated erythropoietin in the selected solution is increased compared with the originally entered the mix,
C) the introduction of a dedicated monopegylated erythropoietin from stage b) to a second cation exchange chromatography column under conditions suitable for binding mentioned monopegylated erythropoietin with cation exchange resin contained in the specified second column, where the cation contained in such second column, of the same type as the cation in what erway column
g) isolation of purified monopegylated erythropoietin in a form which is substantially homogeneous, mentioned the second cation exchange chromatography column by using the method of continuous elution with a continuous increase of the ionic strength of the running buffer.

2. The method according to claim 1, characterized in that said cation exchanger is sulfopropyl cation exchange resin.

3. The method according to claim 1, characterized in that the said speed increasing ionic strength at the stage b) of the method is a three-stage increase in ionic strength.

4. The method according to claim 3, characterized in that the selected at stage b) monopegylated secrete erythropoietin on the second step of the method of stepwise elution.

5. The method according to claim 4, characterized in that on said stage b) paliperidone secrete erythropoietin after the first increase of the ionic strength of the running buffer, monopegylated secrete erythropoietin after the second increase of the ionic strength of the running buffer, and naegeliana secrete erythropoietin after the third increase of the ionic strength of the running buffer.

6. Method according to any one of claims 1 to 5, characterized in that the difference of concentrations causing elution of salt in stage b) of the method of stepwise elution is 120% or more on each step of the way stupen the addition of elution.

7. Method according to any one of claims 1 to 5, characterized in that the said buffered aqueous solution contains phosphoric acid or a salt thereof, or citric acid or a salt thereof, or a histidine or salts thereof as a buffer substance.

8. Method according to any one of claims 1 to 5, characterized in that on said stage d) monopegylated erythropoietin isolated from the second cation exchange chromatography column under the action of a linear gradient, starting from potassium phosphate buffer with a concentration of about 100 mm, containing about 50 mm sodium chloride at pH of about 3.0, and ending with potassium phosphate buffer with a concentration of about 100 mm, containing 500 mm sodium chloride at pH of 3.0, where the change in the concentration of sodium chloride linearly during the time of the transmission volume of eluent is equal to ten volumes of the column.

9. The method of obtaining monopegylated erythropoietin, comprising the following stages:
and tahilramani erythropoietin,
b) cleaning monopegylated erythropoietin using two consecutive cation exchange chromatography steps, where the first and second cation exchange chromatographic stages use the same type of cation exchange resin, these successive stages are carried out using different methods of elution and include the following steps:
i) introduction Sabo arenoso aqueous solution, containing a mixture of mono-, poly - and naegeliana erythropoietin, as well as forms with low molecular weight in the first cation exchange chromatography column under conditions suitable for binding mentioned monopegylated erythropoietin with cation exchange resin contained in the specified first column
ii) selection monopegylated erythropoietin from the first cation exchange chromatography column by using the method of stepwise elution with a stepwise increase of the ionic strength of the running buffer, where the relative content monopegylated erythropoietin in the selected solution is increased compared with the originally entered the mix,
iii) the introduction of a dedicated monopegylated erythropoietin from step ii) to a second cation exchange chromatography column under conditions suitable for binding mentioned monopegylated erythropoietin with cation exchange resin contained in the specified second column, where the cation contained in such second column, of the same type as the cation exchange resin in the first column
in the selection monopegylated erythropoietin from the second cation exchange chromatography column in a form which is substantially homogeneous, using the method of continuous elution with a continuous increase of the ionic strength of the running buffer.

10. The method according to Liu the WMD one of claims 1 to 5 and 9, characterized in that the second cation exchanger belongs to the same type as the first cation exchange resin, but is not the same of his faction.

11. Method according to any one of claims 1 to 5 and 9, characterized in that the said remainder of the PEG is characterized by a molecular weight of 20-35 kDa in the case of a linear PEG and 40 kDa in the case of a branched PEG.

12. Method according to any one of claims 1 to 5 and 9, characterized in that the said monopegylated erythropoietin get in shape, which is substantially homogeneous, containing more than 95% by area monopegylated erythropoietin according to the exclusion HPLC.

13. Method according to any one of claims 1 to 5 and 9, characterized in that the said monopegylated secrete erythropoietin on the first cation exchange chromatographic stage with a purity of more than 60% by area according to the exclusion HPLC.

14. Method according to any of p-5 and 9, characterized in that the said buffered aqueous solution contains about 100 mm potassium phosphate buffer and is characterized by a pH of 3.0.

15. Method according to any one of claims 1 to 5 and 9, characterized in that the pH value of the solutions mentioned chromatographic stages is about 3,0.

16. Method according to any one of claims 1 to 5 and 9, characterized in that the salt that causes elution pegylated erythropoietin from the cation exchange chromatography to the of look, is sodium citrate, or sodium chloride, or potassium chloride.

17. Method according to any one of claims 1 to 5 and 9, characterized in that the said erythropoietin is characterized by the amino acid sequence of SEQ ID NO:1 or number 2.



 

Same patents:

FIELD: medicine.

SUBSTANCE: invention describes Mus museums hybrid cultivated cell strains of Russian National Collection of Industrial Microorganisms H-117, Russian National Collection of Industrial Microorganisms H-116, Russian National Collection of Industrial Microorganisms H-115, Russian National Collection of Industrial Microorganisms H-113 and Russian National Collection of Industrial Microorganisms H-114 - producers of the monoclonal antibodies showing various specificity to human recombinant erythropoietin (EPO) and enabling distinguishing between a glycolised form of EPO and accompanying hypo- and deglycolised impurities. Estimating the strain efficacy and the produced antibody specificity is ensured by immune-enzyme assay with using a number of specificity markers: EPO recombinant protein; hypoglycolised EPO (o-glycolised) produced by natural antigen periodate oxidation; deglycolised EPO (de-EPO) produced by enzymatic treatment of recombinant EPO (PNGasa).

EFFECT: hybridoma produced monoclonal antibodies with said specificity are required for recovery of glycolised forms of EPO, and the quantitative and qualitative analysis of EPO mixtures.

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SUBSTANCE: by recombinant method obtained is fused protein, which contains natural molecule of human erythropoetine with cysteine residue near its C-end and Fc fragment of humal IgG, containing hinge region, N-end of said Fc fragment is connected to said C-end of said erythropoetine molecule, and said Fc fragment is natural, excluding mutation, consisting in substitution of cysteine residue in said hinge region, located the nearest of all to said erythropoetine molecule, with non-cysteine residue, which resulted in the fact that first cysteine residue of said hinge region, located the nearest of all to said N-end, is separated, by, at least, 12 or 17 amino acids from said cysteine residue of said erythropoetine molecule. Obtained peptide is used for stimulation of erythropoesis in mammal.

EFFECT: invention makes it possible to obtain fused protein, which possesses erythropoetine activity, has prolonged time of half-life in vivo in comparison with native human erythropoetine.

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FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and immunology. The invention discloses erythropoetin which is conjugated with polyethylene glycol, where polethylene glycol is bonded to erythropoetin via an aromatic azo group.

EFFECT: invention increases circulation time of erythropoetin in blood while preserving its biological activity.

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SUBSTANCE: invention relates to the bio-organic chemistry and relates to the novel erythropoietin conjugate, the method of production and its pharmaceutical composition. The hydroxyethylamylum and erythropoietin conjugate containing one or several hydroxyethylamylum molecules, is invented. Each hydroxyethylamylum molecule is conjugated with erythropoietin via hydrocarbon fragment. The said hydroxyethylamylum has the molecular mass from 1 to 300 kDa and shows the correlation of the C2:C6-substitution in the range of 2-20 by hydroxylethyl groups. The method for producing the hydroxyethylamylum and erythropoietin conjugate is invented; in accordance with the method, the sialic acid is not necessarily removed, partially or totally, with use of enzyme and/or chemical way, and the erythropoietin end saccharide link is oxidised not necessarily partially or totally to receive the erythropoietin which is able to react with the modified hydroxyethylamylum. Into hydroxyethylamylum, the free hydrazide, hydroxylamine, thyol or semicarbazide functional group is entered to receive the modified hydroxyethylamylum which is conjugated with erythropoietin which is able to react with the modified hydroxyethylamylum, and the hydroxyethylamylum and erythropoietin conjugate containing one or several hydroxyethylamylum molecules, is obtained. The pharmaceutical composition contains the hydroxyethylamylum and erythropoietin conjugate with the erythropoietin activity, and the pharmaceutically acceptable solvent, adjuvant and/or carrier.

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Thrombopoietin // 2245365

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SUBSTANCE: invention describes homogenous polypeptide ligand mpI representing polypeptide fragment of the formula: X-hTPO-Y wherein hTPO has amino acid sequence of human fragments TPO (hML); X means a amino-terminal amino-group or amino acid(s) residue(s); Y means carboxy-terminal carboxy-group or amino acid(s) residue(s), or chimeric polypeptide, or polypeptide fragment comprising N-terminal residues of amino acid sequence hML. Also, invention relates to nucleic acid encoding polypeptide and expressing vector comprising nucleic acid. Invention describes methods for preparing the polypeptide using cell-host transformed with vector, and antibodies raised against to polypeptide. Invention describes methods and agents using active agents of this invention. The polypeptide ligand mpI effects on replication, differentiation or maturation of blood cells being especially on megacaryocytes and progenitor megacaryocyte cells that allows using polypeptides for treatment of thrombocytopenia.

EFFECT: valuable medicinal properties of polypeptide.

21 cl, 92 dwg, 14 tbl, 24 ex

The invention relates to the field of biotechnology and can be used to obtain a fused protein with increased eritropoetinovmi activity

The invention relates to a conjugate of a glycoprotein erythropoietin, which has at least one free amino group and has biological activity in vivo, leading to increase production of reticulocytes and red blood cells by the bone marrow cells, and which are selected from the group comprising human erythropoietin and its analogues, which have the sequence of human erythropoietin modified by the addition of from 1 to 6 glycosylation sites or a rearrangement of at least one glycosylation site

The invention relates to a fused protein with in vivo increased activity of erythropoietin

FIELD: chemistry.

SUBSTANCE: disclosed is a method of purifying antibodies by adding a negatively charged polyelectrolyte such as polyvinyl sulphonic acid, polyvinyl sulphonate, polystyrene sulphonic acid or polyacrylic acid, to a mixture containing an antibody and extracting the obtained precipitate containing the antibody. The invention can further be used in extracting antibodies from body fluids and purification thereof.

EFFECT: improved method.

13 cl, 13 ex, 13 tbl, 38 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, particularly a method for production and purification of human recombinant growth hormone (rHGH). The presented method for production and purification of rHGH involves dissolution of inclusion bodies in 2 M urea at pH=11.0 followed by renaturation in a buffer solution 20 mM "Трис"-HCl at pH=8.0. Hydrogen peroxide is used as an rHGH sulphhydryl group oxidiser. N-terminal methionine is split from leucinic aminopeptidase. Chromatographic purification of rHGH is enabled with two-stage ion-exchange chromatography on the sorbent Q Sepharose FF and the stage of purification by hydrophobic chromatography on the sorbent Butyl Sepharose 4 FF. Gel filtration on Sephadex G-25 and ion-exchange chromatography on Q Sepharose FF, pH=6.5. It is followed by ion-exchange chromatography on Sephacryl S-100HR.

EFFECT: invention enables producing a preparation of high-purity rHGH protein of high compendial grade applicable for preparing drug preparations.

3 dwg, 2 tbl, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention concerns biochemistry and medicine area. What is presented is a visualisation agent representing a conjugate of structure I as described in the patent claim. The visualisation agent refers to marked cMet-binding peptides. These peptides include a mark with an optical reporter group applicable for visualisation in vivo with the use of light within the range of wave length in the spectrum of 600-1200 nm. There are also presented a pharmaceutical composition for optical visualisation, containing the visualisation agent, a kit for preparing it and a method for optical visualization of a mammalian body in vivo. What is also presented is a method for managing the patients suffering colorectal cancer, involving the stage of optical visualisation in vivo.

EFFECT: presented visualisation agent possess higher cMet-binding affinity and selective cell targeting in vivo.

28 cl, 2 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: invention discloses versions of a method of cleaning a liquid composition which contains recombinant factor VII from viruses. The composition to be cleaned contains at least 5% recombinant polypeptide of factor VII in activated form. Cleaning is carried out using a nanofilter with pore size of at most 80 nm.

EFFECT: methods enable to obtain a clean recombinant polypeptide of factor VII without breakdown of its active form.

34 cl, 1 dwg, 6 ex

FIELD: medicine.

SUBSTANCE: what is presented is a method of preparative recovery of basic proteins from supramolecular structures of Escherichia coli growing population. The Escherichia coli cells are preserved in buffered 80-90% glycerol at -25°C. Then the sediment cells are washed in 3% triton X-100. It is followed by sediment extraction in salts of increasing concentrations: 0.14 M, 0.35 M; 2 M NaCl, 6 M in guanidine hydrochloride with 0.1% β-mercaptoethanol. Basic proteins are recovered from the prepared fractions by means of ion-exchange chromatography with amberlite resin IRC-50 of discontinuous gradient of guanidine hydrochloride: 6%, 8.9%, 10.6%, 13% on 0.1 M potassium phosphate buffer pH 6.8.

EFFECT: method enables producing fractions enriched by basic proteins with the use of a microamount of protein of Escherichia coli cell suprastructures.

7 dwg, 1 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry. A method of cleaning thrombin solution from infectious particles is provided. Macromolecules are added to the starting thrombin solution. The obtained solution is then passed through a nanofilter to obtain a thrombin solution free from infectious particles. Said macromolecule is not a nonionic surfactant, is different from thrombin and can be selected from a polymer containing at least 3 monomers of sugar, amino acids, glycols, alcohols, lipids or phospholipids. A thrombin-containing solution obtained using said method is also provided.

EFFECT: invention increases efficiency of removing infectious particles from thrombin solution; thrombin output reaches 93% using said method.

20 cl, 2 dwg, 13 tbl

FIELD: medicine.

SUBSTANCE: invention relates to chromatographic purification of polypeptides. Disclosed is a method of producing monopegylated erythropoietin, involving chromatographic purification thereof and regeneration of the cation-exchange chromatographic column after elution of the monopegylated erythropoietin. Elution of the adsorbed monopegylated erythropoietin from the column is carried out with a buffered aqueous solution containing sodium chloride in concentration of at least 500 mM. The column is then washed with purified water, 0.5 M sodium hydroxide solution is then fed into the column and the column is rewashed. A solution containing 0.5 M sodium dihydrophosphate and 1 M phosphoric acid is fed into the column and the column is washed. 0.5 M sodium hydroxide solution is fed into the column for at least 4 hours and the column is finally regenerated by washing it with purified water.

EFFECT: invention provides high purity of monopegylated erythropoietin.

10 cl, 2 dwg, 1 tbl, 1 ex

FIELD: chemistry.

SUBSTANCE: disclosed is an E.coli strain which produces a recombinant protein p30 of the African swine fever virus. The strain is homogeneous, stable during passage and culturing in liquid and solid culture media and is resistant to chloramphenicol.

EFFECT: invention can be used to produce a recombinant protein p30 of African swine fever virus for diagnostic purposes.

1 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a method of purifying a cyclic or an acyclic peptide selected from a group comprising eptifibatide, exenatide, atosiban and nesiritide or combination thereof, from a mixture containing at least one impurity, involving contacting said mixture with a reverse phase HPLC matrix and an ion-exchange chromatography matrix and obtaining a purified peptide product with purity of at least 96% and, preferably, at least about 99%.

EFFECT: obtaining a purified peptide product.

11 cl, 4 tbl, 18 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a method of purifying a cyclic or an acyclic peptide selected from a group comprising eptifibatide, exenatide, atosiban and nesiritide or combination thereof, from a mixture containing at least one impurity, involving contacting said mixture with a reverse phase HPLC matrix and an ion-exchange chromatography matrix and obtaining a purified peptide product with purity of at least 96% and, preferably, at least about 99%.

EFFECT: obtaining a purified peptide product.

11 cl, 4 tbl, 18 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to pharmacology and medicine, and concerns an antibactrial agent representing a nanodiamond conjugate to amikacin of particle size 2-10 nm with the amikacin content up to 40 wt %, as well as a method for preparing it.

EFFECT: preparing a new antibacterial agent.

3 cl, 6 dwg, 1 tbl, 1 ex

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