Method of purifying cyclic or acyclic peptide

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

 

The technical field to which the invention relates.

The invention relates, inter alia, to the field of purification of peptides, in particular cyclic or acyclic peptides, analogs or derivatives. More specifically, the invention relates to a simplified or streamlined method of purification of cyclic peptides from the composition containing the indicated peptide and at least one related impurity, chromatographic methods, allowing to obtain the desired end product with high yields, selectivity and purity. An improved method, in particular, suitable for eptifibatida, exenatide, atosiban, nesiritide and related derivatives and analogs. The resulting polypeptides have a high degree of purity that is at least 96% and preferably is at least about 99%.

The level of technology

A substantial aspect in obtaining a recombinant (genetically engineered) peptides including cyclic peptides, their derivatives and analogues intended for therapeutic use for the treatment of humans or animals, is considered the treatment method is intended to obtain a product with high selectivity, yield and degree of purity so that the desired product essentially does not contain p is imesa extraneous proteins, which may be formed during the synthesis process.

In the process of synthesis of peptides are formed of various kinds of impurities, for example, the diastereomers, the products of hydrolysis of labile amide bonds, deletion of the sequence generated mainly in solid-phase peptide synthesis, peptides implementation and by-products, in particular polymorphic form, formed by removing the protective group at the last stage of the synthesis. These are often some by-products associated with the formation of cyclic peptides containing disulfide bonds [Bodansky et al., Principles of Peptide Synthesis; Springer-Verlag; Berlin, 1993]. In the art there is a need to develop a chromatographic purification methods that could be used on a large scale with a minimum number of stages required for the separation of the desired peptide contained in the mother solution, from a complex mixture of related impurities. In fact, most of the impurities formed during the synthesis process, it is impossible to remove a single chromatographic method, but this can be achieved by a combination of several methods, such as chromatography with reversed-phase and cation-exchange chromatography, which allows to obtain the drug, drug prescription in buffer solution described in us is oasam the invention.

In the claimed invention uses two chromatographic method, namely chromatography with reversed-phase and cation-exchange chromatography.

To obtain the desired end result in regard to the purity and yield of the product uses a number of chromatographic methods. Chromatography with reversed phase is one of the most effective ways of cleaning, the main principle of the separation of which is based on hydrophobic interactions. Reversed-phase liquid chromatography (RP-LC) and reversed-phase high-performance liquid chromatography (RP-HPLC) is widely used for the purification of molecules such as peptides and proteins produced either synthetically or by recombinant means. Methods OF LC and RP-HPLC can effectively divide closely related impurities, these methods are being used to clean a variety of molecules (Lee et al., "Preparative HPLC," 8th Biotechnology Symposium, Pt. 1, 593-610 (1988)). In addition, PF-LC and RP-HPLC have been used successfully for the purification of molecules, in particular proteins on an industrial scale (Olsen et al., 1994, J. Chromatog. A, 675, 101).

The principle of ion exchange chromatography (ZIOC) includes two different approaches: anion and cation exchanges in accordance with the charge of the ligands on the ion-exchange resin. Traditional ZIOC cleaning method usually involves several stages: stage trims, Nan is placed or download washing, elution and regeneration (see Remington''s Pharmaceutical Sciences, Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, or Remington: The Science and Practice of Pharamacy, 19th Edition (1995)).

Each parameter chromatographic procedure plays an important role in getting the desired protein product. For large-scale purification of peptides by reversed-phase resins using various chromatographic medium (matrix). The most common environments include resins with C-4, C-8 and C-18 alkyl chains attached to the surface of the silicon dioxide. One of the important parameters include the shape and size of the resin particles of the stationary phase. Other significant variables include the type of buffer system, flow rate, pH, etc. Despite improved methods of purification of peptides, some of the purified peptides still contain unacceptable counterions in undesirable quantities. In the context of this proposed principle is referred to alternative cleaning method, which allows us to overcome the challenges in this field of technology.

US 2006148699 relates to a method of purification of peptides based on RP-HPLC, which includes a step of washing the column with an aqueous solution pharmaceutically acceptable salt containing a counterion, and elution of the peptide from the column with a mixture of organic solvents and acids, containing what she pharmaceutically acceptable counterion, if the pH of an aqueous solution is at least 6. In the subsequent dependent claims, the invention also more specifically claimed cyclic, acyclic peptides and eptifibatide.

B WO 2005100388 disclosed RP-HPLC purification method of the basis 4 using acetonitrile gradient mixture, which allows you to extract the product with a purity of 97.5%.

WO 2005019262 refers to the use of resin based on a copolymer of styrene and divinylbenzene for RP-HPLC purification glucagonomas peptides.

In the art there is a need for efficient chromatographic method for effective separation of molecules, such as cyclic peptides, after solid-phase synthesis with the aim of obtaining the final peptide products of high purity. This need could be met if the method allows to reproduce, as far as possible, the yield, purity, performance and operating conditions of the chromatographic process in which the elution is carried out using a specific solvent mixture, in a certain pH range and taking into account other relevant factors. The working technique can successfully be used for the industrial division.

Disclosure of inventions

The main objective of the present invention is to provide an str is both purification of the peptide from the mixture, containing at least one related impurity.

Another objective of the present invention is to provide a method of purification by reversed-phase high-performance liquid chromatography and ion exchange chromatography.

Another objective of the present invention is to provide a method of purification of peptides, which can be cyclic or acyclic and selected from the group consisting of eptifibatida, exenatide, atosiban or nesiritide and related analogs and derivatives.

Accordingly, the present invention relates to a method of purifying a peptide from a mixture containing at least one related impurity, while this method involves the step of contacting a mixture of peptides by matrix columns for reversed-phase high-performance liquid chromatography and/or matrix for ion-exchange chromatography to obtain a purified peptide; method of purification of peptides using reversed-phase high-performance liquid chromatography of a mixture containing at least one related impurity, while this method involves the following stages: filling columns for reversed-phase high-performance liquid chromatography polymer resin, attached to the silicon dioxide with subsequent equilibration of the column with buffer solution of the organic acid, containing 5% of the polar solvent; downloads peptide compositions containing at least one related impurity, to the chromatographic column with a flow rate of at most 100-400 cm/h; washing the column with the same buffer solution used in stage (a); and elution of the purified product from the column with a linear concentration gradient (from 8 to 14%) to obtain the purified peptide product; and also relates to a method of purification of the peptide by using ion-exchange chromatography from a mixture containing at least one related impurity, while this method involves the following stages: trim columns for cation exchange chromatography (cation-exchange column) aqueous buffer solution of a weak acid; downloads purified peptide on the column for reversed-phase high-performance liquid chromatography; washing the column and elution of the peptide buffer solution used in stage (a) obtaining a purified peptide product; the method of purification of the peptide from a mixture containing at least one related impurity, while this method involves the following stages: filling columns for reversed-phase high-performance liquid chromatography polymer resin, attached to the silicon dioxide, followed by equilibration of the column with the buffer body is practical acid, containing 5% of the polar solvent; downloads peptide compositions containing at least one related impurity, on the column with a flow rate of at most 100-400 cm/h with subsequent washing of the column with buffer solution used in stage (a); elution of the purified product from the column with a linear concentration gradient (from 8 to 14%), download the product, lirovannomu with columns for reversed-phase high-performance liquid chromatography on a cation exchange column, equilibrated aqueous buffer solutions of a weak acid, with subsequent washing of the column and elution of the peptide product eluting buffer with getting purged peptide product.

Brief description of drawings

Fig 1. Chromatogram showing the purity eptifibatida.

Fig 2. Chromatogram showing the purity atosiban.

Fig 3. Chromatogram showing the purity of nesiritide.

Fig 4. Chromatogram showing the purity of exenatide.

The implementation of the invention

The present invention relates to a method of purification of the peptide from a mixture containing at least one related impurity, while this method involves the step of contacting a mixture of peptides by matrix for reversed-phase high-performance liquid chromatography and/or matrix for ion-exchange chromatography, p is imodasa to obtain a purified peptide.

In another embodiment of the present invention the specified peptide is a cyclic or acyclic peptide selected from the group consisting of eptifibatida, exenatide, atosiban or nesiritide and related analogs or derivatives thereof.

In yet another embodiment, the present invention indicated peptide mixture in contact in any sequence with the specified matrix consisting of a polymer resin, attached to the silicon dioxide.

In yet another embodiment of the present invention, the resin may be selected from the group comprising Sephadex, Sephadex LH20, Sephadex G-25, Sephadex G-10, Sepharose, Superdex, methylacrylate resin, carboxymethyl cellulose, sulfopropyl, carboxymethylstarch, sulforaphanes, solveproblems and carboxymethylcellulose, preferably polystyrene or polydivinylbenzene.

In yet another embodiment of the present invention the particle size and the pore size of the resin granules varies in the range of 1-50 μm and 100-500 Å, respectively.

In yet another embodiment of the present invention, in that way clearing uses a gradient mixture with a concentration of polar organic buffer solvent is from 2 to 30% in the aqueous phase containing a buffer solution of an organic acid.

In another embodiment, the present invention specified buffer solution of an organic acid selected from the group comprising citric acid, acetic acid, perchloric acid and formic acid.

In another embodiment of the present invention the molar concentration of the buffer solution ranges from about 10-50 mm.

In another embodiment of the present invention, the cleaning is conducted at a pH ranging from 2 to 9.

In another embodiment, the present embodiment of the invention the method additionally includes other optional stage exclusion chromatography.

In another embodiment of the present invention by means used in the method is obtained a purified peptide product, the degree of purity of which is from 97 to 100%.

In another embodiment of the present invention, the degree of purity of the product is at least 96%.

The present invention relates to a method of purification of peptides using reversed-phase high-performance liquid chromatography of a mixture containing at least one related impurity, while this method involves the following stages:

a) filling the column for reversed-phase high-performance liquid chromatography polymer resin, keeping the Noi on the silicon dioxide, with the subsequent equilibration of the column with buffer solution of organic acid containing 5% of the polar solvent;

b) loading of the peptide composition containing at least one related impurity, to the chromatographic column with a flow rate of at most 100-400 cm/h;

c) washing the column with the same buffer solution as used in stage (a); and

(d) elution of the purified product from the column with a linear concentration gradient (from 8 to 14%) to obtain the purified peptide product.

The present invention relates to a method of purification of the peptide using ion-exchange chromatography from a mixture containing at least one related impurity, while this method involves the following stages:

a) trim columns for cation exchange chromatography aqueous buffer solution of a weak acid;

b) loading the columns for cation-exchange chromatography purified peptide; and

c) washing the column and elution of the peptide in the same buffer solution as used in stage (a)obtaining a purified peptide product.

The present invention relates to a method of purification of the peptide from a mixture containing at least one related impurity, while this method involves the following stages:

a) filling the column for reversed-phase highly the positive liquid chromatography polymer resin, attached to the silicon dioxide, followed by equilibration of the column with buffer solution of organic acid containing 5% of the polar solvent;

b) loading of the peptide composition containing at least one related impurity, on the column with a flow rate of at most 100-400 cm/h with subsequent washing of the column with the same buffer solution as used in stage (a);

c) elution of the purified product from the column with a linear concentration gradient (from 8 to 14%);

d) download the product, lirovannomu with columns for reversed-phase high-performance liquid chromatography on a cation exchange column, equilibrated aqueous buffer solution of a weak acid; and

e) washing the column and elution of the peptide product eluting buffer to obtain a purified peptide product.

In another embodiment of the present invention, the resin may be selected from the group consisting of Sephadex, methylacrylate resin, carboxymethyl cellulose, carboxymethylstarch, sulfopropyl and sulforaphanes, preferably made of polystyrene or polydivinylbenzene.

In yet another embodiment of the present invention the particle size and the pore size of the resin granules varies in the range of 1-50 μm and 100-500 Å, respectively.

Another option is the implementation of the present invention this method, it also includes other optional stage exclusion chromatography.

In yet another embodiment of the present invention the specified peptide is a cyclic or acyclic peptide selected from the group consisting of eptifibatida, exenatide, atosiban or nesiritide and related analogs or derivatives thereof.

In yet another embodiment of the present invention the polar buffer solvent is acetonitrile.

In yet another variant implementation of the present invention a buffer solution of an organic acid selected from the group consisting of citric acid, acetic acid and formic acid.

In yet another embodiment of the present invention the molar concentration of the used buffer solution ranges from about 10-50 mm.

In yet another embodiment of the present invention, the cleaning is conducted at a pH ranging from 2 to 9.

In yet another embodiment of the present invention by means of the used methods is obtained a purified peptide product, the degree of purity of which is from 97 to 100%.

In yet another embodiment of the present invention, the degree of purity of the peptide, such as eptifibatide, exenatide, atosiban and nesiritide is at least 96%.

You izlojennye and other objectives of the present invention are achieved in a dedicated way of obtaining and purification of cyclic or non-cyclic peptide compounds formed after the solid-phase synthesis.

The method of purification of peptides includes the chromatographic purification by reversed-phase high-performance liquid chromatography using a polar buffer solvent in a concentration of from 2 to 20%, preferably acetonitrile in an aqueous phase containing the organic acid buffer with a pH from 2 to 5.

The purpose of the present invention is to provide a chromatographic medium/solvent system, which is carried out by reversed-phase high-performance liquid chromatography and cation exchange chromatography.

In a broad sense, the present invention relates to a RP-HPLC chromatographic method of purifying a peptide from a mixture containing a specified peptide and related impurities, which involves the following stages: separation of the indicated peptide and specified related impurities contained in the above-mentioned mixture by elution on a column for RP-HPLC, filled with a polymer resin, equilibrated buffer solution of organic acid with 5% content of the polar solvent, download solution of the peptide on the column with the desired flow rate of at most 360 cm/h, wash the column with 50 mm solution of organic acid with a low content (5%) of the polar organic solvent elution peptide cont the KTA specified combination of buffers with a linear concentration gradient from 8%to 14%.

Specialists in the art will understand that there are different variables that can be adjusted in the process of conducting chromatographic methods of the present invention. This includes loading and elution, such as ionic strength, composition of buffer, pH, temperature, addition of small amounts of organic solvent, etc. However, these parameters are adjustable in the usual way and specialists in the art can easily determine optimum conditions.

Before explaining at least one variant embodiment of the invention in more detail on the example of the illustrative drawings, experiments, results and methods should be understood that the invention is not limited to the details of construction and arrangement of the components set forth in the following description or illustrated in the drawings, in experiments and/or results.

Other variants of the invention or the invention may be embodied in practice or carried out in various ways. Assume that, as such, the definitions used in this document are given in the broadest sense, and that the options for implementation are exemplary and not exhaustive. Also we must understand that the wording and terminology, ispolzuemye in this document, given the purpose of description and should not be considered as restrictive.

This invention relates to methods and techniques used in the purification of cyclic/acyclic peptides with biological activity similar to the activity of natural peptides, and more specifically, to methods that use RP-HPLC for the separation of active peptide compounds from other substances, which do not have this activity and therefore can be considered as impurities. Purified peptide product purchasing prescription solution using cation-exchange chromatography or a combination of cation exchange and exclusion chromatography.

In one aspect the present invention provides a method of purification of the peptide, polypeptide or protein obtained by solid-phase synthesis, with the specified method includes the initial stage of purification of the peptide sample by reverse-phase chromatography on a column of polymer resin in the chromatographic conditions sufficient to obtain the peptide with a purity of about 99%, followed by concentration lirovannomu peptide by cation-exchange chromatography in a buffer solution to prepare the finished product.

Another important advantage of the method is divided into the I according to the present invention is it can gradually scale suitable and reproducible manner. Furthermore, the method of the present invention gives products that are superior to the products obtained by other known up to the present time the methods of cleaning and are formed with a higher output.

Elution of the peptide in solution for the preparation of medicinal products (hereinafter - prescription solution") has advantages compared to traditional methods. Traditional methods include lyophilization of the pure peptide and re-dissolution of lyophilized powder before adding tools.

Elution of the peptide prescription solution eliminates the need for the use of freeze drying and re-dissolving.

Thus, when used either separately or in conjunction with standard methods of extraction and chromatography was carried out the extraction methods of the present invention allow the cyclic peptides of the claimed invention with high yield and high purity with fewer stages compared with what is required when using traditional methods.

A feature of the invention in another aspect is a method of purification protein, which includes the stage of loading a mixture containing the compound on the column DL the RP-HPLC and elution of the sample, containing the peptide with an organic solvent, the interaction of the sample with the ion exchange resin under conditions providing a binding compound with the resin, and washing the organic solvent remaining in the column, the aqueous buffer solution.

For the effective implementation of the present invention for effective cleaning requires the selection of the correct combination used chromatographic column, the pH and ionic strength of the buffer.

Each parameter chromatographic procedure plays an essential role to obtain the desired protein product. The buffer system used for cleaning, can improve the separation of impurities and interested molecule due to the hydrophobicity of the compounds. At the specified pH value of the buffer system aluinum impurities before, during gradient elution. This is due to the fact that impurities are less hydrophobic compared with the target molecule under specified conditions.

In the present invention are applied buffered solutions containing polar solvent as the organic eluents for RP-HPLC purification of cyclic peptides, analogs or derivatives whose pH is in the range from 2 to 8. The present invention provides improved separation efficiency, higher chistotoi convenience for industrial use compared to the current level of technology in the field of RP-HPLC purification of cyclic peptides using the above solvent system. Unexpectedly, the separation of the target cyclic peptide compounds and related impurities is improved due to the new methodology used in the present invention, which provides a more pure cyclic peptide products.

Together with the hydrophobicity of the compounds on the separation effect of the pH buffer system. The change in pH that occur at various stages of chromatographic separation affects the mobility of the compounds on the column.

The granules used for the separation are a copolymer of styrene and divinylbenzene with a particle size of 10 μm and pore size 300 Å. Downloadable sample has the ability to contact the granules, because the size of the pores is governed by the surface area available for molecules. The granules have a high pH-resistant, because they consist of a polymer, and due to the size of particles it is possible to achieve better separation.

The composition of the prescription solution

Eptifibatide

1. For injection: 1 ml contains 2 mg eptifibatida, the 5.25 mg citric acid solution with a pH of 5.25, adjustable by adding acetic acid, and water (remaining amount).

2. For infusion: 1 ml contains 0.75 mg eptifibatida, the 5.25 mg citric acid solution with a pH of 5.25, adjustable by adding acetic acid, and water (remaining amount).

Atosiban

1. For injection: 1 ml contains 7.5 m is atosiban, 50 mg of mannitol, 4.5 mg of citric acid solution with a pH of 4.5, adjustable by the addition of HCI and water (remaining amount).

Definitions of terms

In the description and presentation of the formulas of the present invention will use the following terminology in accordance with the definitions set forth herein.

The term "polypeptide", "protein", "peptide" refers to a polymer of amino acids and does not refer to a specific length of the product; thus, peptides, oligopeptides and proteins are included within the definition of polypeptide. This term also does not refer to or exclude postexplosion modifications of the polypeptide, although chemical or postexplosion modifications of these polypeptides can be included or excluded in the form of specific embodiments. So, for example, modification of polypeptides, which include covalent binding helicoiling, acetyl, phosphate, lipid groups and the like undoubtedly are covered by the term polypeptide. In addition, the polypeptides of these modifications can be defined as individual particles to be included or excluded from the present invention. In one embodiment, the molecule is a polypeptide or its related analogue or derivative. Preferably, the polypeptide is a cyclic peptide. According to another preferred variant implementation of the program the polypeptide is a cyclic peptide. In yet another embodiment, the polypeptide selected from the group comprising eptifibatide, exenatide, atosiban or nesiritide.

The term "cleanup" of the peptide of the composition containing the peptide and one or more contaminants, indicates a higher degree of purification of the peptide contained in the composition, by reducing the content of at least one of the impurities in the peptide composition.

"Impurity" is a substance that differs from the desired polypeptide product or protein of interest. To various kinds of impurities are (but the list is not limited to) the diastereomers, the products of hydrolysis of labile amide bonds, deletion of the sequence generated mainly in solid-phase peptide synthesis, peptides implementation and by-products, in particular polymorphic form, formed by removing the protective group at the last stage of the synthesis.

The term "chromatography" refers to the process by which interested the permeate is separated from other dissolved substances contained in the mixture, due to differences in the speed with which individual solute mixture moving through the stationary phase under the influence of the mobile phase, or due to differences in the processes of binding and elution.

Used in this document, the term"liquid chromatography" refers to such a chromatographic method, in which particles (stationary phase)used to populate the columns have the same small size (from 3 to 50 microns) with a small deviation from the selected size. In this way, the chromatography is typically used relatively high (of the order of 3,4-24,1 MPa) inlet pressure. The term "ion exchange" and "ion exchange chromatography" refers to chromatography method in which interested dissolved substance (e.g. protein)contained in the mixture interacts with the charged compound related (for example, through covalent bonds) with the ion-exchange substance of the solid phase, so that interested permeate nonspecific interacts with charged connection to a greater or lesser extent in comparison with the dissolved impurities or contaminants contained in the mixture. Pollutants dissolved substances contained in the mixture, washed with a column containing the ion exchange substance, faster or slower than interested dissolved substance, or associated or derived from the resin better than interested dissolved substance. To "ion exchange chromatography", in particular, cation-exchange, anion-exchange chromatography and chromatography of the mixed type. By stage cation exchange chromatography can be followed by stage RP-HPLC yonabaru. It is preferable for the phase cation-exchange chromatography followed by other chromatographic stage.

"Cation exchange chromatography is a process in which positively charged ions bind with the negatively charged resin.

Boot buffer" is a buffer that is used to load the compositions containing the molecules of interest polypeptide and one or more impurities, ion-exchange resin. Boot buffer has such conductivity and/or pH at which the molecule of interest polypeptide (and usually one or more impurities) contact with ion exchange resins or by which a protein of interest flows through the column, while the impurities remain bound with resin.

"Polar buffer solvent may be any solvent which dissolves ionic compounds or covalent compounds capable of dissociation of the ions, and is used as a buffer. In the context of the present invention, the preferred polar solvent is acetonitrile.

A typical example of the method of separation and purification of the peptides of the invention obtained by solid-phase synthesis, includes the following stages:

i) filling the column for RP-HPLC polymer resin, equilibrated buffer solution of organic acid containing 5% of a polar solution of the indicator;

ii) loading the peptide composition containing at least one related impurity, on the column with a flow rate less than or equal to about 100-400 cm/h;

iii) washing the column with the same buffer solution as used in stage (i); and

iv) elution of the purified product from the column with a linear concentration gradient from 8%to 14%.

Suirvey purified peptide solution is then loaded on a cation exchange column, which contributes to the concentration of the product. The concentration of the indicated peptide in combination with elution its RX buffer by cation-exchange chromatography comprises the following stages:

a) balancing cation exchange column aqueous buffer solution of a weak acid;

b) loading of the peptide is purified by RP-HPLC on a column;

(C) washing the column and elution of the peptide product buffer solution used in stage (a).

Buffer And is a 1-5% solution of acetonitrile and buffer In - 10-50 mm buffer solution of an organic acid; the sample is loaded with a flow rate of at most 100-400 cm/H. Used the gradients of concentrations vary depending on the sample peptide is subjected to cleaning.

The first stage of the method described in this document includes the purification of molecules from mixtures containing them by retrieve and mixtures on the column for reversed-phase liquid chromatography. The column can be a column of low or high pressure (ghvd), the latter is filled with a medium particle diameter which is less than 20 μm. The column preferably is filled with a medium particle diameter of which is 5-40 μm, more preferably 10-40 μm and, most preferably 10-15 μm. In the context of the present invention, the particle size of the resin filled into the column is 10 μm. Thus, the column preferably is a column for jhud used, in particular, for the purification of peptides. Preferably, the pore size of the column is 100-4000 Å, more preferably 100-500 Å. In the context of the present invention, the pore size of the resin filled in a column, 300 Å. The length of the column is preferably 10-50 cm, more preferably 25-35 see

pH of the eluting buffer can be from 2 to 9, or 3 to 8, from 4 to 8 or 5 to 8, although the pH value or range of pH values when the elution is determined depending on the required peptide and the type carried out by chromatographic method. The appropriate pH range boot, washing or eluting buffer is easily determined by standard methods, while the protein of interest must be extracted in the active form. Examples eluting buffers used for this purpose, carrying the camping citrate or acetate buffer. Matrix columns can be any suitable material, including matrix-based polymeric resins, silicon dioxide or methacrylic resin. Preferably, the matrix is AMBERCHROM HPR10. To cation-exchange resins that can be used to implement the present invention include alphapapillomavirus, hydrogel polymerized ceramic granule, carboxymethylcellulose, hydrophilic spherical polymer beads, carboxymethylstarch, sulfopropyl, sulforaphanes etc. To the currently favored cation exchange resins include alphapapillomavirus and hydrogel polymerized ceramic granules, while alphapapillomavirus is currently the most preferred cation exchange resin for use in the implementation of the present invention due to its affordability and excellent technical characteristics. Exclusion resins used in this invention are the Sephadex LH-20, Sephadex G-25, Sephadex G-10. The flow rate is usually 20-400 cm/h or 4-40 volume of the column (s)/h depending on whether chromatography acidic or neutral. Preferably, the peptide is loaded onto the column with a flow rate of at most 360 cm/h

In the case of chromatographic under the Oia by RP-HPLC loading capacity of the peptide on the column is usually 2-15 g/l based on the content of the molecules and impurities. The capacity of ion-exchange columns is at most 70 g/l at the stage of concentration.

These and other non-restrictive embodiments of the present invention easily understandable to the ordinary practitioner in the art upon reading the disclosure and the accompanying claims. It should be understood that the invention is not limited to individual described methods and processes, of course, possible variations in the desired protein/peptide products and methods. It should also be understood that as used in this document terminology is given only for the purpose of describing specific embodiments and not to limit.

It should be understood that the use of the above purification method described in the examples, to achieve separation with high resolution in combination with the components used to separate, making it particularly effective to obtain the desired peptide is simple, convenient and inexpensive way.

The techniques of the present application is additionally illustrated by the following examples. However, it should be understood these examples as limiting the scope of the present invention. The following examples represent preferred embodiments of the present invention.

Example 1

Triptorelin eptifibatida with the degree number is the notes 66%, obtained by solid-phase synthesis, purified on a column filled with a polymer resin. Triptorelin eptifibatida first dissolved in a mixture of acetonitrile and 50 mm acetic acid (1:1)to give a clear solution. The resulting solution was further diluted with 50 mm acetic acid, so that the concentration of acetonitrile is 5%, and the concentration eptifibatida is less than 2 g/L. Before loading on the column, the solution is filtered.

A column filled with resin Amberchrom HPR10 (particle size of 10 μm and a pore size 300 Å), balance solution with low content (5%) of acetonitrile in 50 mm acetic acid. The filtered solution eptifibatida load on the column with a flow rate of at most 360 cm/h Loading of the peptide on the column hold up until its concentration will not be at most 10 g/l resin. After loading the column was washed with a solution with a low content (5%) of acetonitrile in 50 mm acetic acid. Pure product elute column with a linear concentration gradient (8-14%) of acetonitrile (buffer B) in 50 mm acetic acid (buffer A) in the amount of 25 OK. The pressure drop along the column during the purification process is 23-26 MPa (bar). Eptifibatide obtained by this method has a purity of 98.7 per cent and is obtained with a yield of 54%. Conditions for HPLC analysis eptifibatida presented in tab is itzá below.

Table 1
Conditions analysis
The concentration gradientTime (min)%
Buffer And0.1% TFUK + water020
The buffer0.1% TFUK + acetonitrile1530
ColumnS3 ExsilODS (80 Å, 5 μm)2550
The flow velocity1 ml/min2680
The temperature of the column25°C2720
Wavelength220 nm3220

The purified solution eptifibatida loaded on cation-exchange column for concentrating eptifibatida and ale is testing it in the RX buffer, who could imagine a concentrate of finished dosage forms. The eluate can be diluted to the required concentration and fill it in bottles in the form of finished dosage forms.

Cation-exchange column balance 27 mm citric acid with a pH of 2.7. Purified eptifibatide loaded onto cation exchange column after dilution with water in the ratio 1:1 to a concentration of at least 65 g/l basis. The column was washed with 27 mm citric acid with a pH of 2.7. Elution spend 27 mm citric acid with a pH of 5.25. The resulting eluate with a concentration of at least 9 g/l diluted to the required concentration and filled into vials in the form of finished dosage forms. The pressure drop along the column during the process ranges from 0.5 to 0.7 MPa (bar) at a flow rate of 180 cm/h Eptifibatide obtained by this method has a purity of 98.6% of and is obtained with a yield of 100%.

Example 2

Triptorelin eptifibatida purity of 69.5%, obtained by solid-phase synthesis, purified on a column filled with a polymer resin. Triptorelin eptifibatida first dissolved in a mixture of acetonitrile and 50 mm acetic acid (1:1)to give a clear solution. The resulting solution was further diluted with 50 mm acetic acid, so that the concentration of acetonitrile is 5%, and the concentration eptifibatida on the information least 2 g/L. Before loading on the column, the solution is filtered. the pH of sodium acetate was adjusted to 3.0 by addition of acetic acid.

A column filled with resin Amberchrom HPR10 (particle size of 10 μm and a pore size 300 Å), balance 10 mm sodium acetate solution (pH 3.0) with low content (5%) of acetonitrile. The filtered solution eptifibatida load on the column with a flow rate of at most 360 cm/h Loading of the peptide on the column hold up until its concentration will not be at most 10 g/l resin. After loading the column was washed with 10 mm sodium acetate solution (pH 3.0) with low content (5%) of acetonitrile. Pure product elute column with the implementation of a linear concentration gradient (9-12%) of acetonitrile (buffer B) in 10 mm sodium acetate solution (pH 3.0) (buffer A) in the amount of 25 OK. The pressure drop along the column during the purification process is 25-29 MPa (bar). Eptifibatide obtained by this method has a purity of 98.6% of and obtained access to 43.0%.

The purified solution eptifibatida loaded on cation-exchange column for concentrating eptifibatida and elution his prescription in the buffer, which could provide a concentrate of finished dosage forms. The eluate can be diluted to the required concentration and fill it in bottles in the form of finished dosage forms.

Cation-exchange column balance 27 mm citric acid with a pH of 2.7. Purified eptifibatide loaded onto cation exchange column after dilution with water in the ratio 1:1 to a concentration of at least 65 g/l basis. The column was washed with 27 mm citric acid with a pH of 2.7. Elution spend 27 mm citric acid with a pH of 5.25. The resulting eluate with a concentration of at least 9 g/l diluted to the required concentration and filled into vials in the form of finished dosage forms. The pressure drop along the column during the process ranges from 0.5 to 0.7 MPa (bar) at a flow rate of 180 cm/h Eptifibatide obtained by this method has a purity of 98.6% of and is obtained with a yield of 100%.

Example 3

Triptorelin eptifibatida purity of 69.5%, obtained by solid-phase synthesis, purified on a column filled with a polymer resin. Triptorelin eptifibatida first dissolved in a mixture of acetonitrile and 50 mm acetic acid (1:1)to give a clear solution. The resulting solution was further diluted with 50 mm acetic acid, so that the concentration of acetonitrile is 5%, and the concentration eptifibatida is at most 2 g/l Before loading on the column, the solution is filtered.

A column filled with resin Amberchrom HPR10 (particle size of 10 μm and a pore size 300 Å), balance solution low with what uranium (5%) of acetonitrile in 10 mm citric acid (pH of 2.5). The filtered solution eptifibatida load on the column with a flow rate of at most 360 cm/h Loading of the peptide on the column hold up until its concentration will not be at most 10 g/l resin. After loading the column was washed with a solution with a low content (5%) of acetonitrile in 10 mm citric acid (pH of 2.5). Pure product elute column with a linear concentration gradient (9-12%) of acetonitrile (buffer B) in 10 mm citric acid (pH 2,5) (buffer A) in the amount of 25 OK. The pressure drop along the column during the purification process ranges from 22 to 28 MPa (bar). Eptifibatide obtained by this method has a purity of 98.6% of and is obtained with a yield of 57%.

The purified solution eptifibatida loaded on cation-exchange column for concentrating eptifibatida and elution his prescription in the buffer, which could provide a concentrate of finished dosage forms. The eluate can be diluted to the required concentration and fill it in bottles in the form of finished dosage forms.

Cation-exchange column balance 27 mm citric acid with a pH of 2.7. Purified eptifibatide loaded onto cation exchange column after dilution with water in the ratio 1:1 to a concentration of at least 50 g/l basis. The column was washed with 27 mm citric acid with a pH of 2.7. Elution spend 27 mm Limon the th acid with a pH of 5.25. The resulting eluate with a concentration of at least 9 g/l diluted to the required concentration and filled into vials in the form of finished dosage forms. The pressure drop along the column during the process ranges from 0.5 to 0.7 MPa (bar) at a flow rate of 180 cm/h Eptifibatide obtained by this method has a purity of 98.6% of and is obtained with a yield of 100%.

Example 4

Triptorelin eptifibatida with purity of 66%, obtained by solid-phase synthesis, purified on a column filled with a polymer resin. Triptorelin eptifibatida first dissolved in a mixture of acetonitrile and 50 mm acetic acid (1:1)to give a clear solution. The resulting solution was further diluted with 50 mm acetic acid, so that the concentration of acetonitrile is 5%, and the concentration eptifibatida is at most 2 g/l Before loading on the column, the solution is filtered.

A column filled with resin Amberchrom HPR10 (particle size of 10 μm and a pore size 300 Å), balance solution with low content (5%) of acetonitrile in 0.05% perchloric acid (pH 1,70). The filtered solution eptifibatida load on the column with a flow rate of at most 360 cm/h Loading of the peptide on the column hold up until its concentration will not be <10 g/l resin. After loading the column was washed with a solution with a low content is m (5%) of acetonitrile in 0.05% perchloric acid (pH 1,70). Pure product elute column with the implementation of a linear concentration gradient (8-12%) of acetonitrile (buffer B) 0.05% perchloric acid (pH 1,70) (buffer A) in the amount of 25 OK. The pressure drop along the column during the purification process is 28-32 MPa (bar). Eptifibatide obtained by this method has a purity of 96.6 percent and is obtained with a yield of 40%.

The purified solution eptifibatida loaded on cation-exchange column for concentrating eptifibatida and elution his prescription in the buffer, which could provide a concentrate of finished dosage forms. The eluate can be diluted to the required concentration and fill it in bottles in the form of finished dosage forms.

Cation-exchange column balance 27 mm citric acid with a pH of 2.7. Purified eptifibatide loaded onto cation exchange column after dilution with water in the ratio 1:1 to a concentration of at least 50 g/l basis. The column was washed with 27 mm citric acid with a pH of 2.7. Elution spend 27 mm citric acid with a pH of 5.25. The resulting eluate with a concentration of at least 9 g/l diluted to the required concentration and filled into vials in the form of finished dosage forms. The pressure drop along the column during the process ranges from 0.5 to 0.7 MPa (bar) at a flow rate of 180 cm/h Eptifibatide obtained by this method has a purity of 98.6% of and is obtained with a yield of 100%.

Example 5

Triptorelin eptifibatida purity of 69.5%, obtained by solid-phase synthesis, purified on a column filled with a polymer resin. Triptorelin eptifibatida first dissolved in a mixture of acetonitrile and 50 mm acetic acid (1:1)to give a clear solution. The resulting solution was further diluted with 50 mm acetic acid, so that the concentration of acetonitrile is 5%, and the concentration eptifibatida is at most 2 g/l Before loading on the column, the solution is filtered.

A column filled with resin Amberchrom HPR10 (particle size of 10 μm and a pore size 300 Å), balance 10 mm solution of sodium formate (pH 3.0) with low content (5%) of acetonitrile.

The filtered solution eptifibatida load on the column with a flow rate of at most 360 cm/h Loading of the peptide on the column hold up until its concentration will not be at most 10 g/l resin. After loading the column was washed with 10 mm solution of sodium formate (pH 3.0) with low content (5%) of acetonitrile. Pure product elute column with the implementation of a linear concentration gradient (5-17%) of acetonitrile (buffer B) in 10 mm solution of sodium formate (pH 3.0) (buffer A) in the amount of 25 OK. The pressure drop along the column during the purification process is 30-33 MPa (bar). Eptifibatide received on this way is, has a degree of purity of 96.8 per cent and is obtained from the output of 54.0%.

The purified solution eptifibatida loaded on cation-exchange column for concentrating eptifibatida and elution his prescription in the buffer, which could provide a concentrate of finished dosage forms. The eluate can be diluted to the required concentration and fill it in bottles in the form of finished dosage forms.

Cation-exchange column balance 27 mm citric acid with a pH of 2.7. Purified eptifibatide loaded onto cation exchange column after dilution with water in the ratio 1:1 to a concentration of at least 50 g/l basis. The column was washed with 27 mm citric acid with a pH of 2.7. Elution spend 27 mm citric acid with a pH of 5.25. The resulting eluate with a concentration of at least 9 g/l diluted to the required concentration and filled into vials in the form of finished dosage forms. The pressure drop along the column during the process ranges from 0.5 to 0.7 MPa (bar) at a flow rate of 180 cm/h Eptifibatide obtained by this method has a purity of 98.6% of and is obtained with a yield of 100%.

Example 6

Triptorelin eptifibatida purity of 69.5%, obtained by solid-phase synthesis, purified on a column filled with a polymer resin. Triptorelin eptifibatida first dissolved in a mixture of acetone is trila and 50 mm acetic acid (1:1), obtaining a clear solution. The resulting solution was further diluted with 50 mm acetic acid, so that the concentration of acetonitrile is 5%, and the concentration eptifibatida is at most 2 g/l Before loading on the column, the solution is filtered.

A column filled with resin Amberchrom HPR10 (particle size of 10 μm and a pore size 300 Å), balance solution with low content (5%) of acetonitrile in 10 mm boric acid (pH 4.0). The filtered solution eptifibatida load on the column with a flow rate of at most 360 cm/h Loading of the peptide on the column hold up until its concentration will not be at most 10 g/l resin. After loading the column was washed with a solution with a low content (5%) of acetonitrile in 10 mm boric acid (pH 4.0). Pure product elute column with the implementation of a linear concentration gradient (5-17%) of acetonitrile (buffer B) in 10 mm boric acid (pH 4.0) (buffer A) in the amount of 25 OK. The pressure drop along the column during the purification process is 30-33 MPa (bar). Eptifibatide obtained by this method has a degree of purity 98,0% and is obtained with a yield of 51.0%.

The purified solution eptifibatida loaded on cation-exchange column for concentrating eptifibatida and elution his prescription in the buffer, which could be a concentrate ready lcars the governmental form. The eluate can be diluted to the required concentration and fill it in bottles in the form of finished dosage forms.

Cation-exchange column balance 27 mm citric acid with a pH of 2.7. Purified eptifibatide loaded onto cation exchange column after dilution with water in the ratio 1:1 to a concentration of at least 50 g/l basis. The column was washed with 27 mm citric acid with a pH of 2.7. Elution spend 27 mm citric acid with a pH of 5.25. The resulting eluate with a concentration of at least 9 g/l diluted to the required concentration and filled into vials in the form of finished dosage forms. The pressure drop along the column during the process ranges from 0.5 to 0.7 MPa (bar) at a flow rate of 180 cm/h Eptifibatide obtained by this method has a purity of 98.6% of and is obtained with a yield of 100%.

Example 7

Atosiban

The crude salt atosiban purity of 73.5%, obtained by solid-phase synthesis, purified on a column filled with a polymer resin. The crude salt atosiban first dissolved in a mixture of acetonitrile and 50 mm acetic acid (1:1)to give a clear solution. The resulting solution was further diluted with 50 mm acetic acid, so that the concentration of acetonitrile is 5%, and the concentration atosiban is at most 2 g/l Before loading the solution ochiltree is up.

A column filled with resin Amberchrom HPR10 (particle size of 10 μm and a pore size 300 Å), balance solution with low content (5%) of acetonitrile in 50 mm acetic acid. The filtered solution atosiban load on the column with a flow rate of at most 360 cm/h Loading of the peptide on the column hold up until its concentration will not be at most 10 g/l resin. After loading the column was washed with a solution with a low content (9%) of acetonitrile in 50 mm acetic acid. Pure product elute column with the implementation of a linear concentration gradient (9-12%) of acetonitrile (buffer B) in 50 mm acetic acid (buffer A) in the amount of 25 OK. The pressure drop along the column during the purification process is 26-32 MPa (bar). Atosiban obtained by this method has a purity of 98.6% of and is obtained with a yield of 57%. Conditions Ehud analysis atosiban presented in the table below.

Table 2
Conditions analysis
The concentration gradientTime (min)%
Buffer And0,18% et 3 n + water, pH to titsa to the value of 2.3 adding ORA 024
The bufferacetonitrile15.038
ColumnSupelco (180 Å, 5 μm)15.150
The flow velocity1 ml/min19.050
The temperature of the column25°C19.124
Wavelength220 nm25.024

Example 8

The crude salt atosiban purity of 73.5%, obtained by solid-phase synthesis, purified on a column filled with a polymer resin. The crude salt atosiban first dissolved in a 5% solution of acetonitrile in 50 mm acetic acid, obtaining a clear solution with a concentration of at most 2 g/l Before loading on the column, the solution is filtered.

A column filled with resin Amberchrom HPR10 (particle size of 10 μm and a pore size 300 Å), balance solution with low content (5%) of acetonitrile in 50 mm UKS the red acid. The filtered solution atosiban load on the column with a flow rate of at most 360 cm/h Loading of the peptide on the column hold up until its concentration will not be at most 10 g/l resin. After loading the column was washed with a solution with a low content (5 and 9%) of acetonitrile in 50 mm acetic acid. Pure product elute column with the implementation of a linear concentration gradient (9-13%) of acetonitrile (buffer B) in 50 mm acetic acid (buffer A) in the amount of 25 OK. The pressure drop along the column during the purification process is 26-32 MPa (bar). Atosiban obtained by this method has a purity of 99.6 percent and is obtained from the output 71,0%.

The purified solution atosiban loaded on cation-exchange column for concentration. Cation-exchange column (TO) balance 5 mm acetic acid with a pH of 3.3. Cleaned atosiban loaded onto cation exchange column after dilution with water in the ratio 1:1. The loading of the peptide on the column hold up until its concentration will not be at most 50 g/l resin. The column was washed with 5 mm acetic acid with a pH of 3.3. Elution spend 500 mm solution of ammonium acetate with a pH of 7.8. The concentration of the obtained eluate is at least 15 g/L. the pressure Drop along the column during the process ranges from 0.2 to 0.3 MPa (bar) at speed through the eye 60 cm/H. Atosiban obtained by this method has a purity of 99.6 percent and is obtained with a yield of 80%.

The eluate, washed with cation-exchange column, enter on exclusion column, replacing the buffer in acetic acid, to obtain a concentrate of finished dosage forms. The eluate can be diluted to the required concentration formulation ingredients and fill it in bottles in the form of finished dosage forms.

Exclusion column balance solution of acetic acid with low concentration (2-5 mm). Sample volume (eluate after KOS)entered on the column is 30% of the volume of the column. The eluate from the column is collected so that the concentration atosiban is at least 15 g/L. the Process is carried out at a flow rate of 15 cm/h and the pressure drop along the column at most 3 MPa (bar). The eluate, washed from the column, is a concentrate that is diluted to the required concentration by adding the components of the formulation, and fill in a bottle.

Example 9

The crude salt atosiban purity 84,1%, obtained by solid-phase synthesis, purified on a column filled with a polymer resin. The crude salt atosiban first dissolved in a 5% solution of acetonitrile in 50 mm acetic acid, obtaining a clear solution with a concentration of at most 2 g/l Before loading on the column races the thief filtered.

A column filled with resin Amberchrom HPR10 (particle size of 10 μm and a pore size 300 Å), balance 10 mm sodium acetate solution (pH 3.0) with low content (5%) of acetonitrile. The filtered solution atosiban load on the column with a flow rate of at most 360 cm/h Loading of the peptide on the column hold up until its concentration will not be at most 10 g/l resin. After loading the column was washed with 10 mm sodium acetate solution (pH 3.0) with low content (5%) of acetonitrile. Pure product elute column with the implementation of a linear concentration gradient (13-20%) of acetonitrile (buffer B) in 10 mm sodium acetate solution (pH 3.0) (buffer A) in the amount of 20 OK. The pressure drop along the column during the purification process is 19-22 MPa (bar). Atosiban obtained by this method has a purity of 99.6 percent and is obtained from the output 94,0%.

The purified solution atosiban loaded on cation-exchange column for concentration. Cation-exchange column (TO) balance 5 mm acetic acid with a pH of 3.3. Cleaned atosiban loaded onto cation exchange column after dilution with water in the ratio 1:1. The loading of the peptide on the column hold up until its concentration will not be at most 50 g/l resin. The column was washed with 5 mm acetic acid with a pH of 3.3. Elution spend 500 mm concrete is ammonium acetate with a pH of 7.8. The concentration of the obtained eluate is at least 15 g/L. the pressure Drop along the column during the process ranges from 0.2 to 0.3 MPa (bar) at a flow rate of 60 cm/h, Atosiban obtained by this method has a purity of 99.6 percent and is obtained with a yield of 80%.

The eluate, washed with cation-exchange column, enter on exclusion column, replacing the buffer in acetic acid, to obtain a concentrate of finished dosage forms. The eluate can be diluted to the required concentration formulation ingredients and fill it in bottles in the form of finished dosage forms.

Exclusion column balance solution of acetic acid with low concentration (2-5 mm). Sample volume (eluate after KOS)entered on the column is 30% of the volume of the column. The eluate from the column is collected so that the concentration atosiban is at least 15 g/L. the Process is carried out at a flow rate of 15 cm/h and the pressure drop along the column at most 3 MPa (bar). The eluate, washed from the column, is a concentrate that is diluted to the required concentration by adding the components of the formulation, and fill in a bottle.

Example 10

The crude salt atosiban purity of 81.5%, obtained by solid-phase synthesis, purified on a column filled with a polymer resin. The crude salt atosiban in which the Achal dissolved in a 5% solution of acetonitrile in 50 mm acetic acid, getting a clear solution with a concentration of at most 2 g/l Before loading on the column, the solution is filtered.

A column filled with resin Amberchrom HPR10 (particle size of 10 μm and a pore size 300 Å), balance solution with low content (5%) of acetonitrile in 10 mm citric acid (pH 3.0). The filtered solution atosiban load on the column with a flow rate of at most 360 cm/h Loading of the peptide on the column hold up until its concentration will not be at most 10 g/l resin. After loading the column was washed with a solution with a low content (5%) of acetonitrile in 10 mm citric acid (pH 3.0). Pure product elute column with the implementation of a linear concentration gradient (12-20%) of acetonitrile (buffer B) in 10 mm citric acid (pH 3.0) (buffer A) in the amount of 25 OK. The pressure drop along the column during the purification process is 22-23 MPa (bar). Atosiban obtained by this method has a degree of purity 99.8% and is obtained from the output 73,0%.

The purified solution atosiban loaded on cation-exchange column for concentration. Cation-exchange column (TO) balance 5 mm acetic acid with a pH of 3.3. Cleaned atosiban loaded onto cation exchange column after dilution with water in the ratio 1:1. The loading of the peptide on the column hold up until its concentration is the situation will not be at most 50 g/l resin. The column was washed with 5 mm acetic acid with a pH of 3.3. Elution spend 500 mm solution of ammonium acetate with a pH of 7.8. The concentration of the obtained eluate is at least 15 g/L. the pressure Drop along the column during the process ranges from 0.2 to 0.3 MPa (bar) at a flow rate of 60 cm/h, Atosiban obtained by this method has a purity of 99.6 percent and is obtained with a yield of 80%.

The eluate, washed with cation-exchange column, enter on exclusion column, replacing the buffer in acetic acid, to obtain a concentrate of finished dosage forms. The eluate can be diluted to the required concentration formulation ingredients and fill it in bottles in the form of finished dosage forms.

Exclusion column balance solution of acetic acid with low concentration (2-5 mm). Sample volume (eluate after KOS)entered on the column is 30% of the volume of the column. The eluate from the column is collected so that the concentration atosiban is at least 15 g/L. the Process is carried out at a flow rate of 15 cm/h and the pressure drop along the column at most 3 MPa (bar). The eluate, washed from the column, is a concentrate that is diluted to the required concentration by adding the components of the formulation, and fill in a bottle.

Example 11

The crude salt atosiban purity 80.2%of the obtained firm is asnam synthesis, purified on a column filled with a polymer resin. The crude salt atosiban first dissolved in a 5% solution of acetonitrile in 50 mm acetic acid, obtaining a clear solution with a concentration of at most 2 g/l Before loading on the column, the solution is filtered.

A column filled with resin Amberchrom HPR10 (particle size of 10 μm and a pore size 300 Å), balance solution with low content (5%) of acetonitrile in 0.05% perchloric acid (pH 1,70). The filtered solution atosiban load on the column with a flow rate of at most 360 cm/h Loading of the peptide on the column hold up until its concentration will not be at most 10 g/l resin. After loading the column was washed with a solution with a low content (5%) of acetonitrile in 0.05% perchloric acid (pH 1,70). Pure product elute column with the implementation of a linear concentration gradient (12-20%) of acetonitrile (buffer B) 0.05% perchloric acid (pH 1,70) (buffer A) in the amount of 25 OK. The pressure drop along the column during the purification process is 23-24 MPa (bar). Atosiban obtained by this method has a purity of 99.6 percent and is obtained from the output 94,0%.

The purified solution atosiban loaded on cation-exchange column for concentration. Cation-exchange column (TO) balance 5 mm acetic acid with a pH of 3.3. Ocimene is atosiban loaded onto cation exchange column after dilution with water in the ratio 1:1. The loading of the peptide on the column hold up until its concentration will not be at most 50 g/l resin. The column was washed with 5 mm acetic acid with a pH of 3.3. Elution spend 500 mm solution of ammonium acetate with a pH of 7.8. The concentration of the obtained eluate is at least 15 g/L. the pressure Drop along the column during the process ranges from 0.2 to 0.3 MPa (bar) at a flow rate of 60 cm/h, Atosiban obtained by this method has a purity of 99.6 percent and is obtained with a yield of 80%.

The eluate, washed with cation-exchange column, enter on exclusion column, replacing the buffer in acetic acid, to obtain a concentrate of finished dosage forms. The eluate can be diluted to the required concentration formulation ingredients and fill it in bottles in the form of finished dosage forms.

Exclusion column balance solution of acetic acid with low concentration (2-5 mm). Sample volume (eluate after KOS)entered on the column is 30% of the volume of the column. The eluate from the column is collected so that the concentration atosiban is at least 15 g/L. the Process is carried out at a flow rate of 15 cm/h and the pressure drop along the column at most 3 MPa (bar). The eluate, washed from the column, is a concentrate that is diluted to the required concentration by adding the components of the formulation, is filled into the vial.

Example 12

The crude salt atosiban purity of 73.5%, obtained by solid-phase synthesis, purified on a column filled with a polymer resin. The crude salt atosiban first dissolved in a 5% solution of acetonitrile in 50 mm acetic acid, obtaining a clear solution with a concentration of at most 2 g/l Before loading on the column, the solution is filtered.

A column filled with resin Amberchrom HPR10 (particle size of 10 μm and a pore size 300 Å), balance solution with low content (5%) of acetonitrile in 0.05% perchloric acid (pH 3.0). The filtered solution atosiban load on the column with a flow rate of at most 360 cm/h Loading of the peptide on the column hold up until its concentration will not be at most 10 g/l resin. After loading the column was washed with a solution with a low content (5%) of acetonitrile in 0.05% perchloric acid (pH 3.0). Pure product elute column with the implementation of a linear concentration gradient (12-20%) of acetonitrile (buffer B) 0.05% perchloric acid (pH 3.0) (buffer A) in the amount of 25 OK. The pressure drop along the column during the purification process is 26-32 MPa (bar). Atosiban obtained by this method has a degree of purity of 99.4% and is obtained from the output 71,0%.

The purified solution atosiban loaded on cation-exchange column for holding the koncentrirovannaya. Cation-exchange column (TO) balance 5 mm acetic acid with a pH of 3.3. Cleaned atosiban loaded onto cation exchange column after dilution with water in the ratio 1:1. The loading of the peptide on the column hold up until its concentration will not be at most 50 g/l resin. The column was washed with 5 mm acetic acid with a pH of 3.3. Elution spend 500 mm solution of ammonium acetate with a pH of 7.8. The concentration of the obtained eluate is at least 15 g/L. the pressure Drop along the column during the process ranges from 0.2 to 0.3 MPa (bar) at a flow rate of 60 cm/h, Atosiban obtained by this method has a purity of 99.6 percent and is obtained with a yield of 80%.

The eluate, washed with cation-exchange column, enter on exclusion column, replacing the buffer in acetic acid, to obtain a concentrate of finished dosage forms. The eluate can be diluted to the required concentration formulation ingredients and fill it in bottles in the form of finished dosage forms.

Exclusion column balance solution of acetic acid with low concentration (2-5 mm). Sample volume (eluate after KOS)entered on the column is 30% of the volume of the column. The eluate from the column is collected so that the concentration atosiban is at least 15 g/L. the Process is carried out at a flow rate of 15 cm/h and pressure drop of the SAR is ü column at most 3 MPa (bar). The eluate, washed from the column, is a concentrate that is diluted to the required concentration by adding the components of the formulation, and fill in a bottle.

Example 13

Nesiritide

The crude salt of nesiritide purity of 59.0%, obtained by solid-phase synthesis, purified on a column filled with a polymer resin. The crude powder of nesiritide first dissolved in a 10% solution of acetonitrile in 50 mm acetic acid, obtaining a clear solution with a concentration of at most 2 g/l Before loading on the column, the solution is filtered.

A column filled with resin Amberchrom HPR10 (particle size of 10 μm and a pore size 300 Å), balance 10 mm sodium acetate solution (pH 3.0) with low content (5%) of acetonitrile. The filtered solution nesiritide load on the column with a flow rate of at most 360 cm/h Loading of the peptide on the column hold up until its concentration will not be at most 10 g/l resin. After loading the column was washed with 10 mm sodium acetate solution (pH 3.0) with low content (5%) of acetonitrile. Pure product elute column with the implementation of a linear concentration gradient (5-15%) of acetonitrile (buffer B) in 10 mm sodium acetate solution (pH 3.0) (buffer A) in the amount of 25 OK. The pressure drop along the column during the purification process, is 28-na (bar). Nesiritide obtained by this method has a degree of purity of 92.5% and is obtained with a yield of 50.0%. Conditions for HPLC analysis of nesiritide presented in the table below.

Table 3
Conditions analysis
The concentration gradientTime (min)%
Buffer And0.1% TFUK + water010
The buffer0.1% TFUK + acetonitrile20.025
ColumnSymmetry C18 (300 Å, 5 μm)25.050
The flow velocity1.2 ml/min25.180
The temperature of the column60°C28.080
Wavelength220 nm 28.110
32.010

Example 14

The crude salt of nesiritide purity of 59.0%, obtained by solid-phase synthesis, purified on a column filled with a polymer resin. The crude powder of nesiritide first dissolved in a 10% solution of acetonitrile in 50 mm acetic acid, obtaining a clear solution with a concentration of at most 2 g/l Before loading on the column, the solution is filtered.

A column filled with resin Amberchrom HPR10 (particle size of 10 μm and a pore size 300 Å), balance solution with low content (5%) of acetonitrile in 10 mm citric acid (pH 3.0). The filtered solution nesiritide load on the column with a flow rate of at most 360 cm/h Loading of the peptide on the column hold up until its concentration will not be at most 10 g/l resin. After loading the column was washed with a solution with a low content (5%) of acetonitrile in 10 mm citric acid (pH 3.0). Pure product elute column with the implementation of a linear concentration gradient (5-15%) of acetonitrile (buffer B) in 10 mm citric acid (pH 3.0) (buffer A) in the amount of 25 OK. The pressure drop along the column during the purification process is 28-33 MPa (bar). N is seretid, obtained by this method has a purity of 97.2 per cent and is obtained from the output of 33.0%.

Example 15

The crude salt of nesiritide purity of 59.0%, obtained by solid-phase synthesis, purified on a column filled with a polymer resin. The crude powder of nesiritide first dissolved in a 10% solution of acetonitrile in 50 mm acetic acid, obtaining a clear solution with a concentration of at most 2 g/l Before loading on the column, the solution is filtered.

A column filled with resin Amberchrom HPR10 (particle size of 10 μm and a pore size 300 Å), balance 10 mm solution of sodium formate (pH 3.0) with low content (5%) of acetonitrile. The filtered solution nesiritide load on the column with a flow rate of at most 360 cm/h Loading of the peptide on the column hold up until its concentration will not be at most 10 g/l resin. After loading the column was washed with 10 mm solution of sodium formate (pH 3.0) with low content (5%) of acetonitrile. Pure product elute column with the implementation of a linear concentration gradient (12-20%) of acetonitrile (buffer B) in 10 mm solution of sodium formate (pH 3.0) (buffer A) in the amount of 25 OK. The pressure drop along the column during the purification process is 24-28 MPa (bar). Nesiritide obtained by this method has a purity of 98.7 per cent and get to the exit 18,0%.

Example 16

Exenatide

Triptorelin of exenatide with purity of 56%, obtained by solid-phase synthesis, purified on a column filled with a polymer resin. Triptorelin of exenatide first dissolved in a mixture of acetonitrile and 50 mm acetic acid (1:1)to give a clear solution. The resulting solution was further diluted with 50 mm acetic acid, so that the concentration of acetonitrile is 5%, and the concentration of exenatide is at most 2 g/l Before loading on the column, the solution is filtered.

A column filled with resin Amberchrom HPR10 (particle size of 10 μm and a pore size 300 Å), balance solution with low content (5%) of acetonitrile in 10 mm citric acid (pH 5.0). The filtered solution exenatide load on the column with a flow rate of at most 360 cm/h Loading of the peptide on the column hold up until its concentration will not be at most 5 g/l resin. After loading the column was washed with a solution with a low content (5%) of acetonitrile in 10 mm citric acid (pH 5.0). Pure product elute column with the implementation of a linear concentration gradient (30-33%) of acetonitrile (buffer B) in 10 mm citric acid (pH 5.0) (buffer A) in the amount of 20 OK. The pressure drop along the column during the purification process is 15-25 MPa (bar). Exenatide obtained by this method, it is no degree of purity of 92.2% and is obtained with a yield of 49%. Conditions for HPLC analysis of exenatide presented in the table below.

Table 4
The concentration gradient
Conditions analysisTime (min)%
Buffer And0.1% TFUK + water025
The buffer0.1% TFUK + acetonitrile1035
ColumnSymmetry C18 (300 Å, 5 μm)2040
The flow velocity1.2 ml/min2170
The temperature of the column40°C2370
Wavelength220 nm2425
2825

The eluate obtained by the above method, diluted three times with water, which contributes to its binding to the column. The eluate of exenatide obtained by the method described in example 1, with the degree of purity 92,0% diluted three times with water, which contributes to its binding to the column. A column filled with resin Amberchrom HPR10 (particle size of 10 μm and a pore size 300 Å) from the company Rohm and Haas, balance solution with 10% acetonitrile in 50 mm acetic acid. The obtained sample exenatide purity 92% load on the column with a flow rate of at most 360 cm/h Loading of the peptide on the column hold up until its concentration will not be at most 5 g/l resin. After loading the column was washed with 20% solution of acetonitrile in 50 mm acetic acid. Pure product elute column with the implementation of a linear concentration gradient (25-28%) of acetonitrile (buffer B) in 50 mm acetic acid (buffer A) in the amount of 20 OK. The pressure drop along the column during the purification process is 20-25 MPa (bar). Exenatide obtained by this method has a purity of 99.6 percent and is obtained with a yield of 50%.

Example 17

Triptorelin of exenatide purity 46,2%, obtained by solid-phase synthesis, purified on a column filled floor is measured with resin. Triptorelin of exenatide first dissolved in a mixture of acetonitrile and 50 mm acetic acid (1:1)to give a clear solution. The resulting solution was further diluted with 50 mm acetic acid, so that the concentration of acetonitrile is 5%, and the concentration of exenatide is at most 2 g/l Before loading on the column, the solution is filtered.

A column filled with resin Amberchrom HPR10 (particle size of 10 μm and a pore size 300 Å), balance 10 mm sodium acetate solution (pH 4.0) low (10%) of acetonitrile. The filtered solution exenatide load on the column with a flow rate of at most 360 cm/h Loading of the peptide on the column hold up until its concentration will not be at most 10 g/l resin. After loading the column was washed with 10 mm sodium acetate solution (pH 4.0) low (10%) of acetonitrile. Pure product elute column with the implementation of a linear concentration gradient (28-33%) of acetonitrile (buffer B) in 10 mm sodium acetate solution (pH 4.0) (buffer A) in the amount of 20 OK. The pressure drop along the column during the purification process, 18-20 MPa (bar). Exenatide obtained by this method has a degree of purity of 94.2 percent and is obtained with a yield of 57%.

The eluate of exenatide with purity of 94.2 percent, obtained by the method of the above example, diluted three times with water, that SP is contributes to its binding to the column.

A column filled with resin Amberchrom HPR10 (particle size of 10 μm and a pore size 300 Å) from the company Rohm and Haas, balance solution with 10% acetonitrile in 50 mm acetic acid. The obtained sample exenatide purity 94.2% of load on the column with a flow rate of at most 360 cm/h Loading of the peptide on the column hold up until its concentration will not be at most 10 g/l resin. After loading the column was washed with 20% solution of acetonitrile in 50 mm acetic acid. Pure product elute column with the implementation of a linear concentration gradient (25-28%) of acetonitrile (buffer B) in 50 mm acetic acid (buffer A) in the amount of 20 OK. The pressure drop along the column during the purification process is 20-25 MPa (bar). Exenatide obtained by this method has a purity of 99.6 percent and is obtained with a yield of 50%.

Example 18

Triptorelin of exenatide purity 46,2%, obtained by solid-phase synthesis, purified on a column filled with a polymer resin. Triptorelin of exenatide first dissolved in a mixture of acetonitrile and 50 mm acetic acid (1:1)to give a clear solution. The resulting solution was further diluted with 50 mm acetic acid, so that the concentration of acetonitrile is 5%, and the concentration of exenatide is at most 2 g/l Before loading the solution of filtrowa.

A column filled with resin Amberchrom HPR10 (particle size of 10 μm and a pore size 300 Å), balance 10 mm solution of sodium formate (pH 4.0) low (10%) of acetonitrile. The filtered solution exenatide load on the column with a flow rate of at most 360 cm/h Loading of the peptide on the column hold up until its concentration will not be at most 10 g/l resin. After loading the column was washed with 10 mm solution of sodium formate (pH 4.0) low (10%) of acetonitrile. Pure product elute column with the implementation of a linear concentration gradient (27-35%) of acetonitrile (buffer B) in 10 mm solution of sodium formate (pH 4.0) (buffer A) in the amount of 20 OK. The pressure drop along the column during the purification process is 24-29 MPa (bar). Exenatide obtained by this method has a purity of 94.9% and is obtained with a yield of 52%.

The eluate of exenatide purity of 94.9%, obtained by the method of the above example, diluted three times with water, which contributes to its binding to the column. A column filled with resin Amberchrom HPR10 (particle size of 10 μm and a pore size 300 Å) from the company Rohm and Haas, balance solution with 10% acetonitrile in 50 mm acetic acid. The obtained sample exenatide purity 94.2% of load on the column with a flow rate of at most 360 cm/CH Sarasqueta column on hold until until its concentration will not be at most 10 g/l resin. After loading the column was washed with 20% solution of acetonitrile in 50 mm acetic acid. Pure product elute column with the implementation of a linear concentration gradient (25-28%) of acetonitrile (buffer B) in 50 mm acetic acid (buffer A) in the amount of 20 OK. The pressure drop along the column during the purification process is 20-25 MPa (bar). Exenatide obtained by this method has a purity of 99.6 percent and is obtained with a yield of 50%.

1. The method of purification of cyclic or non-cyclic peptide selected from the group of peptides comprising eptifibatide, exenatide, atosiban and nesiritide or their combination, of a compound containing at least one impurity, comprising contacting this mixture with the matrix for RP-HPLC and matrix for ion-exchange chromatography and obtaining the purified peptide product with a purity of at least 96%, which fill the columns for RP-HPLC polymer resin, which balance the buffer solution of organic acid containing 5% acetonitrile, download the peptide mixture containing at least one impurity, the column a flow rate of at most approximately 100÷400 cm/h, the column is washed with the same buffer solution, and that when used at the stage of filling the columns for RP-HPLC, elute ocimene the peptide column with a linear concentration gradient from 8%to 14%, balance column for cation-exchange chromatography of aqueous buffer solution of a weak acid, upload the peptide is purified by RP-HPLC on a column for cation exchange chromatography, washed with column and elute the peptide product of the buffer solution used for equilibration of the column for cation-exchange chromatography, and receive the purified peptide with a purity of at least 96%.

2. The method according to claim 1, in which the specified polymer resin selected from the group comprising Sephadex, methylacrylate resin, carboxymethyl cellulose, carboxymethylated, sulfopropyl, sulforaphanes and a polymer resin, attached to the silicon dioxide.

3. The method according to claim 1, in which the specified polymer resin is polystyrene or polydivinylbenzene.

4. The method according to claim 1, wherein the particle size of the granules of the specified polymer resin is 1÷50 µm.

5. The method according to claim 1, wherein the pore size of the granules of the specified polymer resin is 100÷500 Å.

6. The method according to claim 1, which further conduct exclusion chromatography.

7. The method according to claim 1, in which the specified organic acid selected from the group comprising citric acid, acetic acid, perchloric acid and formic acid.

8. The method according to claim 1, in which the molar concentration of the specified buffer solution organic to the slots is 10÷50 mm.

9. The method according to claim 1, which indicated a weak acid selected from the group comprising citric acid and acetic acid.

10. The method according to claim 1, in which the molar concentration of the specified buffer solution of a weak acid is 5 mm.

11. The method according to claim 1, in which cleaning is performed at pH from 2 to 9.



 

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FIELD: chemistry.

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3 cl, 2 tbl, 8 ex

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20 cl, 1 dwg, 14 tbl, 15 ex

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26 cl, 22 ex

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39 cl, 44 dwg, 4 tbl, 29 ex

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19 cl, 6 dwg, 6 tbl, 4 ex

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6 cl, 7 dwg, 4 tbl, 14 ex

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17 cl, 2 tbl, 8 ex

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3 cl, 3 ex, 3 dwg

FIELD: chemistry.

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EFFECT: invention enables to reduce the level of protein S at least twofold.

30 cl, 24 ex

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