Method of purifying human interferon beta

FIELD: chemistry, biochemistry.

SUBSTANCE: invention relates to biotechnology. Method of purifying human interferon beta from a recombinant culture which contains human interferon beta includes carrying out affinity chromatography and cation-exchange chromatography. Affinity chromatography includes adsorption of the culture containing interferon beta, in a balanced chromatograph column for affinity chromatography, rinsing the column using a liquid buffering solution A with a pH 6.5-7.5, containing 30-60 wt % of propylene-glycol, and liquid buffering solution B with a pH 6.5-7.5, containing 10-30 wt % of propylene-glycol and 1-2 M NaCl, with elusion fraction, containing human interferon beta, with a buffering solution with pH 6.5-7.5, containing 40-60 wt % propylene-glycol and 1-2 M NaCl. Solution obtained is subjected to diafiltration and cation-exchange chromatography is carried out, elute the interferon-containing fraction at a pH 5-7 with the gradient of concentration of NaCl within the limits 0-400 mM followed by concentration and gel-filtration of the product.

EFFECT: obtaining interferon beta, characterised with a high level of purity (99%) with the simplification of the technology of separation.

4 cl, 4 dwg, 4 ex

 

The technical field to which the invention relates.

The present invention relates to a method of purifying human interferon beta from a culture containing recombinant interferon beta, human, using affinity chromatography and cation exchange chromatography.

The level of technology

Interferons in the broad sense of the word are extracellular messaging, mediating reactivity of the host body, and evolutionary conservative families of proteins that are released in relatively small quantities of cells. Interferons are exempt from producing interferons cells in response to stimulation by viruses, double-stranded RNA, various microorganisms or cytokines, such as tumor necrosis factor (TNF) or interleukin 1 (IL1), and then contacting the interferon receptors, localized on the surface of neighboring cells. After that, interferons induce the synthesis of different proteins, so that the reactivity and the homeostasis of the host body are supported by the serial signal transmission cells. Thus, interferons act in the body as antiviral, antiproliferative and immune signaling proteins and have a direct antiproliferative effect on cancer cells, resulting attract attention as therapeu the practical agents [Postka S., Langer J.A. and Zoon KS (1987) Interferons and their actions, Armu. Rev. Biochem. 56: 727-777].

Interferons belong to the class of spiral physiologically active substances. According to the physical-chemical characteristics and functional activity distinguish two classes of interferons: type 1 and 2. Interferons alpha, beta, Tau and Epsilon are representatives of interferon type 1 [S. Weissman and Weber H. (1986) The Interferon genes, Prog. Nucleic Acid Res. Mol. Biol. 33: 251-300], and interferon gamma is representative of the type 2 interferons. Among them, interferon beta, related to type 1 interferons are proteins, demonstrating the species specificity. Interferon beta is also referred to as interferon fibroblasts according to their source and rn-stable interferon according to their biological characteristics. Interferon beta bind to the same receptors on the surface of cells, and interferons alpha, belonging to the type 1 interferons, and then induce the transcription of antiviral factors in response to the activation of the signaling pathways of the cell.

Interferon beta are glycoproteins (about 20% sugar residues) with a molecular weight of about 20 kDa and a single-chain protein consisting of 166 amino acid residues. It is known that a single site of N-glycosylation plays a role in increasing the stability or solubility, i.e. to improve the Phi is ICO-chemical functions, rather, what is involved in the biological activity or antigenicity [Karpusas M, Whytty A., Runkel L, Hochman and P. The structure of human interferon-β: implications for activity CMLS, 54: 1203-1216 1998].

Advances in genetic recombinant technology has made possible the determination of the amino acid sequence of human interferon beta, as well as cloning and expression of human interferon beta in E. coli [Taniguchi, Gene 10: 11-15, 1980]. Moreover, it was also reported the expression of interferon beta in the ovary cells of Chinese hamster (Chinese hamster ovary (CHO) cells) [USP 4,966,843, USP 5376567, and USP 5795779].

Currently, interferon beta produced by the technology of genetic recombination and commercially available under the trade names Betaseron®, Avonex®and Rebif®. It is known that recombinant interferon beta are effective in slowing the development of multiple sclerosis in patients with symptoms of illness and pain with the disease. Moreover, recombinant interferon beta are widely used as therapeutic agents in multiple sclerosis and at the same time are effective in non-specific regulation of immune response the human immune response to viral infection and in inhibiting proliferation of cancer cells.

Currently available treatment technology of recombinant interferon beta, expressed in CHO cells consist of 3-5 cleaning procedures, including primary cleaning method of affinity chromatography (USP 4278661, USP 4289689, USP 4541952, USP 4808523 etc), metal-chelate chromatography (USP 4257938, USP 4359389, USP 4541952, USP 5244655, etc), chromatography on porous glass with controlled pore size (CPG, controlled pore glass chromatography) (USP 4359389, USP 5066786, USP 5244655 etc), or chromatography on Concanavalin And (USP 4289689, USP 4658017 etc) followed by cation exchange chromatography or reversed-phase chromatography.

In the above-described conventional technologies for cleaning metal-chelate chromatography may cause environmental pollution due to the use of heavy metals. Chromatography on porous glass or chromatography on Concanavalin And have low specificity. That is, chromatography on Concanavalin And based on selective binding of many sugar free protein present in the cell culture of Cho, which leads to low specificity. Chromatography on porous glass allows the separation of molecules by size after binding to protein. However, the efficiency of the separation and purity of interferon beta lower than affinity chromatography (for example, by chromatography on a column of Blue-Separate).

Moreover, the conventional methods of purification using affinity chromatography include washing and elution with ethylene glycol using monoclonal is antibodies and/or resins, containing dyes. However, affinity chromatography using monoclonal antibodies requires a separate stage of removal deglycosylation forms of interferon beta, which makes mass production difficult. In particular, it is used for washing and elution ethylene glycol is very toxic to the body, which limits the use of this cleaning method.

At the same time, U.S. Patent No. 4483849 discloses a method of purification and stabilization of interferon beta using propylene glycol instead of toxic glycol in affinity chromatography. The method disclosed in this patent document includes applying interferonsource culture on the affinity column with a dye, such as balanced Affi-Gel Blue, washing the column with 1.0 M NaCl buffer/PO4and 1.0 M NaCl buffer/PO4containing 40% propylene glycol, and then the elution of interferon 50% propylene glycol. Although described in this patent document, the process includes flushing the column and the elution, the peak of the desired end product and the peak of the impurities are superimposed on each other, which reduces the purity of the product.

Disclosure of inventions

The present invention provides a method of purification of interferon beta, which includes obtaining a high purity product primary treatment of interferon beta with improvements is tavanai affinity chromatography using non-toxic propylene glycol, followed by cation exchange chromatography.

Thus, the present invention provides a method of purification of human interferon beta from recombinant cultures containing interferon beta person, including affinity chromatography and cation exchange chromatography, which includes washing and elution of a specific buffer solution.

According to one aspect of the present invention proposes a method of purification of human interferon beta from a culture containing recombinant interferon beta, human, using affinity chromatography and cation exchange chromatography, in which affinity chromatography involves the adsorption of the culture containing the human interferon beta, pre-equilibrated column for affinity chromatography, subsequent washing of the column with the buffer for equilibration, wash column wash buffer solution with a pH of 6.5-7.5, containing 30-60% (by weight) of propilenglikola, and wash column wash buffer solution with a pH of 6.5-7.5, containing 10-30% (by weight) propylene glycol and 1-2 M NaCl, and the elution fractions containing the human interferon beta, a buffer solution with a pH of 6.5-7.5, containing 40-60% (by weight) of propylene glycol and 1-2 M NaCl.

In the purification method of the present invention limitiruyuschie examples of cultures containing recombinant human interferon beta used as the e sample, include producing interferon beta cells and strains. For example, recombinant interferon beta, human culture can be obtained in a known manner described in Carter and Horoszewicz, Pharm. Ther. 8, 359-377, 1980; Strander and Cantell, Ann. Med. Exp. Fenn. 44, 265-273, 1966; Wheelock, Science, 149, 310-311, 1965, and the like. Preferably, the culture containing recombinant human interferon beta, is a serum-free culture derived from ovarian cells of the Chinese hamster (Cho), producing recombinant human interferon beta

In the purification method of the present invention used for affinity chromatography the chromatographic column can be a normal affine column with a dye, for example, column (e.g. column XK-50 column, Amersham biosciences, Sweden), filled with Blue-Sepharose 6 (Amersham biosciences, Sweden), or column of Affi-Gel Blue (Bio-Rad, America). The buffer solution used for equilibration of the column for affinity chromatography may be a buffer solution of sodium phosphate-EDTA (pH about 7.2). Affinity column can be equilibrated with 3 column volumes (CV) buffer solution for balancing, for example, the linear speed of about 15-30 cm/h

In the purification method of the present invention affinity chromatography involves the adsorption of culture containing interferon beta, balanced column for affinity chromatography and remove nespec is physically associated protein by rinsing with buffer solution to trim.

Affinity chromatography also includes a multi-stage leaching, i.e. the wash column wash buffer solution with a pH of 6.5-7.5, containing 30-60% (by weight) of propylene glycol and wash column wash buffer solution with a pH of 6.5-7.5, containing 10-30% (by weight) of propylene glycol and 1 to 2 M NaCl. Preferably, affinity chromatography also includes flushing the column wash solution With a pH of 6.5-7.5, containing 1-2 M NaCl. Preferably, each washing is carried out using 2-4 column volumes (CV) of each buffer solution

In the purification method of the present invention is not limited by the sequence of use of the washing buffer solutions. For example, the flushing can be carried out using a wash buffer solution and then washing buffer solution or using a wash buffer solution, and then washing buffer solution A. in Addition, the flushing can be carried out using a wash buffer And wash buffer solution and then washing buffer solution or using wash buffer, wash buffer solution and then wash buffer A. Wash using wash buffer solution And effectively removes impurities with vysokoshirotnoi, rinsing with washing buffer To remove hydrophilic contamination and washing with washing buffer solution To remove protein impurities.

Removing interferon beta with a column can be carried out by elution fractions containing the human interferon beta, a buffer solution with a pH of 6.5-7.5, containing 40-60% (by weight) of propylene glycol, preferably 50% (by weight), and 1-2 M NaCl.

Preferably, each used for washing or elution buffer may be a sodium phosphate buffer solution or a potassium phosphate buffer solution.

In the purification method of the present invention, the washing buffer solution containing about 50% propylene glycol, can effectively remove the peaks of pollution, in contrast to the method disclosed in US Patent No. 4483849, in which the washing and elution are carried out stepwise gradient of the concentration of propylene glycol.

In the purification method of the present invention after the above-described affinity chromatography is cation exchange chromatography. Preferably before conducting cation-exchange chromatography obtained by elution in affinity chromatography, the solution is subjected to diafiltration on the membrane for ultrafiltration with limit bandwidth based on the molecular weight of 10000. If diafiltration interferon beta with a relatively high concentration of the second salt may be communicated to the appropriate salt concentration.

Cation exchange chromatography was carried out as follows: the sample obtained after diafiltration, was applied on the column, and then the fraction containing the human interferon beta, suirable at pH 5-7 by the concentration gradient of NaCl. In more detail, the fractions containing interferon beta, suirable linear concentration gradient, starting with the sodium phosphate buffer solution (pH 5-7), which is balanced buffer solution to NaCl-containing sodium phosphate buffer solution (pH 5-7).

Column for cation exchange chromatography can be column (HC-50, 150 ml CV, Amersham biosciences, Sweden) and CCM-Sepharose FF (Amersham biosciences, Sweden) and can be balanced about 3 CV sodium phosphate buffer solution at a flow rate of 5 ml/min. by cation-exchange chromatography the sample obtained by diafiltration, was passed through the equilibrated column at a suitable flow rate and suirable linear salt concentration gradient of NaCl-containing sodium phosphate buffer solution (approximately 12 CV). The result of this was removed interferon beta, devoid of sugar chains, and selectively received interferon beta with a sugar chain.

The fraction containing interferon beta obtained by cation exchange chromatography can then be replaced by a new buffer solution. C the exchange to a new buffer solution can be performed by gel filtration or concentration and diafiltration.

For example, in the case of gel-filtration fractions containing interferon beta obtained by cation-exchange chromatography, concentrated to, for example, about 200-1000 µg/ml, dialist against 10-50 mm sodium acetate buffer solution (pH 3.5˜5,5) and applied to a chromatographic column for gel filtration (e.g., Sephacryl S-200, Amersham biosciences), equilibrated 10-50 mm, preferably 20 mm sodium acetate buffer solution (pH 3.5˜5,5). Then 10-50 mm sodium acetate buffer solution (pH 3.5˜5,5) is passed through the column at a suitable flow rate, thus changing the desired solution of proteins and separation, and destruction of polymers.

A block diagram illustrating the purification method of the present invention, is shown in figure 1.

Brief description of figures

Figure 1 is a block diagram of the purification method of the present invention.

Figure 2 represents a C4 RP-HPLC (reverse phase high-performance liquid chromatography) analytical chromatogram of interferon beta, aliremove when affinity chromatography according to the purification method of the present invention.

Figure 3 represents a C4 RP-HPLC analysis chromatogram of interferon beta, aliremove without washing with 50% propylene glycol.

Figure 4 represents a C4 RP-HPLC analysis chromatogram suiruev what about the solution after gel filtration.

The implementation of the invention

Hereinafter the present invention will be described in more detail using examples. However, the following examples are given solely as an illustration and, therefore, the present invention is not limited to them.

Example 1: affinity chromatography

Blue-Sepharose 6 (Amersham biosciences, Sweden) in the amount of 350 ml was introduced into the column HC-50 (Amersham biosciences, Sweden) for the manufacture of columns for affinity chromatography. To balance the speakers through it missed in sufficient quantity 20 mm sodium phosphate buffer solution containing 1 mm EDTA. After this was passed through the column 25 l of serum-free cell culture Chinese hamster ovary (Cho)containing interferon beta, at a flow rate of 5-10 ml/min, after which the column was washed with 3 column volumes (CV) solution to trim.

About 3 CV of 20 mm sodium phosphate buffer solution (pH of 7.2)containing 50% propylene glycol was passed through the column at a flow rate of 5 ml/min to remove impurity proteins, after which the column was washed about 3 CV of buffer solution for balancing. Then about 3 CV of 20 mm sodium phosphate buffer solution (pH of 7.2)containing 2 M NaCl was passed through the column at a flow rate of 5 ml/min to remove impurity proteins. And, finally, about 3 CV of 20 mm sodium phosphate buffer Rast is ora (pH 7,2), containing 2 M NaCl and 20% propylene glycol was passed through the column at a flow rate of 5 ml/min to remove impurity proteins.

About 3 CV of buffer solution for elution (20 mm sodium phosphate buffer solution containing 2 M NaCl and 50% propylene glycol, pH 7,2) was passed through the column at a flow rate of 5 ml/min, to obtain a solution containing interferon beta. The purity of the obtained aliremove solution was tested C4 HPLC analytical chromatography, the results of which are presented in figure 2. According to figure 2, the purity of interferon beta was about 85% or more.

In the control experiment affinity chromatography was performed according to the above Protocol, except that the wash column 20 mm sodium phosphate buffer solution (pH of 7.2)containing 50% propylene glycol was omitted. The purity of the obtained elution solution was analyzed using C4 HPLC analytical chromatography, the results of which are presented in figure 3. As can be seen from figure 3, in the absence of leaching of 20 mm sodium phosphate buffer solution (pH of 7.2)containing 50% propylene glycol, significantly reduced the purity of interferon beta.

Example 2: cation-exchange chromatography

The solution containing interferon beta obtained according to the present invention and example 1 were subjected to diafiltration, and who uses the system for ultrafiltration (cutoff molecular weight of 10000) and then was applied to a column (XK-50, 150 ml CV, Amersham biosciences, Sweden) CCM-Sepharose FF (Amersham biosciences, Sweden) at a flow rate of 1.5 ml/min and Then the column was balanced using approximately 3 CV of 50 mm sodium phosphate buffer solution (pH 6,7) at a flow rate of 5 ml/min, the Elution of interferon beta was carried out using a linear concentration gradient from 50 mm sodium phosphate buffer solution (pH of 6.7), 50 mm sodium phosphate buffer solution (pH of 6.7)containing 400 mm NaCl in order thus to obtain fractions of interferon beta-related sugar chains.

Example 3: gel filtration

The solution containing interferon beta obtained in example 2 was concentrated to 200 μg/ml of the Resulting solution was applied on a column of Sephacryl S-200 (1700 ml, HC-50/100, Amersham biosciences, Sweden), equilibrated to 20 mm sodium acetate buffer solution (pH 4.0) to obtain a solution containing interferon beta.

Example 4: reversed-phase liquid chromatography high resolution (RP-HPLC)

Each of the solutions obtained in examples 1, 2 and 3, was applied to the column C4 RP-HPLC (Vydac 214TP54, inner diameter 4.6 mm×25 cm in length, particle size 5 μm, pore size of 300 Å) at a flow rate of 1 ml/min then passed through the column 20 CV of a solution of acetonitrile containing 0.1% triperoxonane acid, in the form of a linear gradient from 30% acetonic the sludge, containing 0.1% triperoxonane acid, 80% acetonitrile containing 0.1% triperoxonane acids for chromatographic analysis of a painting.

The result of analysis by gel-filtration chromatography is shown in figure 4. From figure 4 it can be seen that the present invention allows to receive interferon beta high purity.

Industrial applicability

According to the purification method of the present invention interferon beta can be purified to high purity, making 99% or more, when using non-toxic propylene glycol and improved affinity chromatography.

1. The method of purification of human interferon beta from a culture containing recombinant human interferon beta, including affinity chromatography and cation exchange chromatography, where affinity chromatography includes:

the adsorption of culture containing interferon beta, balanced column for affinity chromatography followed by washing equilibrating buffer solution;

wash column wash buffer solution with a pH of 6.5-7.5,

containing 30-60 wt.% propylene glycol and wash buffer solution with a pH of 6.5-7.5, containing 10-30 wt.% propylene glycol and 1-2 M NaCl; and

the elution fractions containing the human interferon beta, buffer solution is m with a pH of 6.5-7.5, containing 40-60 wt.% propylene glycol and 1-2 M NaCl,

the solution obtained after affinity chromatography, subjected to diafiltration obtained, and applied to a column for cation-exchange chromatography, and the fraction containing the human interferon beta, elute at pH 5-7, using solutions with a concentration gradient of NaCl from 0 to 400 mm,

moreover, the solution obtained by cation-exchange chromatography, concentrated and subjected to gel filtration.

2. The method according to claim 1, wherein the stage further includes washing the wash column wash buffer solution With pH of 6.5-7.5, containing 1-2 M NaCl.

3. The method according to claim 1 or 2, characterized in that each buffer solution used for washing and elution, is a sodium phosphate buffer solution or a potassium phosphate buffer solution.

4. The method according to claim 1 or 2, characterized in that diafiltration performed using ultrafiltration membranes with limit bandwidth based on the molecular weight of 10000.



 

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SUBSTANCE: invention relates to biotechnology. Method of purifying human interferon beta from a recombinant culture which contains human interferon beta includes carrying out affinity chromatography and cation-exchange chromatography. Affinity chromatography includes adsorption of the culture containing interferon beta, in a balanced chromatograph column for affinity chromatography, rinsing the column using a liquid buffering solution A with a pH 6.5-7.5, containing 30-60 wt % of propylene-glycol, and liquid buffering solution B with a pH 6.5-7.5, containing 10-30 wt % of propylene-glycol and 1-2 M NaCl, with elusion fraction, containing human interferon beta, with a buffering solution with pH 6.5-7.5, containing 40-60 wt % propylene-glycol and 1-2 M NaCl. Solution obtained is subjected to diafiltration and cation-exchange chromatography is carried out, elute the interferon-containing fraction at a pH 5-7 with the gradient of concentration of NaCl within the limits 0-400 mM followed by concentration and gel-filtration of the product.

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