Method of purifying human interferon beta

FIELD: chemistry, biochemistry.

SUBSTANCE: invention relates to biotechnology. Method of purifying human interferon beta from a recombinant culture containing human interferon beta, which includes carrying out affinity chromatography and is treated to phase high-efficiency liquid chromatography (HELC). Affinity chromatography adsorption of the culture, which contains interferon beta, in a balanced chromatograph column, 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 phase HELC is carried out, elute the interferon-containing fraction with a gradient of concentrated ethanol from 30-50% to 65-90% with a pH 2.5, followed by concentration and gel-filtration of the product.

EFFECT: obtaining interferon beta characterised with a high level of purity (99%) with a simplified method of separation.

4 cl, 5 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 reversed-phase high-performance liquid 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 p is and a steel attention as therapeutic agents [Postka S., Langer J. A. and Zoon, K. (1987) Interferons and their actions, Annu. 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 [With Weissman and Weber N. (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) [USP4,966,843, USP5,376,567 and USP5,795,779].

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 the cells Is About, consist of 3-5 cleaning procedures, including primary cleaning method of affinity chromatography (USP4,278,661, USP4,289,689, USP4,541,952, USP4,808,523 etc), metal-chelate chromatography (USP4,257,938, USP4,359,389, USP4,541,952, USP5,244,655 etc), chromatography on porous glass with controlled pore size (CPG, controlled pore glass chromatography) (USP4,359,389, USP5,066,786, USP5,244,655 etc), or chromatography on Concanavalin AND (USP4,289,689, USP4,658,017 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 has 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 etrange Olam using monoclonal 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. 4,483,849 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 the interferon-containing 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 using uselen the nd affinity chromatography using non-toxic propylene glycol, followed by reversed-phase high-performance liquid chromatography (RP-HPLC).

Thus, the present invention provides a method of purification of human interferon beta from recombinant cultures containing interferon beta person, including affinity chromatography and RP-HPLC, 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 RP-HPLC, in which affinity chromatography includes: adsorption culture containing 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 propylene glycol 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 containing interferon beta human faction 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 a sample, include producing interferon bet the cells and strains. For example, recombinant interferon beta, human culture can be obtained by a known method 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 producing interferon beta human recombinant cells Chinese hamster ovary (Cho).

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/hour.

In the purification method of the present invention affinity chromatography involves the adsorption of culture containing interferon beta, balanced affinity column and remove non-specific bound protein by rinsing with buffer solution for uranous is found.

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 high what agroforesty, 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 U.S. Patent No. 4,483,849, 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 carried out RP-HPLC. It is preferable to conduct the RP-HPLC 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 salt concentration can be increased to appropriate to the concentrations of salt.

RP-HPLC 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 2-5 gradient of the concentration of ethanol containing HCl. In more detail, the column was balanced with 0.1% HCl containing 0.1-20%, preferably 5% or less propylene glycol, after which the sample obtained by diafiltration containing 0.1-20%, preferably 5% or less of propylene glycol was applied to the column. Then the column was washed with 0.1% model HC1, and the fractions containing interferon beta, suirable linear concentration gradient from 30-50%, preferably 45% ethanol containing 0.1% HCl 65 to 90%, preferably 70% ethanol containing 0.1% HCl.

Column RP-HPLC can be Protein C4 (particle size of 10 μm, a pore size of 30 Å, Vydac) and can be balanced about 5 CV of 0.1% solution model HC1 containing propylene glycol. In RP-HPLC the sample obtained by diafiltration, was passed through the equilibrated column at a suitable flow rate, washed with 3 CV or more buffer solution of 0.1% HCl and was suirable linear concentration gradient of ethanol by approximately 10-20 CV, containing 0.1% model HC1, so as to divide the protein impurities and target proteins.

The fraction containing interferon beta obtained by RP-HPLC, can then be replaced on but the first buffer solution. Replace 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 RP-HPLC, 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 to 5.5) is passed through the column at a suitable flow rate, providing, thus, the change of solution of the desired protein and the separation and disposal 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 whom romawki with 50% propylene glycol.

Figures 4A and 4B are respectively C4 RP-HPLC analysis chromatogram buffer solution for gel filtration and C4 RP-HPLC analysis chromatogram of the solution, aliremove 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 the Colo 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 solution (pH of 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 integration of the Feron beta.

Example 2: reverse-phase high-performance liquid chromatography (RP-HPLC)

Interferon beta-containing solution obtained by the present invention in example 1, subjected diafiltration using ultrafiltration system (cutoff molecular weight of 10,000), was applied to the column for RP-HPLC (Protein C4 (a particle size of -10 μm, pore size of 30 Å, Vydac) at a flow rate of 2 ml/min and Then the column was washed for about 3 CV of 0.1% model HC1 buffer solution (pH 2,1). The elution of interferon beta was performed using 0.1% solution model HC1 (a) and solution (B) IS 0.1% model HC1 in 90% ethanol, using a linear concentration gradient from 45% solution (C) up to 80% solution (C) (20 CV) to separate the impurity proteins from the target proteins.

Example 3: gel filtration

The solution containing interferon beta obtained in example 2 was concentrated to 200 μg/ml and contained in concentrate the ethanol was replaced by 500 times or more than 20 mm sodium acetate buffer solution (pH 4.0). 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 3.0) to obtain a solution containing interferon beta.

Example 4: analysis by RP-HPLC

Each of the solutions obtained in examples 1, 2 and 3, was applied to the column C4 RP-HPLC (Vydac TR, inner diameter 4.6 m is x 25 cm in length, the particle size 5 µm, pore size 300 Ĺ) 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% acetonitrile containing 0.1% triperoxonane acid, 80% acetonitrile containing 0.1% triperoxonane acids for chromatographic analysis of a painting.

Figures 4A and 4B respectively show a C4 RP-HPLC analysis chromatogram buffer solution for gel-filtration chromatography and C4 RP-HPLC analysis chromatogram of the solution, aliremove after gel-filtration chromatography. From figures 4A and 4B 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 reversed-phase high-performance liquid chromatography (RP-HPLC), where affinity chromatography includes:

the adsorption of culture, the content is soup interferon beta, on 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 the 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, a buffer solution with a pH of 6.5-7.5, containing 40-60 wt.% propylene glycol and 1-2 M NaCl,

moreover, the solution obtained after affinity chromatography, subjected to diafiltration received a sample applied to the column for RP-HPLC, and then the fraction containing the human interferon beta, elute at pH 2-5, using solutions with a concentration gradient of ethanol from 30-50% of 65 to 90%,

moreover, the solution obtained RP-HPLC, 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 pre is Elam bandwidth based on the molecular weight of 10000.



 

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