KR20090081288A - A method for purifying recombinant human Interferon beta - Google Patents

Abstract

A method for refining a human interferone beta is provided to efficiently suppress the coagulation during the refining process with high purity and easily obtain an activated interferone beta 1b. A method for refining a human interferone comprises: a step of smashing a cell which contains a recombinant interferone beta 1b to obtain an envelope; a step of washing the envelop with buffer containing urea and resolving with DTT and amphoteric ionic surfactant; a step of performing cationic exchange chromatography; a step of adding oxidant in an isolated interferone beta 1b fraction and induce the disulfide bond; and a step of performing cation exchange column and gel filtration chromatography of interferone beta 1b to obtain the interferone beta 1b of high purity.

Description

A method for purifying recombinant human Interferon beta}

The present invention relates to a method for purifying recombinant interferon beta 1b from E. coli , which produces recombinant human interferon beta 1b.

There are currently eight types of human interferons (IFNs), which are classified into two types according to physicochemical and functional characteristics. Type 1 interferons are alpha-, beta-, kappa-, delta-, epsilon-, tau- and omega-interferon (Oritani and Tomiyama, International journal of hematology, 80, 325-331, 2004; Hardy et al., Genomics, 84, 331-345, 2004), type 2 interferons include gamma-interferon (Gray, PW, Nature) , 298, 859-863, 1982). Interferon alpha is produced in leukocytes or lymphocytes, interferon beta is produced in fibroblasts, and interferon gamma is produced in immunolymphocytes.

Interferon (IFNs) inhibit antiviral replication, thereby exhibiting antiviral activity, inhibiting cell proliferation and regulating immune responses. Interferon beta is a cytokine (cytokine) produced in response to viral infections and the like, in particular as a treatment for multiple sclerosis (MS). Multiple sclerosis is a central nervous system disorder that involves the destruction of myelin enveloping axons of central nervous system cells in the brain and spinal cord. Multiple sclerosis patients suffer from several disorders due to decreased or blocked neurotransmission. Depending on the site, symptoms such as motor paralysis, speech disorders, consciousness disorders, and thinking disorders may be present.

Currently, there are two types of interferon beta for the treatment of multiple sclerosis. Firstly, pharmaceutical interferon beta 1a is produced from Chinese hamster ovary (CHO) containing the human interferon beta gene and consists of 166 amino acid residues. It is a glycosylated protein that is 25 kD in size. Secondly, interferon beta 1b is a protein consisting of 165 amino acid residues produced from E. coli, which lacks sugars, lacks amino acid 1 methionine residues, and cysteine residue 17 serine. It is substituted by. Currently available interferon beta 1a includes Rebif (Serono) and Avonex (Avonex, Biogen), and interferon beta 1b includes betaceron (Betaseron, Schering).

When recombinant proteins are expressed in microorganisms using genetic recombination techniques, the expression of proteins in the form of inclusion bodies is a well known phenomenon, although the information on its formation mechanism is limited [Williams, DC et al, Science, 215, 687-689 (1982). This phenomenon has been reported to occur depending on the culture conditions not only when inducing the expression of heterogeneous protein in the microorganism, but also when the cytoplasmic protein is expressed [Cheng, Y. Y., Biochem. Biophys. Res. Commun., 111, 104-111 (1983); Bowen, G. A., J. Biol. Chem. 265, 16760-16766 (1990). It has also been reported that the formation of inclusion bodies can be increased by raising the culture temperature [Schein, C. H. and Noteborn, M., Bio / Technology, 6, 291-294 (1988); schein, C. H. Curr. Opinnion Biotechnol., 2, 746-750 (1991).

However, the protein expressed in the form of inclusion bodies in the cell does not have the original structure and at the same time loses its activity [Kiefhaber, T., Roudolph et al., Bio / Technology 9, 825-829 (1991)]. Purification of interferon beta 1b expressed in the form of intracellular inclusion bodies in bacteria inevitably involves dissolution of the inclusion bodies and the protein refolding process to achieve the three-dimensional structure of the protein. Complete dissolution of proteins affects refolding yield, so for complete dissolution, anions such as chaotropic reagents such as urea and guanidine-HCl and sodium dodecyl sulfate A large amount of anionic detergent is used. In order to refold the protein after dissolving the protein in the inclusion body form, the chaotropic agent and the surfactant, which are protein solubilizers, are removed. In this case, the non-glycosylated interferon is very unstable and precipitation occurs due to the hydrophobic aggregation. There is a problem. In many cases, a high concentration of SDS is used in the process of dissolving and purifying interferon beta in inclusion body form, but there is a limitation that the use of a purification column is extremely limited due to the SDS included in all processes during purification, and the removal of SDS is not easy. There is a disadvantage. To overcome this limitation, zwitterionic detergents have been used to increase solubility and to purify several types of columns simultaneously (DEAN RH, J. I & C. Res., 15: 31-37). (1995), while the limitation of column purification was overcomeable, there was a disadvantage in that the efficiency and purity of refolding were lowered.

In order to overcome these problems of refolding and purification, the present inventors adsorb the protein to the cation exchange resin, and in the state where the protein is adsorbed, lowers the concentration of the zwitterionic surfactant to reduce precipitation and further add an oxidizing agent. The present invention was completed by confirming that the refolding yield of the existing E. coli-derived interferon beta purification process was reduced and the limitations of the column process selection were simultaneously overcome and the problems of the existing process could be solved. .

It is an object of the present invention to provide an improved method for purifying interferon beta 1b which improves protein refolding yield required for purification of interferon beta 1b and overcomes the limitations of column purification.

As one aspect for achieving the above object, the present invention,

(a) disrupting cells containing recombinant interferon beta 1b to obtain inclusion bodies;

(b) washing the inclusion body obtained in step (a) with a buffer containing urea and dissolving it with dithiothreitol (DTT) and zwitterionic detergent;

(c) filtering the solution obtained in step (b) and then performing cation exchange chromatography to induce refolding;

(d) adding an oxidizing agent to the interferon beta lb fraction separated in step (c) to induce disulfide bonds to complete refolding; And

(e) performing a cation exchange column and gel filtration chromatography on the refolded interferon beta1b in step (d) to obtain a high purity interferon beta1b.

In the purification method according to the present invention, a sample used as a starting material may be produced by culturing genetically recombined cells. Since interferon beta 1b is expressed in inclusion bodies in such cells, the first step of the purification method according to the present invention is to disrupt these cells to obtain inclusion bodies. At this time, in order to crush the cells, any known cell crushing method can be used appropriately selected according to the conditions without limitation, and preferably cells can be crushed by ultrasound.

In the present invention, the inclusion body obtained by such a crushing step is then washed by the wash buffer, which wash buffer preferably comprises urea. After washing, the inclusion body is dissolved to obtain interferon beta in the inclusion body, and the solubilizer used here preferably comprises a dithiothreitol (DTT) and a zwitterionic detergent. The amphoteric ionic surfactant used in the present invention is not limited thereto, but is preferably selected from the group consisting of zwittergent3-08, zwittergent3-10, zwittergent3-12, zwittergent3-14 and zwittergent3-16, and more preferably. Is zwittergent3-14. Preferred amphoteric ionic surfactant concentrations are 1 to 5 (w / v)%, more preferably 2.5%. In the dissolution step, the pH is preferably 11.0 to 12.0 and the dissolution time is 1 to 10 minutes.

The dissolution process is terminated when the pH is lower than the above range, the preferred pH is about 9.0 pH. To lower the pH, but not limited thereto, preferably 1 M Tris-HCl, pH 8.0 buffer may be used.

After the process of dissolving the inclusion body is terminated, the supernatant is collected by centrifugation and filtered, and the chromatography is performed with the filtrate. Filtration can be carried out with an appropriate filter, preferably with a 0.2 μm filter.

In the purification method according to the present invention, the filtrate obtained in the filtration step is diluted and cation exchange chromatography is performed with this diluent to adsorb interferon beta 1b to the cation exchange resin. At this time, the interferon beta 1b is preferably diluted with a minimum dilution factor so that the cation exchange resin can be adsorbed well, and in one embodiment of the present invention, the dilution water is diluted 1 to 3 times with tertiary distilled water.

The functional group of the cation exchange chromatography used in the present invention is not only carboxymethyl (CM-) carboxy (C-), which is a weak cation, but also strong cation. Phosphorus sulfo (S-), sulfomethyl (SM-), sulfoethyl (SE-), sulfopropyl (SP-) and phospho (P-) can be used in various ways, and as a column resin, Sepharose ( Sepharose, Sephadex, Agarose, Sephacel, Sephacel, Polystyrene, Polyacrylate, Cellulose and Toyoperl can be used in various ways. have. In a preferred embodiment, the purification method of the present invention can generally use an SP column, for example, a column filled with an SP FF or an SP HP (GE Healthcare) resin.

In the present invention, cation exchange chromatography is preferably performed at pH 6.0 to 9.0, more preferably at pH 8.0. In this process, interferon beta 1b is adsorbed to the column, DTT is removed, and the adsorbed interferon beta 1b. Will lead to refolding.

In addition, the cation exchange column used in the cation exchange chromatography of the present invention may be equilibrated with a buffer prior to loading the eluate.

Although the equilibration buffer for balancing the cation exchange resin can be variously performed according to conditions, it is preferable to include an amphoteric ionic surfactant, and the concentration of the amphoteric ionic surfactant included in this case is preferably 0.01 ( w / v)% to 0.1 (w / v)%, more preferably 0.05 (w / v)%. More preferably, it is composed of 20 mM Tris-HCl with zwitterionic surfactant, 0.05% zwitterionic surfactant and buffer having a pH of 8.0.

After injecting and adsorbing an interferon beta solution into the cation exchange resin equilibrated by the buffer, the concentration of the amphoteric ionic surfactant can be lowered by flowing the equilibration buffer. Preferably, the equilibration buffer can be flowed in three column volumes. Adsorbed interferon 1b is eluted using a buffer containing NaCl and an amphoteric ionic surfactant. In a specific embodiment of the present invention, interferon is linearly flowed into a 10-fold column volume from 0 to 20% in a 20 mM Tris-HCl, pH 8.0, 1 M NaCl buffer containing 0.05% of an amphoteric ionic surfactant. Beta 1b eluted.

Interferon beta 1b expressed in inclusion bodies in bacteria by genetic engineering methods needs to be oxidized to refold its physiological activity, ie to induce disulfide bonds of interferon beta 1b to complete refolding. In the present invention, oxidation is induced by adding an oxidizing agent to the eluate of interferon beta obtained in the cation exchange chromatography step.

Oxidizing agents that can be used in the present invention O-iodosobenzoic acid, sodium peroxide, cystine, cystamine, oxidized glutathione (GSSG) , O-phenanthroline and CuCl ₂ Etc., but is not limited thereto. Preferably, O-iodosobenzoic acid may be used. The oxidant is preferably used in the same molar ratio of interferon beta 1b. In the oxidation process, the pH of the interferon beta 1b eluate may be preferably adjusted to pH 9.0, and NaOH or the like may be used to adjust the pH. The oxidation reaction time is preferably 6 hours to 24 hours.

In the purification method of the present invention, after the oxidation step, the eluate of interferon beta 1b can be purified by cation chromatography and gel filtration chromatography to finally obtain high purity interferon beta 1b having a very high refolding rate.

Cationic chromatography is performed in the same manner as in step (c), that is, by equilibrating the column and then performing cation chromatography to adsorb interferon beta to the column, and then adsorbed interferon beta 1b to NaCl and an amphoteric ionic interface. Elution is carried out using a buffer containing the active agent. In one specific embodiment, the oxidized interferon beta 1b solution was diluted 2.5-fold and injected into a 5 mL SP HP (Hitrap, GE Healthcare) column equilibrated with 20 mM Tris-HCl, pH 8.0, 0.05% zwitergent 3-14 buffer. Then interferon beta 1b was eluted by linear injection of 20 mM Tris-HCl, 0.05% zwitergent 3-14 detergent, 1 M NaCl buffer for 40 minutes from 0 to 40%.

After performing the cation chromatography, the purified purified interferon beta 1b can be finally obtained by chromatography on an equilibrated gel filtration column. Gel filtration chromatography can effectively remove endotoxins, various reagents and peptides, as well as other proteins having different molecular weights from interferon beta 1b. The gel resin may be Sephacryl S-100 (Sephacryl S-100), Sephacryl S-200 or Sephacryl S-300 (Pharmacia) and Superdex (GE Healthcare) 75, but are not limited thereto. But not preferably Superdex 75.

Purifying the recombinant interferon beta by a series of purification methods according to the present invention, it is possible to efficiently obtain the recombinant interferon beta 1b with very high activity and purity by overcoming the limitation of refolding yield reduction and column process selection at the same time .

In another embodiment, the present invention relates to high purity interferon beta 1b purified by the above purification method.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only for illustrating the present invention, but the scope of the present invention is not limited thereto.

Example  1. Human Interferon Beta 1b  Culture of Producing Bacterial Cells

Recombinant human interferon beta 1b production strain by recombinant DNA technology was inoculated in 500 ml of 2X LB (tryptone 2%, yeast extract 1%, sodium chloride 1%) to which ampicillin was added and shaken for 15 hours at 37 ° C. and 200 rpm. . This was again transferred to 500 ml of 2X LB (tryptone 2%, yeast extract 1%, sodium chloride 1%) with ampicillin and incubated at 600 nm for absorbance between 4.0 and 4.5 (about 3 hours) for seeding (Seed) for fermentation. Used as. Fermentation was carried out using MDL-8C 5L fermentor of Marubishi, batch fermentation (batch fermentation) was a temperature of 37 ℃, stirring speed 500rpm and oxygen 1vvm. The pH was adjusted to 6.90 and NSB was set to 0.2, so that the base solution was added at pH 6.70 or below.

After about 2.3 ~ 3 hours of fermentation progress, the feeding solution was added to the fed batch mode from the time of increase of pH, and proceeded for about 45 hours. In order to cultivate the strain at high cell density, the fermentation was carried out by raising the stirring speed to 600 rpm, and when the growth rate of the strain became more than 80 at 600 nm at about 20 hours after incubation, 0.5M IPTG (iso) Propyl-beta-D-thiogalactopyranoside) was added to the medium at a final concentration of 0.5 mM to induce the expression of the recombinant human interferon beta 1b gene of interest. About 45 hours after the fermentation progress, the cell culture was centrifuged at 700 rpm for 20 minutes using a beckman Aventi J25I centrifuge to obtain bacterial cell precipitates. The resulting cell precipitate of recombinant human interferon beta 1b was stored at -80 ° C until the experiment proceeded to the next step.

Example 2. Destruction of Cells and Protein Lysis

42 g of bacterial cells containing human interferon beta 1b obtained in Example 1 were added to 420 mL of buffer solution (50 mM Tris-HCl, pH 9.0, 1 mM EDTA, 200 mM NaCl, 0.5% Triton X-100). After suspension for an hour, ultrasonic cells were destroyed three times at about 30 minute intervals in an ice bath. Then, a precipitate was collected by centrifugation at 5,500 × g for 30 minutes using a high speed centrifuge (Beckman). The collected precipitate was washed in wash buffer (100 mM Tris-HCl, pH 8.0, 6 M urea and 25 mM dithiothreitol) and then centrifuged again at 5,500 × g. The precipitate was suspended in 80 mL of distilled water, and then DTT was added to 50 mM and stirred at room temperature for 1 hour. 5 M NaOH was added little by little to dissolve the protein to pH 12.0 and stirred for 1 minute. After the same volume of 5% zwitergent 3-14 detergent (Merck) solution was added and stirred again for 5 minutes. Then 1 M Tris-HCl, pH 8.0 solution was added quickly to pH 9.0. The dissolved protein solution was centrifuged at 10,000 × g for 30 minutes to collect only the supernatant, and the collected supernatant was filtered using a 0.2 μm filter.

Example  3. Purification by Cation Exchange Chromatography

The human interferon beta 1b solution obtained in Example 2 was diluted 2.5-fold with tertiary distilled water and then injected into an equilibrium SP HP column with 20 mM Tris-HCl, pH 8.0, 0.05% zwitergent 3-14 detergent buffer. The SP HP column was packed in a 50 mL volume into an XK50 / 25 (GE Healthcare) column and the flow rate was 8 mL / min. After interferon solution was injected into the SP HP column, interferon beta was eluted by linearly injecting 20 mM Tris-HCl, pH 8.0, 0.05% zwitergent, 1 M NaCl buffer from 0% to 20% for 60 minutes.

Example  4. Human Interferon Beta 1b  Oxidation and cation On the chromatogram  Purification by

To the interferon beta 1b solution obtained in Example 3, the protein molar ratio was added to the same oxidizing agent O-iodosobenzoic acid (Sigma) and then NaOH was added to pH 9.0. Then stirred at 4 ° C. for 24 h. 1/6 of the oxidized interferon beta solution was diluted 2.5-fold with 10 mM Tris-HCl, pH 9.0, 0.05% zwitergent 3-14 detergent buffer, followed by 20 mM Tris-HCl, pH 8.0, 0.05% zwitergent 3-14 detergent buffer. Into a 5 mL SP HP (Hitrap, GE Healthcare) column, equilibrated with 20 mM Tris-HCl, 0.05% zwitergent 3-14 detergent, 1 M NaCl buffer linearly from 0 to 40% for 40 minutes. Injection was performed to elute interferon beta 1b. At this time, the flow rate was 5 mL / min. The remaining interferon beta 1b solution was purified by SP HP in this manner.

Example  5. Purification by Gel Filtration Chromatography

The interferon beta 1b solution obtained in Example 4 was concentrated using a membrane having a MWCO of 10.000 (Vivaspin device, Sartorius, Inc.), followed by 50 mM potassium phosphate, pH 6.0, 100 mM NaCl, 0.05% zwitergent 3-14 detergent buffer. Interferon beta was purified by injection into a balanced Superdex 75 column (1.6 cm in diameter, 60 cm in length, GE Healthcare). At this time, the flow rate was 1.5 mL / min. Human interferon purified by gel filtration chromatography was analyzed by SDS-PAGE and shown in FIG. 2.

Example  6. Reverse  Analysis by High Pressure Liquid Chromatography

The interferon beta 1b solution obtained in Example 5 was injected into a C4 column at a flow rate of 1 mL / min, followed by 0.1% trifluoroacetic acid in 40% acetonitrile containing 0.1% trifluoroacetic acid. Analysis was carried out in a linear concentration gradient condition by 13-fold column volume up to 70% acetonitrile. The chromatogram analyzed by the above method is shown in FIG. 3.

Example  7. N-terminal amino acid analysis

The interferon beta 1b solution obtained in Example 5 was electrophoresed on an SDS-PAGE gel and then transferred to a PVDF membrane. The transmembrane was stained with Ponceau S solution and commissioned N-terminal amino acid analysis (5 amino acids) from Korea Research Institute of Basic Science (Seoul Branch). The results were analyzed by Betseron (human interferon beta 1b, Shering). The same result as the amino acid sequence was obtained.

Example  8. Interferon Beta 1b  In Vitro ( in - in vitro Activity measurement

As a method of measuring the potency and pharmacokinetic factors of the interferon beta 1b obtained in Example 5, a method of measuring in vitro cell activity was used. Normally, the in vitro activity of interferon beta is measured by WISH cells (ATCC) to proceed with a test for identifying the cytopathic effect. WISH cells were treated with concentrations of Hanmi interferon beta 1b and interferon beta commercially available substances to measure EC ₅₀ values of protection against Vesicular Stomatitis Virus (VSV) by interferon beta 1a (Avonex). To measure activity in vitro. Table 1 shows the in-vitro cell activity for each material. Herein, interferon beta 1b (Betaseron) and Hanmi-interferon beta 1b are expressed in% relative to interferon beta 1a (Avonex).

Test substance EC ₅₀ S.P (108 IU / mL) In vitro titer (%) Interferon beta 1a (Avonex) 6.13 1.63E + 08 100.0 Interferon beta 1b (Betaseron) 52.150 1.92E + 07 11.8 Hanmi-Interferon beta 1b 46.330 2.16E + 07 13.2

In the case of purifying the interferon beta 1b by the purification method according to the present invention, the refolding can be efficiently induced, the aggregation step of the purification step can be efficiently suppressed, and the purity is increased, and finally, the active type of interferon beta 1b of high purity is easily obtained. The effect can be obtained.

Figure 1 shows a step-by-step process flow chart of the purification method according to the present invention,

Figure 2 shows the interferon beta 1b purified by the purification method of the present invention by SDS-PAGE,

Where MW is the molecular weight label,

Lane 1 is interferon beta 1b purified through the purification method of the present invention,

Figure 3 is an analysis of the interferon beta 1b purified by the purification method of the present invention by C4 RP-HPLC.

Claims (7)

(a) disrupting cells containing recombinant interferon beta 1b to obtain inclusion bodies; (b) washing the inclusion body obtained in step (a) with a buffer containing urea and dissolving it with dithiothreitol (DTT) and zwitterionic detergent; (c) filtering the solution obtained in step (b) and then performing cation exchange chromatography to induce refolding; (d) adding an oxidizing agent to the interferon beta lb fraction separated in step (c) to induce disulfide bonds to complete refolding; And (e) purifying the interferon beta 1b comprising performing a cation exchange column and gel filtration chromatography on the refolded interferon beta 1b in step (d). The zwitterionic detergent of claim 1, wherein the zwitterionic detergent is selected from the group consisting of zwittergent3-08, zwittergent3-10, zwittergent3-12, zwittergent3-14, and zwittergent3-16. Purification method. The method of claim 1, wherein the oxidant is O-iodosobenzoic acid, sodium peroxide, cystine, cystamine, oxidized glutathione (GSSG) ), O-phenanthroline and CuCl ₂ Purification method selected from the group. The method of claim 1, wherein the resin of the cation exchange chromatography has a residue selected from the group consisting of CM (carboxymethyl), SP (sulfopropyl), and S (sulfo). The method of claim 1, wherein the gel filtration chromatography is selected from the group consisting of Sephacryl S200, Sephacryl S300, and Superdex 75. The method of claim 1, wherein the concentration of zwitterionic detergent is 0.01% to 0.1% (W / V) based on the total purification buffer. High purity recombinant interferon beta 1b purified by the method of claim 1.

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