RU2473696C1 - INDUSTRIAL METHOD OF PRODUCING AND PURIFYING RECOMBINANT HUMAN INTERFERON β-1b FROM INCLUSION BODIES - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: disclosed is a method of producing and purifying interferon β-1b. The present method of producing and purifying interferon β-1b involves washing inclusion bodies with 0.3% solution of trifluoroacetic acid in water, followed by centrifuging, extracting protein from the washed inclusion bodies with a solution containing 1-propanol and water in ratio of 1:1, with addition of 0.3% trifluoroacetic acid, purification by reversed-phase chromatography on a C₄ sorbent, refolding in a buffer solution containing 1% sodium laurate, 50 mM Na₂HPO₄ at pH=9.0 with addition of 10 mcM copper sulphate dissolved in ammonia, extracting interferon β-1b from the aqueous solution using 75% phenol solution in water and purification by reversed-phase chromatography on a C₁₈ sorbent. Use of 0.3% trifluoroacetic acid solution for washing inclusion bodies containing β-1b is also disclosed.

EFFECT: invention enables to obtain purified interferon β-1b on industrial scales in an effective amount.

3 cl, 2 dwg, 2 tbl, 3 ex

Description

The invention relates to biotechnology, in particular to an industrial method for producing recombinant human beta-interferon 1b from inclusion bodies and its purification.

Human interferon beta (INF-β) or fibroblast interferon is secreted mainly by fibroblasts and is a glycoprotein with a molecular weight of about 20 kDa. Its structure is referred to as INF-β-1a. The peptide portion of INF-β-1a is represented by 166 amino acid residues. The molecule contains a free sulfhydryl cysteine group at position Cys ¹⁶ and one intramolecular disulfide bond at position Cys ³⁰ -Cys ¹⁴⁰ .

Known INF-β-1a-based drugs are obtained by expression of a natural interferon gene (β in mammalian cells. Recombinant INF-β-1a is completely identical to natural fibroblast interferon [Mazdeh M., Afzali S., Jaafari MR. The therapeutic effect of Avonex, Rebif and Betaferon on EDSS and relapse in multiple sclerosis: a comparative study. ActaMedIran., 2010, Mar-Apr; V. 48 (2), p. 83-88].

Since glycosylation of a protein molecule slightly affects the biological activity of the protein [Plosker G.L. Interferon-β-1b: a review of its use in multiple sclerosis. CNS Drugs, 2011, Jan 1; V.25 (1), p.67-88], instead of the expensive expression in mammalian cells, you can use the expression in bacterial cells, which is more productive and less time consuming. Recombinant interferon beta, obtained in the bacterial system, is a non-glycosylated protein having a substitution of cysteine at position 16 for serine (Figure 1). This replacement eliminates the problem of dimer formation due to improper closure of disulfide bonds [Mark D.F., Lu S.D., Creasey A.A. Yamamoto R., Lin L.S. Site-specific mutagenesis of the human fibroblast interferon gene. Proc. Natl. Acad. Sci USA, 1984, Sep; 81 (18): 5662-6]. This structure of fibroblast interferon is called INF-β-1b.

This protein interacts with specific receptors located on the surface of human cells [Stark G.R., Kerr I.M., Williams B.R., Silver-man R.H., Schreiber R. D. How cells respond to interferons. Annual Review of Biochemistry, 1998, V. 67, p.227-264], inactivates receptors sensitive to γ-interferon, activates the function of T-suppressors, weakens the effect of antibodies to the main components of myelin, inhibits the replication of viruses [Fensteri V., Sen G .FROM. Interferons and viral infections. Biofactors, 2009, Jan-Feb; 35 (1): 14-20].

The main therapeutic effect of INF-β-1b is antiviral and immunomodulatory activity, a decrease in the frequency of exacerbations of multiple sclerosis with remitting course by one third and a decrease in the number of new foci of brain damage. In addition, the frequency of exacerbations in the secondary progressive course of multiple sclerosis decreases and the severity of cerebral atrophy decreases [Vartanian T. An examination of the results of the evidence, incoming, and phase III studies of interferon beta products in the treatment of multiple sclerosis. Clin. Ther., 2003, V.25 (1), p.105-118].

The creation of a producer strain of human interferon beta-1b made it possible to obtain a biologically active protein in an amount necessary for conducting various structural and functional studies, as well as for use in medical practice. Overseas drugs have been created based on recombinant interferon beta-1b (ecstavia, betaferon, etc.) to reduce the incidence of exacerbations of multiple sclerosis, slow the growth of disability, reduce the formation of new foci of brain damage and its atrophy, and also for the treatment of immune diseases.

In this regard, the task of developing effective and cost-effective methods for producing recombinant interferon beta-1b is relevant.

Known methods for producing recombinant interferon beta-1b, but they are all laborious, expensive, described for analytical amounts of protein, and their scaling is ineffective for the production of commercially available drugs [US patent No. 5066786, No. 4485017; Lin L. S, Yamamoto R., Drummond RJ. Purification of recombinant human interferon beta expressed in Escherichia coli. Methods Enzymol, 1986, V.119, p. 183-92, etc.]. All these methods use standard isolation schemes, which include solubilization in buffer systems containing chaotropic agents or ionic detergents in sufficiently high concentrations, which greatly complicate protein purification in subsequent stages.

A known method of purification of interferon beta (US patent No. 5814485), including multi-stage washing of inclusion bodies with urea and non-ionic detergents, as well as solubilization in sodium dodecyl sulfate (SDS). This method allows to obtain a protein with a yield of 0.2 g from 1 g of the target protein extracted from inclusion bodies. The biological activity of the drug in inhibiting the cytopathic effect is 32 × 10 ⁶ IU / mg protein. The disadvantages of this method is the use of multi-stage washing of inclusion bodies, which leads to significant loss of protein, as well as the use of SDS, which is undesirable, because can cause protein denaturation, which complicates subsequent chromatographic purification, and also requires additional complex analysis of the finished product for the content of SDS.

Closest to the claimed method is a method (RF patent No. 2392282), which allows to obtain an intact native recombinant interferon beta-1b, which includes several stages: washing the inclusion bodies with a 2% aqueous solution of zwittergent 3-14, solubilization in 7M guanidine chloride, conducting refolding by adding oxidized and reduced glutathione to the reaction mixture, protein purification using HyperD Ceramic S sorbent, Source S30 ion-exchange sorbent and subsequent chromatographic purification using exclusive gel filtration and gel Sephacryl S-200 HR. This method allows to obtain from 37.6 g of the target protein isolated from inclusion bodies, 3.76 g of recombinant interferon beta-1b with a HPLC purity of 96.5%. The biological activity of the drug in inhibiting the cytopathic effect is 32 × 10 ⁶ ME / mg protein.

The disadvantage of the prototype method is that the use of washing inclusion bodies leads to large losses of protein - up to 40% of the initial amount. Another disadvantage of this method is the need to concentrate the sample using ultrafiltration before the stages of chromatographic purification of the protein, which, when scaling the process, also leads to large losses of protein.

The technical result of the claimed invention is to simplify the technology for the preparation and purification of recombinant human interferon beta-1b.

The specified technical result is achieved by the implementation of the claimed method for the preparation and purification of recombinant human interferon beta-1b, which includes the following successive stages.

Recombinant human interferon beta-1b inclusion bodies are washed with a solution of 0.3% trifluoroacetic acid (TFA). The target protein is extracted with a solution of 1-propanol: water (1: 1) with the addition of 0.3% TFA.

Chromatographic purification of the extracted protein is carried out on a reversed-phase column packed with a YMC-GEL Butyl C ₄ sorbent (YMC, Japan), which is a silica gel with a grafted alkyl silyl group. Sorbed protein is eluted in a concentration gradient from 20 to 50% 1-propanol. The protein load on the sorbent can reach 40 mg of total protein per 1 ml of carrier. The fractions containing the target protein according to the results of analysis on a reversed-phase C ₄ column are combined.

Protein is precipitated from the fraction collected from the reversed-phase column by adding an equal volume of a buffer solution containing 50 mM Na ₂ HPO ₄ and adjusting the pH of the resulting solution to 6.0. The resulting precipitate was dissolved in 200 ml of a buffer solution containing 1% sodium laurate, 50 mm Na ₂ HPO ₄ , pH 7.0, add 10 μm copper sulfate, previously dissolved in ammonia.

The solution is then applied to a column packed with Sephadex G-25 Fine gel (JE Healthcare, USA). Protein is precipitated from the fraction collected from the column by adding sodium dihydrogen phosphate to a total concentration of the reagent in the solution of 50 mM and adjusting the pH of the solution to 6.0.

The resulting precipitate is homogenized in a buffer solution containing 100 mM NaH ₂ PO ₄ , pH 4.2, heated to 65 ° C, an equal volume of a 75% phenol solution preheated to 65 ° C in water is added, incubated for 20 minutes in a thermal shaker at 65 ° C. Then the reaction mixture is cooled to 10 ° C, achieving complete phase separation, and the aqueous phase is removed.

The resulting solution of protein in phenol was diluted 6.5 times with 30% ethanol, TFA was added to a concentration of 0.1% and applied to a reversed-phase column packed with a C ₁₈ sorbent (YMC, Japan). Sorbed proteins elute in a concentration gradient of 1-propanol (from 20 to 50%). The fractions containing the target protein according to the results of analysis on a reversed-phase C ₁₈ column are combined.

A solution of the desired protein is applied to a column packed with Sephadex G-25 Fine gel (JE Healthcare, USA). The concentration of the obtained protein fraction was determined by reverse phase chromatography on a C ₁₈ column.

The result is 0.3 g of recombinant interferon beta-1b with a HPLC purity of 96.5% from 1 g of the target protein obtained from inclusion bodies.

The claimed method has the following advantages.

- The extraction of interferon beta-1b from inclusion bodies in a solution of 50% 1-propanol with the addition of 0.3% trifluoroacetic acid eliminates the steps of washing inclusion bodies with various buffer solutions containing detergents and chaotropic agents, and prolonged centrifugation. This approach helps to significantly reduce protein loss. This extraction allows you to selectively extract the target protein by 75-80%.

- Refolding in a buffer solution of sodium phosphate, sodium laurate and copper sulfate simplifies further protein purification, since sodium laurate is easily removed from the solution by gel filtration on Sephadex G-25 at alkaline pH values.

- The use of copper sulfate, dissolved in ammonia, allows the process of protein oxidation within an hour.

- All stages of chromatographic purification do not require protein concentration.

- Purification of the protein from endotoxins, DNA of the producer strain and proteins of the producer strain is carried out in one stage by extracting the protein from an aqueous solution of a 75% solution of phenol in water. This type of purification allows you to completely remove bacterial endotoxins (to a concentration of less than 1 UE / ml), proteins of the producer strain (to a concentration of less than 0.03 ng / μg interferon beta 1b) and DNA of the producer strain (to a level of not more than 40 pg / mg) .

- The use of phenolic extraction of protein from the aqueous phase with subsequent purification on a reversed-phase sorbent allows you to completely get rid of sodium laurate.

Highly purified protein INF-β-1b, obtained as a result of the application of the claimed method, can be used for the production of drugs in the medical industry.

Brief Description of the Figures

1. Figure 1 is a graphical representation of the primary amino acid sequence of recombinant human interferon-beta 1b. The thickened solid line shows the disulfide bond between Cys ³⁰ and Cys ¹⁴⁰ . The gray color indicates the position of replacing Cys ¹⁶ with Ser ¹⁶ .

Figure 2 presents a graphical image of the HPLC analysis of the main samples taken at each stage of the purification process of interferon β-1b from inclusion bodies:

a - 1-stage: extraction of IFN-β-1b from TB 50% 1-propanol in 0.3% TFA;

b - stage 2: chromatographic purification of IFN-β-1b on an RP sorbent C ₄ 300Å;

in stage 3: the dissolution of IFN-β-1b in a buffer solution containing 1% sodium laurate, 50 mm Na ₂ HPO ₄ at pH 9.0;

d - stage 4: renaturation of IFN-β-1b in a buffer solution containing 1% sodium laurate, 50 mm Na ₂ HPO ₄ at pH 9.0, in the presence of 10 μm copper sulfate, previously dissolved in 30% ammonia;

d - stage 5: purification of IFN-β-1b phenol;

e - stage 6: chromatographic purification of IFN-β-1b on an RP sorbent C ₁₈ 300 Å;

1 - reduced IFN-β-1b; 2 - native IFN-β-1b; 3 - phenol.

The invention is illustrated by the following examples.

Example 1. Obtaining highly purified recombinant human interferon beta-1b.

To 150 inclusion bodies of recombinant human interferon beta-1b, 1000 ml of a solution is added which contains 0.3% trifluoroacetic acid (TFA). After that, inclusion bodies are homogenized in a blender at a speed of 800 rpm for 1 min, transferred to a beaker with a magnetic stirrer and left to mix for 1 h at 4 ° C. Next, the resulting suspension is centrifuged at 4 ° C for 40 minutes at a speed of 10,000 rpm. The resulting precipitate is transferred to a blender, pour 1.0 l of a solution of 1-propanol: water (1: 1) with the addition of 0.3% TFA, homogenize at a speed of 800 rpm for 1 minute, cool the solution at -20 ° C in for 20-30 minutes and set to mix at 4 ° C for 1-2 hours.

The extract obtained is centrifuged at 4 ° C for 40 minutes at a speed of 10,000 rpm. Then the resulting supernatant is filtered through glass fiber and diluted with a solution containing 0.3% TFA to a concentration of 1-propanol 20%.

In the next step, the protein extract is applied to a DAU-100 reverse-phase column (column size 10 × 10 cm) (YMC Co., LTD, Japan), packed with GEL-Butyl C ₄ sorbent (YMC Co., LTD, Japan) and balanced a solution containing 0.1% TFA and 20% 1-propanol. The load on the sorbent can reach 40 mg of total protein per 1 ml of carrier. After applying the protein solution, the column is washed with an initial solution of TFA and 1-propanol until the spectrophotometer shows values equal to the values of the baseline at a wavelength of 280 nm. Sorbed proteins elute in a concentration gradient of 1-propanol (from 20 to 50%) at a wavelength of 280 nm. The fractions containing the target protein according to the results of analysis on a reversed-phase C ₄ column are combined. Protein is precipitated from the combined fraction collected from the reversed-phase column by adding an equal volume of a buffer solution containing 50 mM Na ₂ НРО ₄ , and adjusting the pH of the resulting solution to 6.0.

The protein suspension is then centrifuged using a centrifuge at 4 ° C for 30 minutes at a speed of 14,000 rpm. The resulting precipitate was stirred in 400 ml of the same buffer solution and centrifuged again under similar conditions.

Next, the precipitate was dissolved in 200 ml of a buffer solution containing 1% sodium laurate, 50 mM Na ₂ HPO ₄ , pH 7.0, 10 μM copper sulfate, previously dissolved in ammonia, was added and allowed to mix for 1 h.

Then, the solution was applied to a BPG 200/500 column (column size 20 × 30 cm) (JE Healthcare, USA), packed with Sephadex G-25 Fine gel (JE Healthcare, USA) and balanced with 10 mM NaOH solution. Elution is carried out in isocratic mode at a wavelength of 280 nm Protein is precipitated from the fraction collected from the column by adding sodium dihydrogen phosphate to a total reagent concentration of 50 mM in the solution and adjusting the pH of the solution to 6.0 and centrifuging the protein suspension using a centrifuge at 4 ° C for 30 minutes at a speed of 14000 rpm .

The resulting precipitate is homogenized in a buffer solution containing 100 mM NaH ₂ PO ₄ , pH 4.2 and heated to 65 ° C. An equal volume of a 75% phenol solution in water preheated to 65 ° C is added. Spend incubation for 20 minutes in a thermal shaker at 65 ° C. Then the reaction mixture is cooled to 10 ° C, achieving complete phase separation, and the aqueous phase is removed using a water-jet pump.

The resulting solution of protein in phenol was diluted 6.5 times with 30% ethanol, TFA was added to 0.1% and applied to a DAU-50 reverse-phase column (column size 5 × 20 cm) (YMC Co., LTD, Japan), packed sorbent C ₁₈ (YMC Co., LTD, Japan) and a balanced solution containing 0.1% TFA and 20% 1-propanol. The load on the sorbent can reach 40 mg of total protein per 1 ml of carrier. After applying the protein solution, the column is washed with an initial solution of TFA and 1-propanol until the spectrophotometer shows values equal to the values of the baseline at a wavelength of 280 nm. Sorbed proteins elute in a concentration gradient of 1-propanol (from 20 to 50%) at a wavelength of 280 nm. The fractions containing the target protein according to the results of analysis on a reversed-phase column With ₁₈ are combined.

The target protein solution was applied to a BPG 200/500 column (column size 20 × 30 cm) (JE Healthcare, USA), packed with Sephadex G-25 Fine gel (JE Healthcare, USA) and equilibrated with 10 mM NaOH. Elution is carried out in isocratic mode at a wavelength of 280 nm. The concentration of the obtained protein fraction was determined by reverse phase chromatography on a C ₁₈ column.

The yield of the finished product with 1 g of the target protein obtained by extraction at the first stage from inclusion bodies is 0.3 g of the target protein. Protein purity - not less than 98%. The material balance of the industrial process for the purification of interferon-beta 1b is presented in Table 1.

Table 1 The material balance of the main stages of the industrial purification process IFN-β-1b. CLEANING STAGES IFN-β-1b OUTPUT IFN-β-1b, g OUTPUT IFN-β-1b,% Chromatographic purity IFN-β-1b,% CONTENT Protein E. coli, ng / mg _protein Endotoxins, U / mg _protein Protein extraction from 150 g inclusion bodies 5 one hundred 60-70 > 100 > 250 HPLC C ₄ , YMC four 80 65-75 90-100 50-250 Dissolution of precipitated protein in 1% sodium laurate four 80 Renaturation 2,8 70 Phenol Purification 2,5 fifty 70-75 20-60 2-5 HPLC C ₁₈ , YMC 1.4 28 96-98 5-10 ≤1

Example 2. Analysis of the recombinant protein interferon-beta 1b

The analysis of the obtained interferon-beta 1b is carried out using reverse phase chromatography in a concentration gradient of acetonitrile (from 40 to 70%) in the presence of 0.1% TFA on a C ₄ column, with a sorbent particle size of 5 μm and a sorbent pore size of 300 A (YMC Co ., LTD, Japan). The product elutes at one peak, the purity of the product is at least 98% (figure 2).

The protein gives a positive reaction with monoclonal antibodies to interferon-beta 1b during enzyme-linked immunosorbent assay.

The specific biological activity of interferon-beta 1b was determined in a standard way by the ability to suppress cell division of the Daudi line (human Berkit lymphoma), determined by the change in the intensity of staining of the contents of the plate wells after treatment with a chromogenic substrate. For this, the Daudi cell line is cultured in suspension on medium D, which contains 224 ml of nutrient medium RPMI-1640 (PanEco), 25 ml of fetal calf serum (Highclon, USA), 2.5 ml of glutamine solution (PanEco) and 0.25 ml of a solution of gentamicin, in plastic bottles with an area of 25 cm ² , in an incubator filled with carbon dioxide in a volume of 5%, at a temperature of 37 ± 2 ° C and relative humidity ≥90%. The density of the cell suspension should be in the range of 0.6-1 million cells per ml.

Then, 100 μl of medium D was added to the wells of a 96-well plate, except for the 1st column. Then, 200 μl of the standard solution are added to the four upper wells of the first column (A1 - D1), and 200 μl of the test solution to the four lower wells (E1 - H1).

Next, prepare a series of consecutive twofold dilutions of the standard and test solutions. To do this, transfer 100 μl from each well of the first column to the corresponding wells of the second column (A1 → A2, B1 → B2, H1 → H2), after pipetting, repeat the procedure until the 11th column, from which 100 μl are removed and removed . The last 12th column contains medium without interferon.

After that, 100 μl of a suspension of Daudi cells (300 thousand cells / ml of medium) are poured into all wells of the plate and incubated for 72 hours in a CO ₂ incubator filled with carbon dioxide in a volume of 5% at a temperature of 37 ± 2 ° C and relative humidity ≥90%. Then, 20 μl of AlamarBlu solution (Biosource, USA, Cat. No. DAL1025 or similar quality) was added to each well and incubated for 18 hours under the conditions described above. Interferon inhibits cell proliferation, respectively, the higher its concentration, the less colored the suspension of Daudi cells. The color change is evaluated spectrophotometrically at wavelengths of 530 and 595 nm.

The data obtained are analyzed by the equivalent dose method (GF XI, issue 1, p. 226). The specific biological activity of interferon beta 1b was 95-105% compared to the international standard sample of interferon beta 1b (Interferon Beta Ser 17 Mutein, Human, rDNA derived, NIBC code: 00/574), which is 32 × 10 ⁶ ME / mg

Example 3. Scaling of the purification process of interferon beta-1b.

table 2 CLEANING STAGES IFN-β-1b OUTPUT IFN-β-1b, g OUTPUT IFN-β-1b,% Chromatographic purity IFN-β-1b,% CONTENT Protein E. coli, ng / mg _protein Endotoxins, U / mg _protein Protein extraction from 1500 g inclusion bodies 45-55 one hundred 60-70 > 100 > 250 HPLC C ₄ , YMC 35-40 70-80 65-75 90-100 50-250 Dissolution of precipitated protein in 1% sodium laurate 32-35 64-70 Renaturation 22-26 44-52 Phenol Purification 19-21 38-42 70-75 20-60 2-5 HPLC C ₁₈ , YMC 12.5-14 25-28 96-98 5-10 ≤1

The data shown in Table 2 were obtained on 10 pilot series of the drug.

Claims (3)

1. The method of obtaining and purification of interferon β-1b, including:
1) washing the inclusion bodies with a solution of 0.3% trifluoroacetic acid in water, followed by centrifugation;
2) protein extraction from the washed inclusion bodies with a solution of 1-propanol: water with the addition of trifluoroacetic acid;
3) purification using reverse phase chromatography on a sorbent With ₄ ;
4) refolding in a buffer solution of sodium hydrogen phosphate with the addition of copper salts and sodium laurate;
5) extraction of interferon β-1b from an aqueous solution with phenol;
6) purification using reverse phase chromatography on a sorbent With ₁₈ . 2. A method of obtaining and purifying interferon β-1b, including:
1) washing the inclusion bodies with a 0.3% solution of trifluoroacetic acid in water, followed by centrifugation;
2) protein extraction from the washed inclusion bodies with a solution of 1-propanol: water (1: 1) with the addition of 0.3% trifluoroacetic acid;
3) purification using reverse phase chromatography on a sorbent C ₄ ;
4) refolding in a buffer solution containing 1% sodium laurate, 50 mm Na ₂ HPO ₄ at pH = 9.0 with the addition of 10 μm copper sulfate dissolved in ammonia;
5) extraction of interferon β-1b from an aqueous solution using a 75% solution of phenol in water;
6) purification using reverse phase chromatography on a sorbent C ₁₈ . 3. The use of a 0.3% solution of trifluoroacetic acid for washing inclusion bodies containing interferon β-1b.

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