Method for purification of recombinant interferon β-1b

FIELD: medicine.

SUBSTANCE: proposed method of purification of human recombinant interferon beta-1b (rIFN beta-1b) provides for optimisation of processes of purification of inclusion bodies, their dissolution and refolding of target protein Purification of human rIFN beta-1b is conducted with zwittehent 3-14 as a main detergent for purification and dissolving of inclusion body of Ecoli with the following refolding of rIFN beta-1b. After that prechromatographic processing of a sample is conducted and after that chromatographic processing of Ceramic S and of Source S30 and oxidation using a mixture of cysteamine/cystamine are performed. Then gel-filtration on Superdex 75 Prep grade, chromatographic processing on Source Q30, gel-filtration on Sephacril S200HR are made and finally the effectiveness of purification of the target protein by high performance liquid chromatography is monitored.

EFFECT: raise of the target protein yields and obtaining human rIFN beta-1b which meets the requirements of normative documents with high levels of specific antiviral activity.

5 tbl, 3 ex

 

The invention relates to biotechnology, in particular to a technology for production of recombinant interferon beta-1b of human rights, in particular to a method of cleaning it.

Interferon (IFN) - heterogeneous glycoproteins of the immune system with antiviral, antitumor and immunomodulatory effects and different source of cellular products and manifestations of functional activity. IFN beta from the very first steps of its implementation recombinant drugs was intended mainly for anticancer therapy [S. Angelucci et al. Recombinant human IFN-beta affects androgen receptor level, neuroendocrine differentiation, cell adhesion, and motility in prostate cancer cells. J. Interferon Cytokine Res., 2007, 27(8): 643-52; S. Kohashi et al. Interferon-beta inhibits liver metastases from murine colon 26 carcinoma and its highly metastatic variant. Surg. Today, 2007, 37(6): 474-481]. In addition, preparations of IFN beta in demand for the treatment of viral infections, in particular hepatitis C, vesicular stomatitis and other [I.M. Pedersen et al. Interferon modulation of cellular microRNAs as an antiviral mechanism. Nature, 2007, 449 (7164): 919-22; J. Feher, G. Lengyel. Interferon in the treatment of viral hepatitis. The interferon was discovered 50 years ago. Comander. Hetil., 2007, 148(33): 1539-43; M.D. Trottier, D.S. Lyles, C.S. Reiss. Peripheral, but not central nervous system, type I interferon expression in mice in response to intranasal vesicular stomatitis virus infection. J. Neurovirol., 2007, 13(5): 433-445]. In recent years, the main direction of application of rhIFN beta-1b steel neurological diseases, primarily multiple sclerosis and the other demyelinating diseases of the nervous system, ankylosing spondylitis [M Kremenchutzky, S. Morrow, C. Rush. The safety and efficacy of IFN-beta products for the treatment of multiple sclerosis. Expert Opin. Drug Saf., 2007, 6(3): 279-88; S. Pay et al. Dendritic cell subsets and type I interferon system in Behçet''s disease: does functional abnormality in plasmacytoid dendritic cells contribute to Th1 polarization? Clin. Exp. Rheumatol., 2007, 25 (4 Suppl 45): S34-40]. Marked clinical efficacy of IFN beta-1b in the treatment steroidology.com ulcerative colitis, glomerulonephritis, chronic pancreatitis [S.C. Satchell et al. Interferon-beta Reduces Proteinuria in Experimental Glomerulonephritis. J. Am. Soc. Nephrol., 2007, 18(11): 2875-84; R. Talukdar, R.K. Tandon. Pancreatic stellate cells: a New target in the treatment of chronic pancreatitis. J. Gastroenterol. Hepatol., 2007].

Among the preparations of IFN beta the highest recognition in the global pharmaceutical market has earned recombinant IFN beta-1b, or Betaferon (Schering AG, Germany), and to a lesser extent IFN beta-1A, in particular Avonex (Gedeon Richter, Hungary). In addition to the pharmacological action of each of these drugs, having their their pharmacodynamics and indications for a patient, it should be noted that the drug interferon beta-1b has certain advantages, which identified him greater demand. When assessing these benefits, the researchers note, in particular, its failure, in contrast to IFN beta-1A, to the induction of neutralizing antibodies [R. Barbero et al. Every-other-day interferon beta-1b versus once-weekly interferon beta-1a for multiple sclerosis (INCOMIN Trial) II: analysis of MRI responses to treatment and correlation with Nab. Mult. Scler., 2006, 12(1): 72-76], less expressed the over pain and local reactions at injection interferon beta-1b [K. Baum et al. Comparison of injection site pain and injection site reactions in relapsing-remitting multiple sclerosis patients treated with interferon beta-1a or 1b. Mult. Scler., 2007, 13(9): 1153-1160], as well as differences in technologies for these products: recombinant IFN beta-1A get on the ovary cells of Chinese hamsters, i.e. in eukaryotic system, and recombinant IFN beta-1b can be obtained in prokaryotic system using the producer strain E. coli [by McCormick et al. Human interferon-.beta. (IFN-.beta.) produced in Chinese hamster ovary (CHO) cells. United States Patent 5,795,779 August 18, 1998; A. Whitty et al. Interferon-beta fusion proteins and uses. United States Patent 6,800,735, October 5, 2004].

Technology of obtaining and purification β-1b devoted a lot of patent research, including: US Patent 5,795,779, August 18, 1998 (McCormick et al. Human interferon-.beta. (IFN-.beta.) produced in Chinese hamster ovary (CHO) cells); US Patent 6,323,006, November 27, 2001 (Peregrino Ferreira et al. Recombinant human beta-CIS interferon); US Patent 4,894,330, January 16, 1990 (Hershenson et al. Purification of recombinant beta-interferon incorporating RP-HPLC); US Patent 5,004,605, April 2, 1991 (Hershenson et al. Low pH pharmaceutical compositions of recombinant .beta.-interferon); US Patent 6,923,956, August 2, 2005 (Tschope et al. Liquid interferon-.beta. formulations); RU 2006 123 539 (Park JI-Sook and other Method of purification of interferon beta).

A method of obtaining recombinant interferon beta-1b (IFN beta-1b) man in the insoluble form as inclusion bodies (U.S. Pat. Of the Russian Federation No. 2261913, IPC C12N 15/22, publ. 2005)

Compared with the soluble form of the protein expression in the form of inclusion bodies has a number of advantages - insoluble aggregates be the ka show no toxic effects, practically not attacked by proteases with high output are distinguished by centrifugation. At the same time, there are problems of low output active, correctly folded protein after conducting refolding in the process of cleaning rhIFN beta-1b, which is determined by the spatial structure of the latter [M. Karpusas et al. The crystal structure of human interferon b at 2.2-Å resolution. // Proc. Natl. Acad. Sci. USA. - 1997. Vol.94. P.1 1813-11818].

Known purification method of rhIFN beta-1b and the use of the detergent sodium dodecyl sulphate when it is received in the insoluble form (U.S. Patent 4,530,787, 1985).

However, the relatively low toxicity of this drug using sodium dodecyl sulfate process involves a number of obstacles: this connection causes significant denaturation of the target protein, it cannot be removed from the solution, which in turn makes it impossible for subsequent ion-exchange chromatography, necessary for the further purification process of rhIFN beta-1b.

Known purification method of rhIFN beta-1b, which avoids the aforementioned disadvantages of the use as a detergent of twitterget 3-14, which has no denaturing properties, does not form ionic bonds, is pH-independent, can be removed from the solution, makes it possible to ion-exchange chromatography [Russell-Harde D., M. Knauf, E. Croze The use of Zwittergent 3-14 in the purification of recombinant huan interferon-beta Serl7 (Betaseron). // J. Interferon Cytokine Res. - 1995. - Vol.15(1). - P.31-37].

Known closest to the claimed method for the cleaning of rhIFN beta-1b of the soluble using affinity chromatography, cation exchange chromatography and diafiltration (RF Application No. 2006123543. "Method of purification of interferon beta.

However, this method does not take into account the possibility of obtaining these drugs from the inclusion bodies of E. coli and is not described related features clean it.

The invention solves the problem of optimizing the conditions for the cleaning of rhIFN beta-1b on the stages of washing and dissolution of the inclusion bodies of E. coli producing recombinant interferon beta-1b person, in the process of refolding of the target protein, as well as the oxidation of the latter.

The inventive method of purification of recombinant interferon beta-1b from inclusion bodies of E. coli involves the following basic stages: (1) washing of inclusion bodies; (2) dissolution of inclusion bodies; (3) refolding of the target protein; (4) programehrvatska sample preparation; (5) chromatography on Ceramic S; (6) chromatography on Source S30; (7) oxidation; (8) gel filtration on Superdex 75 Prep grade; (9) chromatography on Source Q30; (10) gel-filtration on Sephacril S200HR; (11) control the efficiency of purification of the target protein by high-performance liquid chromatography (HPLC) with the following standard definition of its protover the red activity.

Distinctive features of the proposed method are:

during the washing of the bodies include a portion of unfrozen bodies include resuspendable in 10 ml of 2% aqueous solution of twitterget 3-14;

the solution for refolding of the target protein contains 20mm Tris-HCl, pH 9.0, 0.2% polyethylene glycol (PEG) 3550, 300 mm NaCl, 2 mm reduced glutathione (GSH), 1 mm oxidized glutathione (GSSG), 2 mm ethylenediaminetetraacetic acid (EDTA), 0.05% twitterget 3-14;

during prednamerennoe sample preparation use the buffer B of the following composition: 25 mm Tris-HCl, pH 8.0, 0.05% twitterget 3-14, 0.5 mm phenylmethanesulfonyl-fluoride (PMSF);

in the process of chromatography use buffers: (1) 25 mm Tris-HCl, pH 8.0, 0.05% twitterget 3-14, 0.5 mm PMSF; (2) 25 mm Tris-HCl, pH 8.0, 50 mm NaCl, 0.05% twitterget 3-14, 0.5 mm PMSF; (3) 25 mm Tris-HCl, pH 8.0, 1 M NaCl, 0.05% Twitterget 3-14, 0.5 mm PMSF;

for oxidation of the target protein using a mixture of group probably facilitates/tsistamin concentration of 1 mm/0.1 mm.

Control of the output and activity of recombinant interferon beta-1b after cleaning showed the following criteria: output 10%, multimer by gel filtration <5%, related proteins by reversed-phase chromatography <5%, related proteins by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (LTOs-page) <5%, proteins producer strain by enzyme-linked immunosorbent analysis (ELISA) &l; 5 ng/mg endotoxin by LAL-test (test using Limulus Amebocite Lisate" - lysate of cells crab horseshoe crabs) <5 in this case/mg (equivalent units of 1 milligram), the specific activity of interferon beta-1b 32 million IU/mg (International Units per 1 milligram).

The invention is illustrated by the examples.

Example 1. Optimization of refolding and oxidation of recombinant interferon beta-1b from inclusion bodies of E. coli using twitterget 3-14

The feasibility of using twitterget 3-14 in the process of dissolution and refolding of recombinant interferon beta-1b from inclusion bodies of E. coli required optimization of conditions for their implementation. With this purpose were developed semi-empirical formulas, and they made selection of the optimum ratio of ingredients in the process of refolding of the target protein and its oxidation of the expected range of values, as presented in tables 1-2 and formulas 1-2.

Table 1
Determination of the total correlation parameters ingredients in the process of refolding of IFN beta-1b
VariableDescriptionThe upper valueThe lower the value of
ApH7,59,0
BThe peg 35500%0,1%
CUrea0 M
DNaCl50 mm300 mm
EThe group probably facilitates/tsistamin1 mm/0.3 mm1 mm/0.1 mm
FTwitterget 3-1425%
GTemperature10°C

Formula 1. The total value of the parameters ingredients with the designation of their interdependence:

Table 2
Determining the ratio of the parameters of the ingredients in the oxidation process IFN beta-1b
Variable DescriptionThe upper valueThe lower the value of
ApH7,59,0
BThe peg 35500%0,1%
DNaCl50 Mm300 Mm
EThe group probably facilitates/tsistamin1 Mm/0.3 Mm1 Mm/0.1 Mm

Formula 2. The ratio of the isolated group of parameters with indication of their interdependence:

In the end, were established following the final execution options refolding and oxidation of IFN beta-1b:

- Twitterget 3-14: 0,05%;

- Tris-HCl 10 mm pH 7.5;

- Temperature: +10°C;

Nacl: 50 mm;

- EDTA-Na: 1 mm;

The group probably facilitates/tsistamin: 1 mm/0.1 mm.

Compliance with these conditions allowed us to achieve a 70% yield of the target protein after refolding and 10% of output after a full cleaning cycle.

Example 2. Purification of recombinant interferon beta-1b from inclusion bodies of E. coli

Washing, dissolution and refolding of inclusion bodies. P is acesse dissolution and refolding of inclusion bodies was used buffer And the following composition: 7 M guanidine chloride, 20 mm dithiothreitol (DTT), 0.5 mm PMSF.

A portion of unfrozen bodies inclusion (500 mg) resuspendable in 10 ml of 2% aqueous solution of twitterget 3-14, the suspension was stirred for 10-15 min at room temperature and centrifuged at acceleration 500g and 4°C for 15 minutes. The supernatant was removed and repeated the procedure centrifugation. Exit by squirrel at this stage ~50-60%.

250 mg laundered Taurus enable resuspendable in 2.5 ml of buffer And the suspension was incubated for 2 hours at 60°C. Nerastvorim material was removed by centrifugation with acceleration 12000g at 4°C, 10 minutes

Preparing solution for refolding (indicated final concentration of the components, the final volume of 50 ml of 20 mm Tris-HCl, pH 9.0, 0.2% of PEG 3550, 300 mm NaCl, 2 mm reduced glutathione (GSH), 1 mm oxidized glutathione (GSSG), 2 mm EDTA, 0.05% twitterget 3-14 and was added dissolved in the buffer And the inclusion body. The resulting solution was mixed and incubated at 10°C during the night.

Prednamerennoe sample preparation. We used the buffer B of the following composition: 25 mm Tris-HCl, pH 8.0, 0.05% twitterget 3-14, 0.5 mm PMSF.

Obtained in solution, the sample was diluted with buffer B 6 times, the precipitation was removed by centrifugation (14000g, 20 min, 4°C). The supernatant was brought to pH and conductivity to the corresponding values of buffer B, after which it was concentrated to recognize the aqueous volume by ultrafiltration Amicon cell (membrane PM-10) and then centrifuged (14000g, 10 min, 4°C).

Chromatographic purification of IFN beta-1b, its oxidation and Gely-filtrate. For the stability of the treatment process at this stage was used buffers: B - 25 mm Tris-HCl, pH 8.0, 0.05% twitterget 3-14, 0.5 mm PMSF; 25 mm Tris-HCl, pH 7.5, 50 mm NaCl, 0.05% twitterget 3-14, 0.5 mm PMSF; G - 25 mm Tris-HCl, pH 7.5, 1 M NaCl, 0.05% twitterget 3-14, 0.5 mm PMSF.

The prepared sample is dissolved inclusion bodies were injected (3 ml/min) column Ceramic HyperD S (Ciphergen, 10/100 mm, maximum pressure of 30 ATM), equilibrated with 10 volumes of buffer B. the Optical density of the eluate was monitored spectrophotometrically at a wavelength of 280 nm. The breakthrough volume of the applied sample (50 ml) were collected and bound peroxidase impurity substances were removed by washing 3 volumes of buffer, the Column was regenerated in 1 volume of 100 mm NaOH and balanced starting buffer (B).

A portion of the sample (10 ml) was applied on the column Source S30 (10/100 mm, maximum pressure of 30 ATM, 2.5 ml/min), equilibrated with 10 volumes of buffer B, collecting the breakthrough. The column was washed 2-5 volumes of buffer B and was suirable bound peroxidase material concentration gradient of NaCl (0-500 mm, 5 volumes column, 3 ml/min). The optical density of the eluate was monitored spectrophotometrically at a wavelength of 280 nm. Collected fraction, published in 150-200 mm NaCl. Before applying the next portion of the sample, the column was regenerated in 1 volume of 100 mm NaOH and balanced starting b is from (B). The procedure was repeated for the joint penetrations with column Source S30.

The collected fractions of rhIFN beta-1b were combined and the resulting solution was added to the group probably facilitates/tsistamin concentrations up to 1 mm/0.1 mm. The sample was incubated over night at 4°C, centrifuged (12000g, 4°C, 10 min) and the supernatant was concentrated to 1.5 ml by ultrafiltration Amicon cell (membrane PM-10).

A sample of oxidized IFN beta-1b (1.5 ml) was injected on the column Superdex 75 Prep grade (16/500 mm, 13000 TT/m, the maximum pressure of 5 ATM), equilibrated with buffer In the Division fought in the buffer B, at a speed of 30 cm/h, the Optical density of the eluate was monitored spectrophotometrically at a wavelength of 280 nm (nanometers). Collected the main fraction (retention time 39,25 min).

A sample of rhIFN beta-1b was applied on the column Source Q30 (10/100 mm, maximum pressure of 30 ATM (atmospheres), 2.5 ml/min), equilibrated with 10 volumes of buffer, collecting the breakthrough. The column was washed 2-5 volumes of buffer and suirable bound peroxidase material concentration gradient of NaCl (50-1000 mm, 10 volumes of column, 4 ml/min). The optical density of the eluate was monitored spectrophotometrically at a wavelength of 280 nm. Collected fraction, published in 200-300 mm NaCl. The column was regenerated by washing in 1 volume of 100 mm HCl. The collected fraction was concentrated to 1 ml by ultrafiltration Amicon cell (membrane PM-10).

Sample β-1b (1 ml) was injected on the Alonso Sephacryl S200HR (16/600 mm, >5000 TT/m, the maximum pressure of 1.5 ATM), equilibrated with buffer C. the Division fought in the buffer at a speed of 15 cm/h, the Optical density of the eluate was monitored spectrophotometrically at a wavelength of 280 nm. Collected the main fraction (retention time to 95.3 min).

Example 3. Monitoring the effectiveness of the method of purification of recombinant interferon β-1b from inclusion bodies of E. coli

The effectiveness of the proposed method of cleaning β-1b was controlled in the process of obtaining 11 batches of sample preparation and subsequent determination of their antiviral activity. Protocols obtain Dauch pilot batches are presented in tables 3 and 4.

Upon receipt of an experimental batch of S4/2401 number of inclusion bodies of E. coli to the washing was 500 mg, after washing - 230 mg. Final content of rhIFN beta-1b 1.88 mg (10%).

In the process of obtaining an experimental batch of S4/1301 number of inclusion bodies of E. coli to the washing was 1000 mg, after washing - 460 mg. Final content of rhIFN beta-1b was 3,76 mg (10%).

Assessment of biological (anti-virus) activity 11 samples of recombinant IFN beta-1b was performed in diploid culture of human fibroblasts M27 (Institute of poliomyelitis and viral encephalitides. Mphangwe RAMS). As a test virus used virus encephalomyocarditis mice (EMC) in an amount of 100 CTD50(cytotoxic dose, call the living cytotoxic effect in 50% of samples of cell cultures). Per unit activity of IFN took the reciprocal of its breeding, in which there is a 50% protection of cells from cytodestructive action caused by the replication of test virus. Control was a sample of drug Betaferon" (Schering AG, USA). The Protocol titration are presented in table 5.

Table 3
The Protocol for production of experimental batches S4/2401 of rhIFN beta-1b
№ p/pVolume, mlStage of the processPurity according to HPLC, %Qty by HPLC, mgThe output stage by HPLC, %Total yield, %LAL, EE/mgProteins of E.coli, ng/mg
120The original body enable-18,8-100%--
29The washed body enable-11,1 59%59%--
3211Solubilization in twittergate 3-14-5,651%30%-29472
440Chromatography on Source S30-4,072%22%-1705,1
540Oxidation91%a 3.996%21%-897,63
615Gel filtration on Superdex 7595.6%of2,462%13%-1532,6
724 Chromatography on Source Q3095,1%2,497%12%-1645,6
88Gel filtration on Sephacril S200HR97,9%2,188%11%-1413,7
950Chromatography on Source C4/G25-1,8871%10%--
10Ready form33,4

Table 4
The Protocol for production of experimental batches S4/1301 of rhIFN beta-1b
№ p/pVolume, mlStage of the processClean wing is X, %Qty by HPLC, mgThe output stage by HPLC, %Total yield, %LAL, EE/mgProteins of E.coli, ng/mg
140The original body enable-37,6-100%--
218The washed body enable-22,259%59%--
3200Refolding-18,282%48%--
4200Desalting/ concentration- 16,390%43%-29472
520Chromatography on Source S30-11,369%30%-1705
65,5Oxidation/concentration59,8to 12.0107%32%-897,6
727Gel filtration on Superdex 7590,78,470%22%-1533
830Chromatography on Source Q3087,96,7 79%18%-1646
97,5Gel filtration on Sephacril S200HR97,0the 4.771%13%-1414
104,5Chromatography on Source C4/G2596,53,7668%10%--
115Ready form33,4
Table 5
The results of the titration of samples Hβ-1b
Samples refnr-1bThe content of IFNIn volumeThe titer of interferon Units/0.2 mlAverage Ude the other activity
No. 10,023 mg0.5 mlof 6.4×105
No. 20,023 mg0.5 ml7,56×105
No. 30,023 mg0.5 mlof 6.4×105
No. 40,023 mg0.5 ml7,56×105
No. 50,023 mg0.5 ml8,2×105
No. 60,023 mg0.5 ml7,56×105 32×106IU/mg
No. 90,023 mg0.5 ml7,56×105
No. 100,023 mg0.5 ml7,56×105
No. 110,023 mg0.5 mlof 6.4×105
Control (Betaferon)0,023 mg0.5 mlof 6.4×10530×106IU/mg

As shown by the research protocols, the proposed optimized purification method allows to obtain the substance of rhIFN beta-1b fully meet the requirements of normative documents, thus resulting rhIFN beta-1b on specific antiviral activity is not inferior to foreign analogue of the Betaferon. In summary, the proposed method of cleaning bodies s is of E.coli allows you to get the substance of rhIFN beta-1b with the following characteristics:

the yield of the target protein: 10%;

- multimer by gel filtration: <5%;

- related proteins by reversed-phase chromatography: <5%;

- related proteins by LTOs-page: <5%;

proteins producer strain by ELISA: <5 ng/mg;

- endotoxin by LAL-test: <5 in this case/mg;

specific activity: 32 million IU/mg.

The method of purification of recombinant interferon β-1b from inclusion bodies of E. coli, including washing of inclusion bodies in 10 ml of 2%aqueous solution of twitterget 3-14 with the subsequent removal of nerastvorimogo material by centrifugation using a wash buffer solution with a pH of 7.5, the dissolution of the inclusion bodies in a buffer containing 7 M handinhand, 20 mm dithiothreitol and 0.5 mm phenylmethanesulfonyl, refolding of the target protein in a solution containing 20 mm Tris-HCl, pH 9,0, 0.2% peg 3550, 300 mm NaCl, 2 mm reduced glutathione, 1 mm oxidized glutathione, 2 mm ethylenediaminetetraacetic acid and 0.05% Twitterget 3-14 followed preromanticism preparation of the sample using a buffer containing 25 mm Tris-HCl, pH 8.0, 0.05% Twitterget 3-14, 0.5 mm phenylmethanesulfonyl, chromatography on Ceramic S using a buffer containing 25 mm Tris-HCl, pH 8.0, 0.05% Twitterget 3-14, 0.5 mm phenylmethanesulfonyl, chromatography on Source S30 using a buffer containing 25 mm Tris-HCl, pH 8,0,0,05% Twitterget 3-14, 0.5 mm phenylmethanesulfonyl, by oxidation with a mixture of group probably facilitates with the applied concentration of 1 mm and 0.1 mm, respectively, by gel-filtration on Superdex 75 Prep grade using a buffer containing 25 mm Tris-HCl, pH 7.5, 50 mm NaCl, 0.05% Twitterget 3-14, 0.5 mm phenylmethanesulfonyl, chromatography on Source Q30 using a buffer containing 25 mm Tris-HCl, pH 7.5, 50 mm NaCl, 0.05% Twitterget 3-14, 0.5 mm phenylmethanesulfonyl and gel-filtration on Sephacril S200HR using a buffer containing 25 mm Tris-HCl, pH 7.5, 50
mm NaCl, 0.05% Twitterget 3-14, 0.5 mm phenylmethanesulfonyl and control the efficiency of purification of the target protein by high-performance liquid chromatography with subsequent standard definition of its antiviral activity.



 

Same patents:

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

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 invention relates to the field of molecular biology, immunology, Microbiology and Virology, and is intended to assess the transcriptional levels of genes activity of interferon (if), IGF-dependent and proliferative cytokines

FIELD: chemistry; biochemistry.

SUBSTANCE: invention pertains to bioengineering. The method involves successive steps for ultrafiltration of a first antibody preparation to obtain a second antibody preparation, diafiltration of the second antibody preparation to obtain an intermediate preparation and second ultrafiltration of the intermediate preparation to obtain a third antibody preparation. All steps are carried out at temperature ranging from approximately 30°C to approximately 50°C.

EFFECT: design of an efficient method of concentrating antibody preparations.

52 cl, 25 dwg, 25 tbl, 12 ex

FIELD: medicine.

SUBSTANCE: there is prepared identification peptide with amino acid sequence: Gly-Pro-Ala-Pro-Gln-Pro-Asp-Glu-Asp-Leu-Lys-Arg-Gln. The prepared peptide is applied to identify, purify and recover the recombinant proteins containing it. For making the recombinant proteins with a polypeptide label, an expression vector pDED is applied. Said vector contains a nucleotide sequence coding amino acid sequence of the identification protein.

EFFECT: invention allows extending range of the methods to identify the recombinant proteins.

14 cl, 6 dwg, 1 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry and can be used for removing proteins and amino nitrogen from aqueous solutions. The method involves adsorption of proteins on a hydroaluminosilicate natural sorbent, which contains such minerals as clay, zeolite, feldspars, mica, calcite, and filtration. Adsorption takes place at pH ranging from 1 to 3 for 1 to 10 minutes.

EFFECT: invention allows for fast and quality deproteinisation of a solution.

3 tbl, 2 ex

FIELD: pharmacology.

SUBSTANCE: invention concerns biotechnology. Method involves: reduction of plasmatic Cohn IV1 fraction; anion exchange chromatography of solution containing alpha-1-antitrypsin on anion exchange gel, preferably DEAE Sepharose® fast flow; concentration of obtained eluate by ultrafiltration; additional chromatography on hydrophobic carrier and processing of fraction containing alpha-1-antitrypsin by material containing immobilised heparin form; further inactivation of viruses covered with lipidic capsule by adding detergents and optional solvent; salting-out of detergents by further salt concentration increase to ≥ 0.5 M; separation of detergents and virus particles by nanofiltration with pore grade of 15-20 nm.

EFFECT: enhanced purity and safety of obtained product.

17 cl, 2 ex

FIELD: medicine.

SUBSTANCE: invention concerns medicine and application of proline-specific endoproteases for peptide and protein hydrolysis. Invention involves application of proline-specific endoprotease with pH optimum under 5.5 for production of diet additive or medicine for in vivo treatment of celiac disease or diseases related to presence of proline-rich peptides in food, or as diet additive or medicine for celiac disease prevention.

EFFECT: enhanced hydrolysis of forage proteins with high proline content.

13 cl, 12 ex,10 tbl, 3 dwg

FIELD: medicine.

SUBSTANCE: substance of polypeptide nature with molecular weight 14350 Da, with N-end amino acid sequence, homologous phospholipase A2 of snake venom, and possessing properties of direct thrombin inhibitor of mixed type as well as antiproliferative action is separated of cobra venom Naja haje by three-stage liquid chromatography.

EFFECT: invention enables to produce selective direct thrombin inhibitor, possessing antiproliferative action.

1 tbl, 3 dwg, 10 ex

FIELD: medicine.

SUBSTANCE: method of obtaining somatotrophic hormone (STH) with decreased content of aggregate of its isoforms involves separation of STH isoforms by means of anion exchange chromatography by using anion exchange resin for decreasing content of the above aggregate up to not more than 10% (wt), on the basis of total mass of the above isoforms and the above aggregate. At that, there performed is loading of the above STH including dephenylalanine and/or trisulphide impurity and/or its aggregate on anion exchange resin chosen from the group including diethylaminoethyl cellulose and Q - sepharose. Loading is performed at the value of loading conductivity of anion exchange resin less or equal to 10 m cm/cm, at pH of anion exchange resin loading of 5 to 10 and at loading of anion exchange STH resin, which includes the above impurity or the above aggregate comprising not more than 10 g of protein/l of the volume filled with anion exchange resin.

EFFECT: invention allows obtaining somatotrophic hormone with decreased content of aggregate of its isoforms, its antagonist with decreased content of aggregate of its isoforms and total content of trisulphide impurity and dephenylalanine impurity.

8 cl, 5 dwg, 8 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: method of obtaining somatotrophic hormone (STH) with decreased content of aggregate of its isoforms involves separation of STH isoforms by means of anion exchange chromatography by using anion exchange resin for decreasing content of the above aggregate up to not more than 10% (wt), on the basis of total mass of the above isoforms and the above aggregate. At that, there performed is loading of the above STH including dephenylalanine and/or trisulphide impurity and/or its aggregate on anion exchange resin chosen from the group including diethylaminoethyl cellulose and Q - sepharose. Loading is performed at the value of loading conductivity of anion exchange resin less or equal to 10 m cm/cm, at pH of anion exchange resin loading of 5 to 10 and at loading of anion exchange STH resin, which includes the above impurity or the above aggregate comprising not more than 10 g of protein/l of the volume filled with anion exchange resin.

EFFECT: invention allows obtaining somatotrophic hormone with decreased content of aggregate of its isoforms, its antagonist with decreased content of aggregate of its isoforms and total content of trisulphide impurity and dephenylalanine impurity.

8 cl, 5 dwg, 8 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: method of obtaining somatotrophic hormone (STH) with decreased content of aggregate of its isoforms involves separation of STH isoforms by means of anion exchange chromatography by using anion exchange resin for decreasing content of the above aggregate up to not more than 10% (wt), on the basis of total mass of the above isoforms and the above aggregate. At that, there performed is loading of the above STH including dephenylalanine and/or trisulphide impurity and/or its aggregate on anion exchange resin chosen from the group including diethylaminoethyl cellulose and Q - sepharose. Loading is performed at the value of loading conductivity of anion exchange resin less or equal to 10 m cm/cm, at pH of anion exchange resin loading of 5 to 10 and at loading of anion exchange STH resin, which includes the above impurity or the above aggregate comprising not more than 10 g of protein/l of the volume filled with anion exchange resin.

EFFECT: invention allows obtaining somatotrophic hormone with decreased content of aggregate of its isoforms, its antagonist with decreased content of aggregate of its isoforms and total content of trisulphide impurity and dephenylalanine impurity.

8 cl, 5 dwg, 8 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: invention refers to the selective method for preparation of "АХЭ" inhibitor - perindopril with usage as initial reagent of the sterospecific amino acid N-/1-(S)-ethoxycarbonylbutyl/-(S)-alanine which is activated by tetramethyl-uronium salts in the presence of tertiary organic base and following interreaction with (2S,3aS,7aS)-octahydroindolo-2-carbonic acid or its ester. After completing of the reaction the protective group is removed by the hydrogenation, interphase hydrogenation or extraction.

EFFECT: obtaining of perindopril with usage of tetramethyl-uronium salts as reagents of coupling reaction.

5 cl, 3 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention pertains to bioengineering. The method involves successive steps for ultrafiltration of a first antibody preparation to obtain a second antibody preparation, diafiltration of the second antibody preparation to obtain an intermediate preparation and second ultrafiltration of the intermediate preparation to obtain a third antibody preparation. All steps are carried out at temperature ranging from approximately 30°C to approximately 50°C.

EFFECT: design of an efficient method of concentrating antibody preparations.

52 cl, 25 dwg, 25 tbl, 12 ex

FIELD: medicine.

SUBSTANCE: there is prepared identification peptide with amino acid sequence: Gly-Pro-Ala-Pro-Gln-Pro-Asp-Glu-Asp-Leu-Lys-Arg-Gln. The prepared peptide is applied to identify, purify and recover the recombinant proteins containing it. For making the recombinant proteins with a polypeptide label, an expression vector pDED is applied. Said vector contains a nucleotide sequence coding amino acid sequence of the identification protein.

EFFECT: invention allows extending range of the methods to identify the recombinant proteins.

14 cl, 6 dwg, 1 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry and can be used for removing proteins and amino nitrogen from aqueous solutions. The method involves adsorption of proteins on a hydroaluminosilicate natural sorbent, which contains such minerals as clay, zeolite, feldspars, mica, calcite, and filtration. Adsorption takes place at pH ranging from 1 to 3 for 1 to 10 minutes.

EFFECT: invention allows for fast and quality deproteinisation of a solution.

3 tbl, 2 ex

FIELD: pharmacology.

SUBSTANCE: invention concerns biotechnology. Method involves: reduction of plasmatic Cohn IV1 fraction; anion exchange chromatography of solution containing alpha-1-antitrypsin on anion exchange gel, preferably DEAE Sepharose® fast flow; concentration of obtained eluate by ultrafiltration; additional chromatography on hydrophobic carrier and processing of fraction containing alpha-1-antitrypsin by material containing immobilised heparin form; further inactivation of viruses covered with lipidic capsule by adding detergents and optional solvent; salting-out of detergents by further salt concentration increase to ≥ 0.5 M; separation of detergents and virus particles by nanofiltration with pore grade of 15-20 nm.

EFFECT: enhanced purity and safety of obtained product.

17 cl, 2 ex

FIELD: medicine.

SUBSTANCE: invention concerns medicine and application of proline-specific endoproteases for peptide and protein hydrolysis. Invention involves application of proline-specific endoprotease with pH optimum under 5.5 for production of diet additive or medicine for in vivo treatment of celiac disease or diseases related to presence of proline-rich peptides in food, or as diet additive or medicine for celiac disease prevention.

EFFECT: enhanced hydrolysis of forage proteins with high proline content.

13 cl, 12 ex,10 tbl, 3 dwg

FIELD: medicine.

SUBSTANCE: substance of polypeptide nature with molecular weight 14350 Da, with N-end amino acid sequence, homologous phospholipase A2 of snake venom, and possessing properties of direct thrombin inhibitor of mixed type as well as antiproliferative action is separated of cobra venom Naja haje by three-stage liquid chromatography.

EFFECT: invention enables to produce selective direct thrombin inhibitor, possessing antiproliferative action.

1 tbl, 3 dwg, 10 ex

FIELD: medicine.

SUBSTANCE: method of obtaining somatotrophic hormone (STH) with decreased content of aggregate of its isoforms involves separation of STH isoforms by means of anion exchange chromatography by using anion exchange resin for decreasing content of the above aggregate up to not more than 10% (wt), on the basis of total mass of the above isoforms and the above aggregate. At that, there performed is loading of the above STH including dephenylalanine and/or trisulphide impurity and/or its aggregate on anion exchange resin chosen from the group including diethylaminoethyl cellulose and Q - sepharose. Loading is performed at the value of loading conductivity of anion exchange resin less or equal to 10 m cm/cm, at pH of anion exchange resin loading of 5 to 10 and at loading of anion exchange STH resin, which includes the above impurity or the above aggregate comprising not more than 10 g of protein/l of the volume filled with anion exchange resin.

EFFECT: invention allows obtaining somatotrophic hormone with decreased content of aggregate of its isoforms, its antagonist with decreased content of aggregate of its isoforms and total content of trisulphide impurity and dephenylalanine impurity.

8 cl, 5 dwg, 8 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: method of obtaining somatotrophic hormone (STH) with decreased content of aggregate of its isoforms involves separation of STH isoforms by means of anion exchange chromatography by using anion exchange resin for decreasing content of the above aggregate up to not more than 10% (wt), on the basis of total mass of the above isoforms and the above aggregate. At that, there performed is loading of the above STH including dephenylalanine and/or trisulphide impurity and/or its aggregate on anion exchange resin chosen from the group including diethylaminoethyl cellulose and Q - sepharose. Loading is performed at the value of loading conductivity of anion exchange resin less or equal to 10 m cm/cm, at pH of anion exchange resin loading of 5 to 10 and at loading of anion exchange STH resin, which includes the above impurity or the above aggregate comprising not more than 10 g of protein/l of the volume filled with anion exchange resin.

EFFECT: invention allows obtaining somatotrophic hormone with decreased content of aggregate of its isoforms, its antagonist with decreased content of aggregate of its isoforms and total content of trisulphide impurity and dephenylalanine impurity.

8 cl, 5 dwg, 8 tbl, 6 ex

Up!