RU2656140C2 - Method for obtaining a hybrid protein containing a fused protein analogue of an interferon gamma conjugated with oligosaccharide - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method comprises conjugating an interferon gamma fused analogue represented by the sequence of SEQ ID NO: 1, with an oligosaccharide-type polymer D3000-7 (Sa-Sp)-(Sp-M) having the structure shown in Fig. 8, where D3000 is the dialdehyde dextran, (Sa-Sp) - Neu5Ac-Lactose-N(CH)-CH(2)-CH(2)-NH(2), (Sp-M) - Maleimide-CH(2)-NH(2), purification of the resulting conjugated interferon gamma fused analogue protein of unconjugated components by the HPLC method with the Sephacryl S400 gel filtration sorbent with the yield of the final product containing impurities not more than 1.2% of the total product mass.

EFFECT: method for producing a fusion protein based on a interferon gamma fused analogue having antiviral activity is proposed.

1 cl, 16 dwg, 2 tbl

Description

The invention relates to the field of biotechnology, in particular to a method for producing a hybrid protein based on a recombinant analog of gamma interferon for the treatment of herpes virus infection by conjugation of a recombinant analog of gamma interferon with an oligosaccharide polymer of nature.

Herpes virus infection (HBV) affects more than 80% of humanity. GVI causes many different diseases that affect various organs and systems of a person. HBV is the second leading cause of viral disease mortality in newborns and young children [Williams E.J., Embleton N.D., Clark J.E. et al. Viral infections: contributions to late fetal death, stillbirth, and infant death. J. Pediatr. 2013, Vol. 163. No. 2. P. 424-428]. The absence of effective vaccines and the impossibility of a complete cure, together with a wide coverage of the population, determine the relevance of herpes virus infection.

The use of interferons is the most promising for the treatment of GVI. Of all types of interferons, interferon gamma is the most effective for the treatment of GVI [Complexity of interferon-g interactions with HSV-1. Nancy J. Bigley // Frontiers in immunology, 2014. doi: 10.3389 / fimmu. 2014.00015].

Existing interferon gamma preparations are based on a molecule expressed in E. coli and have antiviral activity comparable to natural interferon gamma. However, these drugs have a short half-life and an unstable structure.

Human interferon gamma is a glycosylated protein, but when expressed in E. coli, glycosylation of interferon gamma is not possible. N-glycosylation has been shown to provide resistance to hydrolysis by body proteases [N-glycosylation of human interferon-y: glycans at Asn-25 are critical for protease resistance. T. SARENEVA, J. PIRHONEN, K. CANTELL, I. JULKUNEN // Biochem. J. (1995) 308, 9-14].

A preparation based on a recombinant analogue of interferon gamma conjugated to a cysteine residue introduced into the region of the natural glycosylation site with an sialic acid-bearing oligosaccharide should fully retain biological activity and have improved pharmacokinetics compared to native gamma interferon. Such a drug is of great interest for the treatment of GVI, because will overcome the disadvantages of existing drugs.

The presence of sialic acid residues in the oligosaccharide molecule stabilizes the molecule and prevents the formation of antibodies to it. Currently, active development of drugs based on proteins bearing oligosaccharides containing sialic acid residues is underway [Bioconjug Chem. 2012, Aug 15; 23 (8): 1524-33. Glycoengineering approach to half-life extension of recombinant biotherapeutics].

The covalent attachment of oligosaccharides to the substrate is possible in two different ways, one of which is called addition at a single terminal residue, the other can be called multipoint addition. In the first case, oligosaccharides are attached via a terminal reducing residue (e.g., glucose residue). The second method is characterized by attachment to various residues within the oligosaccharide sequence. In nature, there is only one way to attach through the reducing end of the polymer. Until relatively recently, in vitro oligosaccharide-protein conjugates were synthesized by the well-known method of periodically oxidizing vinyl diol groups of internal residues to form two aldehydes that react with amino groups of proteins. This led to the formation of complex polymer molecules, unsuitable for pharmaceuticals.

The use of the aldehyde function of the terminal reducing residue is theoretically possible, but has long been difficult due to the extremely low reactivity of the hemi-acetal present. Methods have been developed that make it possible to obtain an aldehyde group at the reducing end of the oligosaccharide. US Pat. No. 6,653,457 dated 11.25, 2003. describes the preparation of heparin derivatives with an aldehyde group. Using the fact that heparin is a fully sulfated oligosaccharide, methods have been developed for the selective reduction of the terminal hemiacetal with the formation of vinyl diols at this site, followed by their selective oxidation with periodate to form aldehydes capable of reacting with amino groups of various substrates. Heparin as an oligosaccharide has antithrombotic activity and cannot be a universal alternative to pegylation, as the resulting conjugates may have side effects associated with such activity.

In the patent of the Russian Federation POLYEPTIDE AND OLIGOSACCHARIDE CONNUGATES, covalent conjugates of a polypeptide and an oligosaccharide are described in which the polypeptide is conjugated to at least one oligosaccharide-spacer residue, where the oligosaccharide is a synthetic sulfated pentasaccharide residue. Essentially, a synthetic oligosaccharide is a heparin analogue, but its size leads to a therapeutically insignificant activity at concentrations up to 50 nM per dose. In addition to the previous patent, the oligosaccharide is equipped with a spacer. The spacer is an aliphatic group, at the active end of which there can be any functional groups for the formation of covalent bonds with the protein. The resulting conjugates have improved pharmacokinetic properties compared with the corresponding unconjugated polypeptides. Described, for example, conjugates of the peptide with insulin.

The ideas embodied in these two patents cannot be directly transferred to other oligosaccharides, since heparin is a fully sulfated derivative that does not have internal free diol groups capable of periodate oxidation. For the remaining sugars, it was necessary to develop additional methods for the inactivation of such diol groups.

US 7807824 and 7691826 describe methods for preparing polysialic acid conjugates with proteins. Conjugates are prepared by activating a reducing polysialic acid residue and preparing various activated derivatives that react with amino and thio groups of proteins. Obtaining the terminal aldehyde group is achieved by a two-stage method. At the first stage, periodates are oxidized by internal diols, then they are reduced by borohydride with simultaneous reduction of the terminal half-ketal to diol. In a second step, the newly formed diol is selectively oxidized to an aldehyde. The result is a polymer bearing a single reactive group at the reducing end of the oligosaccharide. The subsequent reaction of the aldehyde with heterofunctional reagents allows to obtain various reactive derivatives that selectively react with proteins. Oligosaccharides containing N-oxysuccinimide, maleimide or dithiopyridyl active groups as reactive groups are obtained.

Conjugates are described for amino groups of proteins and for free cysteines. It was shown that such conjugates in their properties are not inferior to similar conjugates with PEG (in terms of half-life and preservation of biological properties), but they have low immunogenicity. The described conjugation methods are rather laborious and lead to products with similar physicochemical properties during the oxidation-reduction chain and, therefore, are difficult to separate by chromatography methods. The scientific literature describes the reactions of the reducing hemiacetal of oligosaccharides with substituted hydroxylamine derivatives (Peri F., Dumy P., Mutter M. Chemo- and stereoselective glycosylation of hydroxylamino derivatives: a versatile approach to glycoconjugates. Tetrahedron 1998; 54: 12269-12278). Such reactions proceed selectively and under mild conditions. Eurasian patent "Conjugate, including protein and polymer or its derivative (variants), method for its preparation, use of conjugate and pharmaceutical composition containing it".

US 7,815,893 discloses the use of hydroxyl starch derivatives as oligosaccharides. The reducing end of the polymer was activated by reaction with O- [2- (2-aminooxyethoxy) ethyl] hydroxylamine. The obtained derivative was treated with hetero-bifunctional reagents and conjugates with proteins were obtained. Received many proteins conjugated with starch and its derivatives.

The oligosaccharide used in the claimed method is close to sugars attached to natural glycoproteins used in pharmaceuticals. Residues of the oligosaccharides of these proteins contain sialic acid located opposite the reducing end. The synthesis of such oligosaccharides and their covalent attachment to a polypeptide substrate will allow one to obtain conjugates with improved properties compared to polysial derivatives or derivatives based on unmodified natural oligosaccharides such as dextran, chitosan or starch.

The invention solves the problem of producing a recombinant protein for the treatment of herpes virus infection by conjugating a recombinant analog of interferon gamma with the sequence SEQ ID NO 1 with a polymer of oligosaccharide nature, having a unique structure, by forming a covalent bond between the polymer molecules and the recombinant protein, and also provides a method for purification of the obtained agent .

Another objective of this work was the creation of an oligosaccharide by the formation of covalent bonds between seven sialic acid molecules (N-acetyl-neuramine-lactose (N5Ac-Lactose)) by means of spacers with a dextran 3000 molecule and activation of the resulting construct (oligosaccharide) to conjugate it with a recombinant analog of interferon gamma of man.

A method for producing a hybrid protein with antiviral activity comprises conjugating a recombinant gamma interferon analogue represented by the sequence SEQ ID NO: 1 with an oligosaccharide polymer of the nature D3000-7 (Sa-Sp) - (Sp-M), where D3000 is dextran dialdehyde, ( Sa-Sp) - Neu5Ac-Lactose-N (CH) -CH (2) -CH (2) -NH (2), (Sp-M) - Maleimide-CH (2) -NH (2), purification of the resulting conjugated recombinant protein - an analog of interferon gamma from unconjugated components by HPLC methods with a Sephacryl S400 gel filtration sorbent with the release of a final product containing impurities more than 1.2% on total product weight.

The technical result of the claimed invention is the creation of a hybrid protein: a recombinant analogue of interferon gamma with SEQ ID NO: 1 conjugated to a polymer of oligosaccharide nature, while the resulting structure of the oligosaccharide: D3000-7 (Sa-Sp) - (Sp-M) leads to stabilization of the molecule and preventing the formation of antibodies to it, i.e. obtaining a substance with antiviral activity.

A brief description of the drawings.

FIG. 1. Electrophoresis of the lysate of cells expressing a recombinant protein containing the sequence of a recombinant analogue of interferon gamma connected to a stabilizing protein - thioredoxin. Track 1 molecular weight standards; track 2 cell lysate; the arrow indicates the position of the recombinant protein - an analog of interferon gamma, connected with a protein-stabilizer in the gel.

FIG. 2. Chromatographic profile of elution of a recombinant protein - an analog of gamma interferon, coupled to a protein stabilizer with Ni-Sepharose. Numbers 1-9 indicate the fractions selected for analysis

FIG. 3. Electrophoresis of fractions containing recombinant protein after chromatography on Ni-Sepharose. Tracks 1-8 - fractions containing the target protein; the arrow indicates the position of the recombinant protein - an analog of interferon gamma, connected with a protein-stabilizer in the gel. FIG. 4. Hydrolysis of a precursor protein coupled to a stabilizer protein with the enterokinase-F enzyme to recombinant IF gamma protein (16868Da) and a stabilizer protein, thioredoxin. FIG. 5. Electrophoresis of a hydrolysis mixture of a recombinant protein and enterokinase at different time intervals. Track 1 - reaction mixture at the beginning of the experiment (time T = 1 min); track 2 — reaction mixture half an hour after the start of the experiment (time T = 30 min); track 3 — reaction mixture one hour after the start of the experiment (time T = 60 min); track 4 - molecular weight standards; the arrows indicate: the position in the gel of a recombinant protein - an analog of interferon gamma connected to a stabilizing protein (a), a pure recombinant protein - an analog of interferon gamma (b) and a pure stabilizer protein (c).

FIG. 6. The chromatographic profile of the elution of the recombinant protein is an analogue of interferon gamma with SP-Sepharose (column HiPrep16 / 10 Q FF).

FIG. 7. HPLC chromatographic profile of elution of a recombinant protein - an analog of interferon gamma from a Kromasil 300-5C18 column. According to the chromatogram, the purity of the recombinant protein is more than 99%.

FIG. 8. Synthesis of activated oligosaccharide D3000-7 (Sa-Sp) -Sp-M.

FIG. 9. Chromatographic elution profile of D3000-7 (Sa-Sp) -Sp-M from a mono-Q preparative column (purification).

Figure 10. Chromatographic elution profile of D3000-7 (Sa-Sp) -Sp-M from a mono-Q analytical column.

FIG. 11 Scheme of pharmaceutical substance.

FIG. 12 Chemical reaction of conjugation of activated oligosaccharide and recombinant protein.

FIG. 13 Chromatographic profile of gel filtration of conjugate of recombinant gamma-activated oligosaccharide via Sephacryl S400. The arrow indicates the yield of the conjugate.

FIG. 14 Mass spectrum of MALD-TOF monoion of a recombinant protein - an analog of interferon gamma.

FIG. 15 Periodic oxidation of the diol groups of dextran 3000 - obtaining DBP.

FIG. 16 Amination of the maleide group — Preparation of Malemid-CH (2) -CH (2) -NH (2) (Sp-M).

Table 1. Molecular weights of the components of the activated oligosaccharide D3000-7 (Sa-Sp) -Sp-M.

Table 2. Antiviral activity of interferon gamma preparations on WISH and DAUDI cells expressed in EC 50 (pM).

DETAILED DESCRIPTION OF THE INVENTION

1. PRODUCTION OF RECOMBINATION PROTEIN - ANALOGUE OF THE GAMMA INTERFERON.

To obtain the recombinant protein represented by the sequence SEQ ID NO 1, a genetic construct was created according to the standard method (Maniatis T., Fritsch E.F., 1982.) based on the commercial plasmid pET32a. The following sequences of regulatory elements (enhancers and promoter) were used to create the plasmid: MxA gene represented by SEQ ID NO 3 and ISG56 gene represented by SEQ ID NO 3. Plasmid transfected cells of E. coli strain BL21 (DE3). The resulting plasmid pET-32a / IF gamma transform competent bacterial cells of the strain Escherichia coli BL21 (DE3) to obtain a soluble fraction of the protein. Strain BL21 (DE3) (Invitrogen) is intended for expression of recombinant proteins. The designation DE3 means that the strain contains the lysogen of phage λ DE3, which carries the gene for T7 RNA polymerase under the control of the lacUV5 promoter. IPTG (isopropyl β-D-1-thiogalactopyranoside) is required to induce the expression of T7 RNA polymerase. The strain Escherichia coli BL21 (DE3) / pET-32a / IF gamma provides fusion protein thioredoxin - interferon gamma. After transfection, the cells were subcultured into a culture flask with LB broth and kanamycin 25 μg / ml. The culture was grown on a shaker at 280 rpm at 37 ° C until the optical density reached OD585 = 0.8E. Protein expression was induced by adding IPTG to a concentration of 1 mM. The culture was additionally cultured for 4 hours, then the cells were precipitated by centrifugation at 5000 g. Cell pellet was lysed in 8 M urea and processed on an ultrasonic disintegrator. The cell lysate was centrifuged at 20,000 g for 2 hours.

The pET-32a construct, carrying the gene encoding the recombinant IF-gamma protein in E. coli cells as a fusion protein with a stabilizing protein thioredoxin, increases the expression level and solubility of the fusion protein.

2. CHROMATOGRAPHIC CLEANING OF RECOMBINANT PROTEIN - ANALOGUE OF THE GAMMA INTERFERON AT SP-SEPHAROSE

Chromatographic purification of a recombinant protein, an analogue of interferon gamma, takes place in one step on an ion-exchange sorbent. The carrier used was SP-Sepharose from GE Helthcare. HiTrap columns were used to standardize the process and its reproducibility. A HiPrepl6 / 10 Q FF chromatographic column was used in the work, the capacity of which was sufficient for the entire volume of the secreted protein. After hydrolysis and heating, the reaction mixture is filtered through a 0.22 μm filter from aggregates of denatured proteins. The filtered mixture is applied to a HiPrep16 / 10 Q FF column, pre-equilibrated with 25 mM NaCl. The target protein was eluted with a linear gradient. Starting buffer: 25mM NaCl, final buffer: 100mM NaCl. The eluate was fractionated. The presence of protein in the fraction was determined by electrophoresis in PAAG. The chromatogram shown in Fig.6.

3. EVALUATION OF RECOMBINANT PROTEIN - ANALOGU OF GAMMA INTERFERON BY METHODS OF CHROMATOGRAPHY AND MASS SPECTROMETRY

Evaluation of the amount of the obtained recombinant protein - an analogue of interferon gamma according to Bradford showed that in the presence of 3212 mg of protein. The purity of the recombinant protein - an analog of interferon gamma was determined by HPLC - chromatography, the authenticity of the rapid method for determining the authenticity of the recombinant analog of interferon gamma as a component of the drug. A solution containing a recombinant protein, an analogue of interferon gamma, was dialyzed against a solution of 0.1% trifluoroacetic acid. 150 μl of the solution was taken and applied to a Kromasil 300-5C18 4.6ID150 column. Analytical chromatography was performed. Eluted with a linear gradient. Starting buffer: 0.1% trifluoroacetic acid, 2% acetonitrile, final buffer: 0.1% trifluoroacetic acid, 80% acetonitrile. Recombinant protein - an analog of interferon gamma descended from the column at a concentration of acetonitrile 32%. According to the chromatogram of FIG. 7 purity of recombinant protein more than 99%.

MALDI-TOF mass spectrometric analysis of the recombinant protein monoion was performed. The results are presented in FIG. fourteen.

A set of several peaks with masses from 16871 to 16899Da was obtained on the mass spectrum. This is due to the fact that for every 1000 units of molecular weight in protein molecules, due to the accumulation of the C13 isotope, the mass of the molecule increases on average by 1 Da, i.e., instead of one mass, we see a set of several masses. The major peak corresponds to a mass of 16897, a "growth" of the peak corresponding to a mass of 16869 is observed, while the peak of 16869, which corresponds to a recombinant protein - an analog of interferon gamma, free of C13 isotopes, is small, because statistically, a protein molecule weighing more than 10 kDa should account for at least one C13 molecule. It can be stated that the mass obtained for the monoion of the recombinant protein, an analogue of interferon gamma, corresponded to the calculated mass (16868 Da).

Based on the results of the work performed, the following conclusions can be drawn.

Recombinant protein analogue of interferon gamma chromatographically purified on SP-Sepharose. Recombinant protein - an analogue of interferon gamma was produced in an amount of 3212 mg. Recombinant protein - an analogue of interferon gamma is defined as a genuine express method for determining the authenticity of a recombinant analogue of interferon gamma. The purity of the recombinant protein, an analogue of interferon gamma, was evaluated by HPLC methods. The authenticity was evaluated by the express method for determining the authenticity of the recombinant analogue of interferon gamma as a component of the drug and the MALDI-TOF mass spectrum of the protein monoion.

4. SYNTHESIS OF ACTIVATED OLIGOSACCHARIDE D3000-7 (Sa-Sp) -Sp-M.

The synthesis of activated oligosaccharide occurs in three stages:

a) the interaction of DBP with Neu5Ac-Lactose-N (CH) -CH (2) -CH (2) -NH (2) (Sa-Sp),

b) the conversion of the COOH group of the terminal glucose residue of the dextran core to the aldehyde group of COH by treatment with ethanolamine;

c) the introduction of the maleimide group into the dextran core of the oligosaccharide due to the interaction of Malemid-CH (2) -CH (2) -NH (2) (Sp-M) with the SON-aldehyde group of the terminal glucose residue.

OXIDATION OF DEXTRAN WITH ALLOCATION OF DAD

Dextran was activated by periodically oxidizing glucose diol groups. The reaction of the periodate with dextran has been well studied and leads to the formation of aldehyde groups capable of reacting with primary amines to form a Schiff base. Anion exchange chromatography on strong anion exchange type Q was used to separate the reaction product from the excess of the oxidizing agent, which made it possible to remove the anions of periodate, iodate, and formate formed during sugar oxidation. The method is very fast, efficient and allows you to get activated polymer with increased stability during storage. Dextran3000 was dissolved in distilled water and mixed with a freshly prepared sodium periodate solution. The resulting solution contains dialdehyde dextran (DBP). This solution was mixed with a suspension of Dowex-1 in acetate form, which absorbs ions and reaction by-products. Dowex-1 is removed by filtration in the solution remains DBP. The chemical reaction equation is shown in FIG. fifteen.

CONJUGATION OF SPACER (SP) FROM THE MALEIMIDE GROUP (M) WITH ISOLATION OF SP-M The maleimide group will be introduced into the oligosaccharide by conjugation of the SMCC derivative with the spacer, as a result of which the maleimide derivative will be formed, carrying the NH2 group, which will conjugate with dextran. The interaction of SMCC with a spacer - obtaining Maleimide-CH (2) - CH (2) -NH (2) (Sp-M) - takes place in a borate buffer. The equimolar samples of SMCC and spacer are dissolved in borate buffer, then mixed. The chemical reaction equation is shown in FIG. 16.

The reaction proceeds by mixing and incubating solutions containing DBP, Neu5Ac-Lactose-N (CH) -CH (2) -CH (2) -NH (2) (Sa-Sp), Maleimide-CH (2) -CH (2 ) -NH (2) (Sp-M) and ethanolamine.

The DBP solution and Sa-Sp are mixed and incubated at 24 ° C for 2 hours, then the ethanol amine solution is added and the mixture is incubated at the same temperature for 10 minutes with stirring, then the Sp-M solution is added and, after stirring, after 30 minutes, the synthesis of activated oligosaccharide takes place to end.

The amount of activated oligosaccharide maleimide-amino-DEXTRAN-7 [NH-CH (2) -CH (2-N (CH) -Lactose-Neu5Ac] (D3000-7 (Sa-Sp) -Sp-M) is determined by the gravimetric method based to determine the weight of the dried residue obtained after evaporation of the sample, 1 ml of a solution of oligosaccharide D3000-7 (Sa-Sp) -Sp-M was placed in a gel filter column PD MiniTrap G-25 (GE Healthcare 28-9180-07), balanced with distilled water. 1 ml of “slip” is collected and its tube is placed, then the tube is weighed. Water is evaporated on a vacuum evaporator at 70 ° C. The tube is weighed again. Weight difference will correspond to the amount of oligosaccharide D3000-7 (Sa-Sp) -Sp-M.

The activated oligosaccharide D3000-7 (Sa-Sp) -Sp-M was obtained in an amount of 5144 mg.

The chemical reaction equation is shown in FIG. 8.

CHROMATOGRAPHIC PROFILE of elution of D3000-7 (Sa-Sp) -Sp-M from a preparative mono-Q column (purification).

The activated oligosaccharide D3000-7 (Sa-Sp) -Sp-M was analyzed by HPLC chromatography on a mono-Q analytical column.

D3000-7 (Sa-Sp) -Sp-M was diluted 10 times with 5 mM NaCl, and loaded onto a mono-Q assay column. Eluted with a linear gradient. Starting buffer: 5 mM NaCl, final buffer: 100 mM NaCl. The target product was obtained at a NaCl concentration of 45 mM. Chromatography is shown in FIG. 10.

Thus, the activated oligosaccharide consists of 7 + 1 spacer molecules, 7 Lactose-Neu5Ac molecules and a maleimide molecule, taking into account mass loss due to the loss of part of the molecules during the formation of covalent bonds, we obtain a set of masses of the components of the activated oligosaccharide.

Table 1.

The calculated mass of all components of the activated oligosaccharide without a dextran molecule is 5409 Da.

5. CONJUGATION OF OLIGOSACCHARID WITH RECOMBINANT ANALOGUE OF THE GAMMA INTERFERON.

In the process of conjugation, a covalent bond is formed between the maleimide group of the activated oligosaccharide and the -SH group of the cysteine molecule of the recombinant gamma interferon analogue. The technology is selected in such a way that the components of this chemical reaction enter the reactor, where conjugation takes place. Conjugation takes place in the presence of 25 mM NaCl. The chemical reaction is shown in FIG. 12.

The activated oligosaccharide is taken in excess. The reaction mixture is incubated for 8 hours, the reaction proceeds with a yield of more than 94%.

Chromatographic profile of gel filtration of conjugate of recombinant gamma-activated oligosaccharide via Sephacryl S400

A dithiotriitol solution is added to the reaction mixture to reduce imines in the activated oligosaccharide molecule. After incubation for 10 minutes, the reaction mixture was applied to a gel filtration column XK 50 * 100 (18-87-68 GE Healthcare) with Sephacryl S400 (17-0506-01 GE Helthcare), previously equilibrated with water.

Fractions containing the desired product were combined. The mass of the recombinant protein was determined according to Bradtford. The total amount was 4247 mg. The data are presented in FIG. 13.

Chromatographic purification of the resulting conjugate. A filtered solution of a pharmaceutical substance is passed through a K5 column with a Sephacryl S400 gel filtration sorbent equilibrated with distilled water. On column K5 of the XP chromatograph, desalination and separation of unreacted components from the product, a pharmaceutical substance, takes place. The first peak is the target product (1.725 L), then the recombinant analogue of interferon gamma and activated oligosaccharide come out, then the salts with the yield of the final product containing impurities of not more than 1.2% of the total mass of the product.

6. RESEARCH ANTI-VIRAL ACTIVITY

Testing of antiviral activity of the drugs was carried out using a cell culture DAUDI and WISH. For this, the cells were seeded in a 96-well plate in an amount of 20 thousand cells / well in a volume of 100 μl / well of the complete Eagle-MEM medium (containing 10% fetal serum 300 mg / L L-glutamine and antibiotic gentamicin 100 mg / ml). Incubated during the day in a CO ₂ incubator at 37 ° C in 5% CO ₂ atmosphere. After incubation, the cell culture was washed from the Igla-MEM serum medium. For this, the old Igla-MEM medium was removed and, without touching the cell monolayer, 100 μl / well of sterile PBS was added with a multichannel pipette, incubated for 5 min, then physiological was selected the solution, without touching the cell monolayer, and 100 μl / well of Eagle-MEM serum-free medium were added. After washing, the studied preparations were introduced into the cell culture — interferon gamma, FS, and LP in tenfold dilutions (0.001 pM, 0.01 pM, 0.1 pM, 1.0 pM) and incubated for 1 day in a CO ₂ incubator at 37 ° C in 5% atmosphere of CO ₂ . After incubation, the VSV strain Indiana virus was introduced into the cell culture at a dose of 100 CPD per well in a volume of 100 μl of serum-free Needle-MEM. Working dilution of the virus was obtained by diluting a laboratory standard with a titer of 104 / ml with two consecutive 10-fold dilutions. After the mixture was added to the wells, the cells were incubated for 1-2 days in a CO ₂ incubator at 37 ° C until a cytopathic effect appeared. The cells of the plate with cells were washed with sterile saline, PBS, two times 200 μl of buffer, and 100 μl of a solution of 0.1 mg / ml MTT dye in Igla-MEM medium was added. Purple crystals of formazan become noticeable after an incubation of 3-5 hours. To dissolve formazan, 100 μl of isopropyl alcohol are added to the cells. The tablet was spectrophotometrically at 590 nm. The results were plotted on a curve and EC 50 - was determined graphically (concentration of a drug having a 50% antiviral effect). The research result is shown in table 2.

For FS and LP, in comparison with the sold interferon gamma, the mechanisms of their antiviral activity were studied in four concentrations (0.001 pM, 0.01 pM, 0.1 pM, 1.0 pM). It was shown that the commercial comparison drug interferon gamma showed antiviral activity against the model virus VSV on WISH and DAUDI cells, as demonstrated by a pronounced increase in optical density depending on the type of cells, the antiviral activity of the developed FS and PL, which is almost equal to the commercial drug for similar inhibition of cytopathic Skog of the virus.

Thus, the recombinant analogue of interferon gamma does not change the lifetime of the conformation (charge) when interacting with the IFNGR1 and IFNGR2 receptors at the same concentration compared to the natural analogue, which in turn leads to the same level of virus inhibition.

Claims (1)

A method for producing a hybrid protein having antiviral activity, comprising conjugating a recombinant gamma interferon analogue represented by the sequence SEQ ID NO: 1 with an oligosaccharide polymer of the nature D3000-7 (Sa-Sp) - (Sp-M) having the structure shown in FIG. 8, where D3000 is dialdehyde dextran, (Sa-Sp) - Neu5Ac-Lactose-N (CH) -CH (2) -CH (2) -NH (2), (Sp-M) - Maleimide-CH (2) -NH (2), purification of the obtained conjugated recombinant protein - an analog of interferon gamma - from unconjugated components by HPLC with a Sephacryl S400 gel filtration sorbent with a yield of the final product containing impurities of not more than 1.2% of the total product weight.

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