Method of purifying and/or extracting biologically active granulocyte colony-stimulating factor

FIELD: chemistry.

SUBSTANCE: extraction is done using affinity chromatography with immobilised metal. The method can be realised in native conditions. Biologically active G-CSF is obtained with purity over 95%. Two more chromatography stages are done, cation-exchange and gel filtration, to remove trace amounts of impurities. The method allows for obtaining G-CSF with high output and over 99% purity.

EFFECT: described method is especially suitable for industrial production of G-CSF.

16 cl, 5 dwg, 5 ex

 

The scope of the invention

The invention relates to a new method of cleaning and/or extraction of biologically active granulocyte colony-stimulating factor (G-CSF) by affinity chromatography with immobilized metal (IMAC).

G-CSF belongs to a group of colony-stimulating factors, which regulate the differentiation and proliferation of hematopoietic predshestvennik cells and activation of Mature neutrophils. G-CSF is used in medicine in the field of Hematology and Oncology. Two types of G-CSF is used to treat: glycosylated form (lenograstim), which is obtained by expression in mammalian cells, and deglycosylated form (filgrastim), which is obtained by expression in bacteriaEscherichia coli (E. coli).

Prerequisites to the creation of inventions

Deglycosylation form G-CSF (filgrastim) and the receipt described in European patent EP 237545 at that time, as glycosylated form of G-CSF (lenograstim) and the receipt described in European patent EP 169566.

Methods for cleaning and/or releasing G-CSF, which is known from the patent and scientific literature, contain various combinations of ion chromatography, chromatofocusing, hydrophobic chromatography, gel chromatography and other methods.

Basically, the first stage of the method of cleaning and/or releasing G-CSF head of the Sith from the host organism for heterologous expression of G-CSF. In the case of expression inE. coliG-CSF was found in the insoluble bodies inclusions. Therefore, conventional methods involve an additional stage of selection of G-CSF from Taurus inclusions, leading to correctly folded (stowed) biologically active form. These stages are usually washing detergents or autroprime substances, the solubilization strong denaturing reagents (e.g., guanidine hydrochloride (GndHCl), urea or detergents with a high concentration (for example, N-lauroylsarcosine (sarkasian) or sodium dodecyl sulfate (SDS)), partial purification solubilizing denatured protein by gel-filtration HPLC with reversed phase (RP-HPLC) and renaturation the dilution of the denaturing reagent or dialysis. In the case of expression of G-CSF in yeast or mammalian cells bullock inclusions or similar patterns find rare, and in these cases the implementation of the method of cleaning and/or selection start right after the secretion of G-CSF. Methods for cleaning and/or releasing G-CSF are described in the following patent applications and patents: European patent EP 169566, EP 237545, EP 215126, EP 243153, U.S. patent 5055555 and international publication WO 0104154. Methods for cleaning and/or releasing G-CSF is also described in the scientific literature: Lu, H.S. et al. inProtein Expr Purif4, 465-472 (1993), Kang, S.H. et al. inBiotechnol. Lett.17, 687-692 (1995), Wingfield P, et al. inBiochem. J.256, 213-218 (1988), Yamasaki, M. et al. inBiosci. Biotechnol. Biochem.62, 1528-1534 (1998), Bae, C.S. et al. inBiotechnol. Bioeng.57, 600-609 (1998).

In the case of some other proteins for the partial purification and renaturation also used IMAC. The first method IMAC described in Porath et al. inNature258, 598-599 (1975) and based on the binding of proteins with immobilized metal ions, which form chelates with various media for the IMAC. Electron-donor groups in the amino acid sequence of the protein responsible for the coordinated linking with the media, especially imidazole ring in histidine residues. The secretion of recombinant proteins with affinity labels to histidine at the N - or C-ends of the protein using IMAC described Hochuli, E. et al. inBio/Technology1321-1325 (1988), Chaga, G. et al. inBiotechnol. Appl. Biochem.29 (part 1), 19-24 (1999) and Jeong, J.K. and Lee S.Y. Protein Expr. Purif., 23: 311-318 (2001). The use of IMAC to study small topographical differences among protein molecules described in SulkowskiTrends Biotechnol.3, 1-7 (1985) and Hemdan et al. inProc. Natl. Acad. Sci. USA86, 1811-1815 (1989).

The method of purification by IMAC some other proteins that contain histidine residues described in U.S. patent 5932102.

Method of purification protein in which amino acids located on the surface of the molecule, capable of binding metal ions described in international publication WO 9012803. In this way the IMAC is used as an additional stage in the Les partial protein purification using some other chromatographic methods. Neither the selection nor the Department of Undenatured or biologically active molecules of G-CSF from denaturirovannykh or biologically inactive molecules of G-CSF under native conditions by IMAC is not described.

Comparative studies on G-CSF, the interaction of its Ser-17 and (His)6-labeled forms with metal ions by means of an affine distribution in relation to the specific complex of the dye-metal ion described in the literature (Zaveckas, M. et al. in J. Chromatogr. A 904, 145-169 (2000)). Based on the evaluation of the chromatographic behavior of bromelain and clean G-CSF for free from metal and containing Hg(II) column with IDA (iminodiacetate), Gelunaite, L. et al. inJ. Chromatogr.A 904, 131-143 (2000)made an attempt to assess the ability of Hg(II)-containing column (sepharose IDA) for IMAC extraction of G-CSF under denaturing conditions from Taurus inclusions, solubilizing detergent.

IMAC immobilized ions Zn(II) or Ni(II) is also used as a method renaturation of interleukin denatured GndHCl, G-CSF and granulocyte colony-stimulating-monocyte-macrophage factor (GM-CSF) (Rozenaite, V. et al. in Poster abstract P-104, Cordoba, Spain, 19-22. April (1998)). Denatured proteins bind to IMAC carrier under denaturing conditions.

A brief description of the invention

The aim of the invention is an improved method of cleaning and/or ejecta is G-CSF and obtaining biologically active G-CSF in high purity and active form, as well as pharmaceutical compositions containing the specified G-CSF.

The present invention relates to a method of cleaning and/or extraction of biologically active G-CSF presented in the independent claim in the claims. In addition, the present invention relates to a method of cleaning and/or extraction of biologically active G-CSF presented in the independent claim in the claims, biologically active G-CSF presented in the independent claim in the claims, the pharmaceutical compositions presented in the independent claim in the claims, and the use of biologically active G-CSF presented in the claims as an independent item. Preferred aspects are presented in the dependent claims.

According to the present invention it has been unexpectedly found that a correctly folded and biologically active molecule G-CSF can be separated from incorrectly folded or biologically inactive molecules of G-CSF under native conditions by applying the IMAC. The method of cleaning and/or extraction of biologically active G-CSF according to the present invention includes the separation of a properly folded and biologically active molecules of G-CSF from incorrectly folded or biologically inactive molecules G-CSF and also from most host proteins by applying IMAC PR is native conditions. According to this method correctly folded and biologically active molecule G-CSF specifically associated with the IMAC carrier at that time, as correctly folded, biologically active forms of G-CSF and most of the impurities remain in the eluate. The method of cleaning and/or selection according to the present invention also includes the separation of biologically active Monomeric form (G-CSF from oligomeric, polymeric and biologically inactive Monomeric form (G-CSF. Biologically active Monomeric form (G-CSF specifically associated with the IMAC carrier at that time, as oligomeric, polymeric and biologically inactive Monomeric form (G-CSF remain largely in the eluate.

The method according to the present invention is an effective stage purification and concentration of correctly folded, biologically active Monomeric form or molecules of G-CSF and can be used as a key stage in General, the method of cleaning and/or releasing G-CSF.

The method of cleaning and/or selection according to the present invention can be applied in the cases when G-CSF after expression of secreted directly through the path of secretion in the environment, or when G-CSF is formed in the form of Taurus inclusions in the cytoplasm, periplasm or any other cell organelle. The method can also be used for purification and/or separation of biologically active G-CSF p is yamo of solubilizing Taurus inclusions and in all cases, where G-CSF was first denaturiruet and then senatoriable. The method of cleaning and/or extraction of biologically active G-CSF according to the present invention can be performed in native conditions throughout the method.

The method of cleaning and/or extraction of biologically active G-CSF according to the present invention leads to the production of biologically active G-CSF with a purity of above 95%. Only two additional chromatographic phase, cation-exchange chromatography and gel filtration, which is used in a preferred aspect of the present invention, can be further used to remove trace amounts of residual impurities (treatment).

Therefore, in General, the method according to the present invention leads to the production of biologically active G-CSF with a purity of above 99%. The method is suitable for obtaining large quantities of biologically active G-CSF and suitable for industrial production of biologically active G-CSF.

Information about the Department biologically active, Monomeric, correctly folded molecules of G-CSF from the oligomer, polymer, or biologically inactive Monomeric form or improperly folded molecules of G-CSF using IMAC at native conditions has not been found either in the scientific or patent literature.

In publications from the previous prior art also does not have information regarding the cleaning and/or extraction of biologically active G-CSF from the raw solution or mixture, containing biologically active molecule G-CSF and impurities, by linking properly folded, biologically active Monomeric form (G-CSF (i.e., those molecules that are already present in the solution or mixture with a carrier for IMAC under native conditions. In addition, the separation of molecules of G-CSF according to their conformational state using the IMAC is not described.

All methods of cleaning and/or releasing G-CSF described in the previous prior art includes several stages. Use only one chromatographic stage for division G-CSF, respectively, of its biological activity and the degree of coagulation and separation from impurities present in the solution or mixture, as well as an efficient method for concentration of G-CSF and obtaining biologically active G-CSF with a purity of above 95%, so that only may require additional treatment, in the prior art are not described.

Detailed description of the invention and its preferred aspects

The present invention relates to the use IMAC as an effective chromatographic method in the method of cleaning and/or extraction of biologically active G-CSF. Correctly folded, biologically active Monomeric form or molecule G-CSF selectively contact the IMAC carrier under native conditions, while incorrectly folded or AG is Egorovna molecule G-CSF and a large part of other impurities, in particular, solubilizing proteins from Taurus inclusions after expression of, for example,E. colimostly not associated with the specified media and washed off, not on. I believe that this feature is due to a specific distribution of natural histidine residues.

The term "native conditions", which is used in this text refers to conditions under which a molecule (G-CSF protein) preserves the native conformation and biological activity.

The term "denaturing conditions" refers to conditions under which the native conformation of G-CSF protein is not stored, the biological activity is changed and not saved.

The term "aggregated molecules", which is used in this text, refers to molecules forming clusters of molecules that are held together by hydrophobic or some other interactions (e.g., disulfide bonds). These molecules are not biologically active.

The term "elution", as used herein, refers to the leaching or extraction of the adsorbed material from the chromatographic column.

The term "eluate", as used herein, refers to the solution, which is obtained by leaching and extraction of the chromatographic column.

The term "calf inclusions", use the given text, refers to insoluble compact units incorrectly folded or partially correctly folded proteins.

The term "solution Taurus inclusions", as used herein, refers to a solution which contains bullock inclusions.

The term "biologically active G-CSF", as used herein, refers to G-CSF, which promotes the differentiation and proliferation of hematopoietic progenitor cells and activation of Mature cells of the hematopoietic system.

The term "biologically active form (or molecule) G-CSF"as used in this text, refers to the form or molecule G-CSF, which is Monomeric and Undenatured state and which exhibits the above-mentioned biological activity.

The term "mixture", as used herein, refers to a substance which is different from the biologically active molecule G-CSF, so that the biologically active molecule G-CSF is not clean. The impurity may be at least one substance from the group consisting of biologically inactive Monomeric form and correctly folded molecules G-CSF, oligomeric and polymeric forms of G-CSF, denaturirovannykh forms of G-CSF and proteins of the host cell. The admixture may also be other substances of the host cell, such as DNA, (lipo)polysaccharides, etc. and additives, which are used when receiving and processing the G-CSF.

The purity specified in the text, refers to the purity, determined by HPLC.

The method of cleaning and/or extraction of biologically active G-CSF according to the present invention, in particular, determine the method comprising the following steps:

a) applying a solution or mixture that contains biologically active form of G-CSF and admixture on the IMAC-media;

b) selective binding of biologically active forms of G-CSF with IMAC carrier may rinsing IMAC-column, and

c) elution of the biologically active forms of G-CSF from the column.

The method according to the present invention can be advantageously carried out under native conditions.

The method of cleaning and/or extraction of biologically active G-CSF according to the present invention may optionally include the further purification of biologically active G-CSF and preferably comprises the following stages, which are performed after the cleaning and/or extraction of biologically active G-CSF by IMAC:

d) cation exchange chromatography and/or

(e) gel-filtration.

Full method according to the present invention leads to the production of biologically active G-CSF, suitable for clinical use in medicine.

Biologically active G-CSF, suitable for clinical use in medicine, can be obtained effective and preferred ability, the BOM purification and/or separation, in which the crude mixture containing G-CSF, under native conditions is subjected to chromatographic stage(s), which consists of the IMAC and possibly at least one of the treatment methods selected from ion-exchange chromatography and gel filtration. The method according to the present invention can also be applied in the case of cleaning and/or releasing derivatives of G-CSF, such as methionyl-G-CSF (Met-G-CSF), glycosylated, enzymatic or chemically modified (e.g., targeted) G-CSF, analogs of G-CSF and fused proteins containing G-CSF.

Significant advantages of the method of cleaning and/or selection according to the present invention are that:

1) the molecules of G-CSF are separated according to their conformational state and therefore according to their biological activity;

2) the method makes possible the binding of biologically active molecules of G-CSF with IMAC carrier in native conditions and subsequently the separation of biologically active molecules of G-CSF from biologically inactive molecules of G-CSF under native conditions;

3) the method makes possible the linking properly folded molecules of G-CSF with IMAC carrier in native conditions followed by the separation of correctly folded molecules of G-CSF from improperly folded molecules of G-CSF under native conditions;

4) the method makes possible the linking biological engineering the active Monomeric form (G-CSF with IMAC carrier in native conditions and subsequently the separation of biologically active Monomeric form (G-CSF from biologically inactive Monomeric form (G-CSF;

5) the method makes possible the binding of Monomeric forms of G-CSF with IMAC carrier and subsequently separating Monomeric forms of G-CSF from oligo - and polymeric forms of G-CSF under native conditions;

6) the method makes possible the separation in native conditions correctly folded Monomeric, biologically active molecules of G-CSF from other proteins and impurities, which are present in the solution, mixture or the environment;

7) thanks to the method according to the present invention can considerably improve the specific activity of the purified G-CSF, for example, to the level of specific activity of at least 1·107IU/mg, more preferably up to the level of specific activity 7-8·107IU/mg, most preferably to the level of specific activity of about 1·108IU/mg, where specific activity is measured by a method based on the stimulation of cell proliferation as described in example 5;

8) thanks to the method according to the present invention can be obtained G-CSF with high yield and with a purity of at least 95% or, in the exercise of further purification by cation-exchange chromatography and gel filtration according to a preferred aspect, even at least 99%, and therefore the method is suitable for industrial production of biologically active G-CSF;

9) a complete method of cleaning and/or extraction of biologically sports the G-CSF according to a preferred aspect, which also includes clearing cation exchange chromatography and gel filtration, does not require any additional stages of purification of G-CSF and may preferably be performed under native conditions throughout.

The full method of cleaning and/or extraction of biologically active G-CSF according to the present invention is most preferably carried out under native conditions.

The method according to the present invention is not a method renaturation of correctly folded molecules G-CSF, but is a method that involves specific binding to IMAC carrier Undenatured or properly folded, biologically active Monomeric molecules of G-CSF, which are already present in the solution, mixture or medium containing crude G-CSF.

The separation of the monomer from oligo - and polymeric forms of G-CSF is such that the biologically active Monomeric form (G-CSF are associated with the IMAC carrier at that time, as oligo - and polymeric forms, which may also be present in aggregate form, remain largely in the eluate.

To separate the Monomeric form (G-CSF from oligo - and polymeric forms of G-CSF instead of the IMAC can be applied to gel filtration. The advantage of the IMAC over the gel filtration is concentrating ability, higher binding capacity and ability and to separate correctly folded Monomeric form (G-CSF from correctly folded Monomeric forms of G-CSF. Gel-filtration cannot be separated correctly folded Monomeric form (G-CSF from correctly folded Monomeric forms of G-CSF. Thus, when using the IMAC, you can achieve the best results.

Another advantage of the method of cleaning and/or extraction of biologically active G-CSF according to the present invention over other methods, known from the previous prior art, lies in the fact that under native conditions, which support throughout the way) because of the way that it is possible to allocate a correctly folded molecule G-CSF from a mixture of different proteins and also from a mixture of molecules of G-CSF present in different conformational States. Therefore, direct selection of G-CSF from (preferably diluted) culture medium or, in particular, from Taurus inclusions under native conditions is possible.

An additional advantage of the method according to the present invention over conventional methods are: the possibility to reduce or eliminate the use of detergents, the opportunity to work in the absence of denaturing reagents that are either toxic or bad for the environment (for example, GndHCl or urea), and the ability to reduce or eliminate the use of buffers and other solutions.

The advantage of the method of cleaning and/or extraction of biologically active is th G-CSF according to the present invention over the methods, employ strong denaturing reagents, is that this method does not require the use of active reducing reagents, such dithiothreitol or beta-mercaptoethanol.

If the secretion of G-CSF directly into the environment, a method of cleaning and/or extraction of biologically active G-CSF according to the present invention enables effective and immediate concentration of biologically active G-CSF from the dilute environments. Biologically active G-CSF high purity receive in the eluate from the IMAC columns.

Since the effective separation of biologically active molecules of G-CSF from a biologically inactive molecule G-CSF and other impurities can be achieved either by separating the biologically active molecule G-CSF from Taurus inclusions, and/or by separating the biologically active molecule G-CSF directly from solution or containing mixture, the method of cleaning and/or releasing G-CSF according to the invention is more cost effective than other methods.

Thus, the purity of G-CSF more than 95% can be achieved using only one stage of cleaning.

The following chromatographic stage(s) is(are) particularly suitable for final treatment (purification) of biologically active G-CSF after elution from IMAC-column:

- cation exchange chromatography and/or;

<> gel-filtration.

Cation exchange chromatography is particularly effective for the removal of trace amounts of nucleic acids, lipopolysaccharides and proteins from the host cells and to remove ion isomers of G-CSF and changed (corrupted) forms of G-CSF with modified values of pI. Gel filtration is particularly effective for the removal of trace amounts of dimers and higher aggregated forms of G-CSF.

The application of an additional two end stages of purification leads to purity of biologically active G-CSF above 99%.

Advantages of the complete method of cleaning and/or extraction of biologically active G-CSF according to a preferred aspect of the invention, which additionally includes cation exchange chromatography and gel filtration are as follows: in addition to the respective stages of pre-processing, such as solubilization and possibly subsequent removal solubilizing reagent by dialysis, ion exchange, ultrafiltration, diafiltration or dilution or the like, the method can effectively include only the above three chromatographic stage; between the three chromatographic stages preferably there are no intermediary stages (like concentration, dialysis, precipitation and so on), and native conditions preferably support for the present method of cleaning and/or selection.

Intermediate stage of concentration will cause the formation of dimers and other aggregates, resulting in reduced output. The full method of cleaning and/or selection can be transferred to the industrial scale for the production of biologically active G-CSF with a purity of at least 99%.

Biologically active G-CSF, the method for treating and/or selection according to the present invention is suitable for preparing a pharmaceutical composition that contains a therapeutically effective amount of a biologically active G-CSF, and suitable for clinical application.

The ability to save the active forms of G-CSF in a short period of purification method and allocation contributes not only to increase output, but also in increasing the purity and efficacy of biologically active G-CSF-containing pharmaceutical compositions.

The term "therapeutically effective amount", as used herein, refers to the amount of biologically active G-CSF, which provides therapeutic effect of biologically active G-CSF.

Suitable pharmaceutically acceptable excipients include suitable diluents, adjuvants and/or carriers used in therapy with G-CSF.

Biologically active G-CSF, which was obtained by the method according to astasia the invention, especially when using additional stages cation-exchange chromatography and gel filtration, can be used for the preparation of drugs, which are designed with readings selected from the group consisting of: neutropenia and associated with neutropenia clinical complications, reduction in hospitalization due to febrile neutropenia after chemotherapy, the mobilization of hematopoietic predshestvennik cells, as an alternative to infusion of donor leukocytes, chronic neutropenia, infections in patients with neutropenia and infections that are not associated with neutropenia, recipients of transplants, chronic inflammatory conditions, sepsis and septic shock, reducing the risk of morbidity, mortality, number of days of hospitalization due infection with neutropenia and infection associated with neutropenia, infection prevention and related infection complications in patients with infection during neutropenia in patients with infection associated with neutropenia, prevention of nosocomial infections and lower mortality rates and frequency of nosocomial infections, enteric introduction newborn, strengthening the immune system of newborns, improve clinical outcome in patients in intensive care units and patients in critical condition, healing and the treatment of wounds to the data sores/burns, intensification of chemotherapy and/or radiotherapy, pancytopenia, increased levels of inflammatory cytokines, reduction of intervals of high doses of chemotherapy with the use of filgrastim prophylaxis, the potentiation of the antitumor effects of photodynamic therapy, prevention and treatment of diseases caused by various cerebral disorders, the treatment of thrombotic disease and its complications and recovery of erythropoiesis after irradiation.

In addition, the factor can be used to treat all other diseases for which G-CSF is shown.

Pharmaceutical composition, containing pure and biologically active G-CSF obtained by the method according to the present invention can also be introduced to patients in therapeutic amount that is effective for the treatment of the aforementioned diseases, by known qualified specialists in this field.

Next will be described the preferred aspects of the implementation IMAC in the method according to the present invention.

The method begins with the application of the sample and binding, at least, biologically active forms of G-CSF protein with IMAC-bearer.

IMAC-carrier is a solid-phase material and chelate compounds of metal ions associated with the solid phase material. Conventional solid-phase m the materials can be materials, used appropriately, such as sepharose (Sepharose), fractogel (Fractogel) and other gel mediums. Metal ions chelate compounds associated with the IMAC carrier, choose a suitable way of metal ions, preferably divalent metals, especially transition metal ions. Metal Hg is the least appropriate, on the basis of its toxicity, and its tendency to leach from the IMAC speakers. Preferred examples of the chelating metal ions associated with the IMAC carrier, are: M(II)-iminodiacetate (IDA), M(II)-nitrilotriacetic acid (NTA), M(II)-carboxymethylstarch etc. where M represents Zn, Cu, Co, Ni, etc. are Particularly effective Zn(II)-IDA, Ni(II)-IDA and Ni(II)-NTA.

To load IMAC-column bullock inclusions preferably presented in the form of a solution or in the case of periodic separation processes or ways of breaking loose layer in the form of a suspension in the presence of detergent or solubilizing reagent with low concentrations. Thus, G-CSF may be in native conditions or when the detergent remain in solution or suspension, or when they are removed using ion exchangers, dialysis, precipitation, etc. Detergent or solubilizers reagent preferably removed before applying the solution or mixture on the IMAC column.

The solution or suspension (or a mixture) tel is C inclusions in the presence of strong denaturing reagents, such as 8 M urea or 6 M GndHCl, or solutions in the presence of denaturing concentrations of detergents (e.g., 1% sarkosyl, 2% akrosil or 1% sodium dodecyl sulphate) can also be used as the source of the sample, if applied to the column, the sample was previously subjected to renaturation, for example, by dilution, dialysis, ultrafiltration or removal of denaturing reagents/detergents.

In the case of the expression of factor secreting systems, such as yeast, fungi or lines of mammalian cells, supernatant or concentrated supernatant or culture medium, a mixture which initially processed, can be applied to the media for the IMAC at pH in the range from 6.5 to 9.0.

The eluate emerging from the IMAC speakers in the first elution, can be used as a coating solution or mixture for IMAC. the pH of the eluate should be brought, for example, by adding NaOH solution or buffer solution with a high pH to a pH in the range from 6.5 to 9.0. Then the eluate is again put on the same IMAC-media chelates, such as Zn(II)-IDA, Ni(II)-IDA, Ni(II)-NTA, or on another IMAC-media. Possible combination with other immobilized metal ions (for example, Zn(II), Cu(II), Co(II), Ni(II), and so on), and it promotes better separation and removal of specific proteins of the host.

Regardless of as what cooked applied the solution, the pH of coating solution should be in the range from 6.5 to 9.0. The preferred pH of coating solution is from 7.0 to 8.5, the most preferred pH ranges from 7.8 to 8.2.

Various buffers, which can maintain the pH in the range from 6.5 to 9.0, can be used for drawing the sample and binding of G-CSF with IMAC carrier. Suitable for these purposes buffers are phosphate, acetate, based on hydroxyethylaminomethyl (Tris/HCl), Tris/acetate, citrate, and other buffers, creating a pH from 6.5 to 9.0. Preferably, use of Tris/HCl buffer.

Buffer, especially Tris/HCl buffer, preferably used at a concentration in the range from 5 to 50 mm, most preferably in the range of from 10 to 40 mm.

After binding with the carrier, the method continues by washing the column and elution of proteins from the column. Elution can be performed using either discontinuous step gradient or linear gradient by lowering the pH or by competitive elution at high pH value (for example, imidazole, histidine, ammonium chloride and the like).

The term "linear gradient", as used in this text refers to washing the chromatographic column with a solution whose composition is changed so that a part of one buffer (or one component buffer) linear led is ensured at the time, as part of another buffer (or other component buffer) linearly decreases.

The term "discontinuous step gradient used in this text refers to washing the chromatographic column with a solution, which consists of a certain part of one buffer (or one component of the buffer) and a specific part of another buffer (or another component of the buffer), within a certain period of time. The proportions of both buffers quickly (suddenly) change, and the column is washed for another certain period of time. The composition of the solution (changing the proportions of the buffer or proportions of components) does not change linearly.

The term "competitive elution", as used herein, refers to elution at pH of the buffer used for binding, where competitive molecules, such as imidazole, histidine, ammonium chloride, etc. in lucianna buffer associated with metal forming chelates with the matrix, and, thus, displace protein molecules.

Preferably, use a discontinuous step gradient, resulting in elution at low pH value. Namely, high pH can lead to activation of the cysteine residues and the formation of dimers. The stability of G-CSF is high at low pH value.

Several lucianic buffers can be used for intermittent stupen is atogo or linear washing and elution, and may be selected from the group consisting of: acetate, Tris/acetate, phosphate, citrate buffer, and other suitable buffers. The range of pH for elution may range from 3.0 to 5.0, preferably 3.5 to 4.5. For discontinuous step gradient of pH changes rapidly from about pH values of the applied solution to about pH values lucynova buffer, for example, from 7.0 to 4.0, and isoelectric point, thus, changes abruptly, which prevents the precipitation of proteins.

Thus, the eluate get Monomeric, biologically active, correctly folded G-CSF with a purity of above 95%.

If desirable, the eluate obtained from IMAC-column, can be applied directly on the cation-exchange chromatographic column without any additional intermediate stages. You can apply various cation-exchange chromatographic media, and they can be selected from the group consisting of: SP Sepharose FF, SP Sepharose HP, CM Sepharose FF, TSK gel SP-5PW, TSK gel SP-5PW-HR, Toyopearl SP-650M, Toyopearl SP-650S, Toyopearl SP-650C, Toyopearl CM-650M, Toyopearl CM-650S, Macro-Prep High S media, Macro-Prep S media, Macro-Prep CM media etc. are Preferably used SP Sepharose FF or TSK gel SP-5PW.

The pH value is applied to a column of the solution to cation exchange chromatography is in the range from 3.0 to 5.8, preferably in the range of from 4.0 to 5.0.

The salt concentration in applied Colo is the top solution for cation exchange chromatography should be low enough to happen binding, which also depends on the pH of the solution.

Different buffers with pH values ranging from 3.0 to 5.8 can be used for applying and binding medium for cation-exchange chromatography and can be selected from the group consisting of: acetate, citrate, Tris/HCl, Tris/acetate, phosphate, succinate, malonate buffer, based buffer 2-(N-morpholinepropanesulfonic) (MES) and other buffers. Preferably, apply sodium acetate buffer.

Acetate buffer can be used at a concentration in the range from 10 to 60 mm, preferably at a concentration in the range from 10 to 30 mm.

When cation exchange chromatography for application to the column followed by washing of the column and elution of proteins from the column. Elution of proteins is due to the higher values of ionic strength after addition of a buffer salt solution with high concentration. You can use discontinuous step gradient, linear gradient, and the appropriate combination of step and linear gradient.

Eluting buffers that can be used for washing and elution, can be selected from the group consisting of: acetate, citrate, Tris/HCl, Tris/acetate, phosphate, succinate, malonate buffer, MES buffer and other suitable buffers with the addition of salts such as NaCl or KCl Ionic strength and salt concentration, with the help of which elution is achieved depends on the pH of buffer solution. The higher the pH of the buffer, the lower the ionic strength required for elution of proteins from the column.

Thus, the eluate get Monomeric, biologically active, correctly folded G-CSF with purity above 98%.

If desirable, the eluate obtained from the IMAC speakers or, preferably, after following cation exchange chromatography column, can be applied directly to the gel-filtration column without any additional intermediate stages.

Various media for gel filtration can be applied can be selected from the group consisting of: Sephacryl S-200HR, Sephacryl S-100HR, Superose 12, Superose 6, Superdex 75, TSK gel G-2500PW, TSK gel G-3000PW, Bio-Gel P-60, Bio-Gel P-100, etc. are Preferably used Superdex 75.

For gel filtration can be applied to a wide range of pH, and therefore, as applied to a column of solution is appropriate eluate obtained from IMAC-column and, preferably, from subsequent cation exchange chromatography. The application of the solution, the binding of the protein with a carrier for gel filtration and elution of the protein can be carried out using the same buffer. Various buffers can be used and can be selected from the group consisting of: citrate, acetate, Tris-buffer, phosphate buffer, and other suitable buffer is in, which can maintain the pH in the range from 3.5 to 8.0. Preferably, use phosphate buffers with pH from 6.0 to 7.0.

Preferably, the phosphate buffers (for the application) can be used at a concentration in the range from 2 to 100 mm, preferably at a concentration in the range between 3 and 10 mm.

The salt concentration in the buffer for gel filtration can be in the range from 30 to 100 mm, preferably approximately 50 mm.

Thus, the eluate get Monomeric, biologically active, correctly folded G-CSF with a purity of above 99% and biological activity of 1×108IU/mg.

The following examples are given to illustrate various aspects of the invention, and they do not seek in any way to limit the present invention.

Description of the drawings

Figure 1 presents the results of the chromatographic separation of proteins from solubilizing Taurus inclusions when using media for IMAC: Zn(II)-IDA Chelating Sepharose fast flow (Pharmacia).

The chromatogram shows the change of absorption at 280 nm (A) (-) and the ratio of buffer P3 (----) depending on time (min.)

Peak A -E. coliproteins and aggregated G-CSF; peak - Monomeric, correctly folded, biologically active G-CSF and trace amounts of proteinsE. coli.

Figure 2 shows the results of polyacrylamide gel electrophoresis in Pris is accordance sodium dodecyl sulfate (SDS-PAGE) of the original sample and samples presented in chromatographic peaks after separation when using Zn(II)-IDA Chelating Sepharose fast flow (Pharmacia), according to figure 1.

Legend:

1. The molecular weight standards (Bio-Rad) and G-CSF (Neupogen) (marked by arrow).

2. The original sample prior to chromatographic separation.

3. Proteins in the peak a in figure 1 (aggregate G-CSF and proteinsE. coli).

4. Proteins in the peak In figure 1 (Monomeric, correctly folded, biologically active G-CSF and trace amounts of proteinsE. coli).

Figure 3 presents the results of the chromatographic separation by using column HK/20 for IMAC with a native Zn(II)-IDA Chelating Sepharose fast flow (Pharmacia).

The chromatogram shows the change of absorption at 280 nm (A) (-) and the proportions of the buffer P6 (----) depending on time (min.)

Peak A -E. coliproteins and aggregated G-CSF; peak - Monomeric, correctly folded, biologically active G-CSF and trace amounts of proteinsE. coli.

Figure 4 presents the results of the chromatographic separation using column XK 16/20 filled with cation exchange media TSK gel SP-5PW (TosoHaas).

The chromatogram shows the change of absorption at 280 nm (A) (-) and the proportion of the buffer R8 (----) depending on time (min.)

The main peak is Monomeric, correctly folded, biologically active G-CSF; small peaks isoforms of G-CSF and trace amounts Bel is s E. coli.

Figure 5 presents the results of the chromatographic separation using column XK 26/70 filled carrier for gel filtration Superdex™ 75 prep grade (Pharmacia).

The chromatogram shows the change of absorption at 280 nm (A) depending on time (min.)

The main peak is Monomeric, correctly folded, biologically active G-CSF.

EXAMPLES

Example 1:Cleaning and/or secretion of biologically active G-CSF by IMAC: Zn-IDA (Chelating Sepharose fast flow)

Chromatographic column (h=10 cm, d=10 mm) was filled with media Chelating Sepharose fast flow (Pharmacia), which were associated ions Zn2+and the column was balanced with 5 column volumes of buffer P2 at a constant flow of 2 ml/min. of Taurus inclusions were solubilizers buffer P1 (0,2% sarkosyl, 40 mm Tris/HCl, pH 8.0), and sarkosyl was removed with an ion exchanger. In the column was brought to 10 ml of solution (17 mg total protein), which contains biologically active G-CSF, at constant flow of 1 ml/min Separation was carried out by using a discontinuous step gradient of buffer P3 at a constant flow rate of 2 ml/min (figure 1). In the first chromatographic peak (peak A) proteins were foundE. coliand a large part of incorrectly folded and aggregated G-CSF. At 100% buffer P3 (0% buffer P2) from the column was suirable mostly pure Monomeric and biological and active G-CSF (7 mg) with purity higher than 95% (figure 2).

The biological activity of the sample from the peak And was determined, as described below in example 5, and it was 1×106IU/mg protein, specific biological activity of the sample from a peak of 0.8 to 1.0×108IU/mg protein, while the biological activity of the standard was 1×108IU/mg protein. Single-stage IMAC initial solution Taurus inclusions leads to the separation of Monomeric forms of G-CSF with a purity of above 95% and biological activity, which is comparable to the activity of the standard.

Example 2:Cleaning and/or secretion of biologically active G-CSF by IMAC: Zn-IDA (Fractogel EMD Chelate)

Chromatographic column (h=2 cm, d=10 mm) was filled with media Fractogel EMD Chelate (Merck), which were associated ions Zn2+. The column was washed with water and balanced with five column volumes of buffer P2 at a constant flow rate of 1 ml/min. of Taurus inclusions were solubilizers buffer P1 and sarkosyl was removed with an ion exchanger. The column was loaded with 3.3 ml solubilizing Taurus inclusions total number of 4 mg. Separation was carried out by a discontinuous step gradient of buffer P3 at a constant flow rate of 1 ml/min (gradient: 0% buffer P3 (100% buffer P2) 13 min, 25% of buffer P3 (75% buffer P2) 12 min, 100% buffer P3 (0% buffer P2) 14 min, 0% buffer P3 (100% buffer P2) 11 min). At 100% buffer P3 was suirable monomers the th, biologically active G-CSF (0.7 mg).

Example 3:Cleaning and/or secretion of biologically active G-CSF by IMAC: Ni-NTA (Superflow)

Chromatographic column (h=10 cm, d=10 mm) was filled with media Ni-NTA Superflow (Qiagen) and was balanced with five column volumes of buffer P4 at a constant flow rate of 2 ml/min. of Taurus inclusions were solubilizers buffer P1 and sarkosyl was removed with an ion exchanger. The column was loaded with 10 ml solubilizing Taurus inclusions (10 mg total protein) at a constant flow rate of 1 ml/min Separation was carried out by a discontinuous step gradient of buffer P5 at a constant flow rate of 2 ml/min (the same gradient as in the separation of the Zn-IDA Chelating Sepharose fast flow presented in figure 1). At 100% buffer P5 (0% buffer P4) was suirable Monomeric, biologically active G-CSF (3.6 mg) with purity higher than 95%. In the first chromatographic peak in addition to proteinsE. colifound only correctly folded and aggregated G-CSF.Monomeric forms of G-CSF from the second peak after several chromatographic separations by Ni-NTA Superflow United, and final clearance (post-treatment) was carried out by cation exchange chromatography and gel filtration.

Example 4:The method of cleaning and/or extraction of biologically active G-CSF, including additional chromatographic stud and

The method of cleaning and/or extraction of biologically active G-CSF was started with the solution Taurus inclusions, which was prepared after the expression of G-CSF inE. coli. Washed calf inclusions in the amount of 4.5 g resuspendable in 225 ml of buffer P1 and left to dissolve at 20°C for 18 hours under careful (light) shaking, using a linear shaker. The sample was diluted with water to obtain the dual volume and ~430 ml Taurus inclusions was obtained with the protein concentration of ~1.4 mg/ml (determined by the method of Bradford on G-CSF as standard). The protein solution was divided into two equal volume, was applied to the chromatographic column HK/20 (Pharmacia), filled with media Chelating Sepharose fast flow (45-165 μm, Pharmacia) to a height of 10 cm (h=10 cm, d=5 cm, V=200 ml), which were associated ions Zn2+. The sample application and elution was carried out at constant flow rate 7 ml/min Column after application of the sample was washed through a discontinuous step gradient (figure 3): 15 min buffer P2, then 45 min with a mixture of buffer P2 and P6 in the volume ratio of 75:25 86 min buffer P6. Monomeric form of biologically active G-CSF was suirable at 100% buffer P6. All factions (two separate)containing Monomeric, biologically active G-CSF, were combined and received 271 ml with a protein concentration of ~0.7 mg/ml of this solution was added EDTA to a final conc is Tracii 2 mm. The solution was diluted three times with 20 mm CH3COOH, pH 4.0 and used as a solution, subjected to cation exchange chromatography.

The eluate from the IMAC speakers in two aliquot was applied to a chromatographic column HK/20 (Pharmacia), filled with chromatographic media SP-5PW (30 μm; TosoHaas) up to a height of 16 cm (h=16 cm, d=1.6 cm, V=32 ml). The sample application and elution from the column was carried out at a constant flow rate of 5 ml/min After application of the sample the column was washed for 11 min buffer P7, followed by elution by a linear gradient buffer of P8 30 min from 0% to 25% of buffer R8 (from 100% to 75% buffer P7). The column was again washed for 16 min with a mixture of buffers P7 and P8 in the volume ratio of 75:25 and then for 22 min buffer R8 (figure 4). Fractions of the main chromatographic peak, which was suirable in the linear part of the linear gradient at ~18% buffer R8 and which contained the correctly folded G-CSF (with purity above 98%), were combined and directly used as solutions for gel-filtration column.

The eluate from the cation exchange chromatography (V=46 ml, a protein concentration of ~2.4 mg/ml) was applied in 5 aliquot to the chromatographic column HK/70 (Pharmacia), filled carrier for gel filtration Superdex 75 (prep grade, 34 μm) (Pharmacia) to a height of 57 cm (h=57 cm, d=2.6 cm, V=300 ml). The separation was carried out in a buffer P9 at a constant ck the grow flow 2.5 ml/min Peak, which corresponds to a protein dimer, clearly separated from the main peak, which corresponds to the Monomeric protein (figure 5). Fractions of the main chromatographic peak were pooled, and the buffer was changed and received 100 mg of pure Monomeric form (G-CSF with a purity of above 99% and biological activity of 1×108IU/mg, which corresponds to the biological activity of the standard.

Example 5:Analysis of biological activity of G-CSFin vitro

The biological activity of G-CSF was determined by the method based on the stimulation of cell proliferation (NFS-60 cells), using the known method (Hammerling, U. et al. inJ. Pharm. Biomed. Anal.13, 9-20 (1995)), and the international standard G-CSF human recombinant G-CSF (88/502 produced by yeast cells; NIBSC, Potters Bar, Hertfordshire, UK; Mire-Sluis, A.R. et al. v.J. Immunol. Methods179, 117-126 (1995).

The compositions of buffers:

P1: 0,2% sarkosyl, 40 mm Tris/HCl, pH 8.0.

P2: 20 mm Tris/HCl, 150 mm NaCl pH 8.0.

P3: 20 mm acetic acid, 150 mm NaCl, pH is brought to 4.5 by addition of 1 M NaOH.

P4: 10 mm Tris/HCl, 200 mm NaCl, pH 8.0.

P5: 20 mm acetic acid, 200 mm NaCl, pH increased to 4.0 by addition of 1 M NaOH.

P6: 20 mm CH3COOH, 150 mm NaCl, pH 4.0.

P7: 20 mm CH3COOH, pH 5.5.

R8: 20 mm CH3COOH, 500 mm NaCl, pH 5.5.

P9: 5 mm Na phosphate, 50 mm NaCl, pH 7.0.

1. The method of cleaning and/or extraction of biologically active G-CSF, which includes:
PR is cooking, mix, which contains biologically active G-CSF in the presence of impurities, and
separation of this mixture by affinity chromatography with immobilized metal (IMAC),
which includes the following stages:
application of this mixture, which contains biologically active G-CSF in the presence of impurities on the media for IMAC
selective binding of biologically active forms of G-CSF with the carrier for IMAC and
elution of the biologically active forms of G-CSF from the column with the carrier for IMAC with obtaining biologically active G-CSF, where the IMAC is performed using chelate compounds, metal ion associated with the carrier for IMAC, where the metal ion is not Hg.

2. The method according to claim 1, where the mixture contains a mixture, which represents at least one substance from the group consisting of biologically inactive Monomeric form and correctly folded molecules G-CSF, oligomeric and polymeric forms of G-CSF, denaturirovannykh forms of G-CSF, proteins of the host cells and other impurities (components) of the host cell;
and IMAC carried out so that the impurity is mainly associated with the carrier for IMAC and eluted from the column for IMAC before elution of the biologically active forms of G-CSF.

3. The method according to claim 1 or 2, where the biologically active G-CSF is at least one selected from the group consisting of negligible is consistent G-CSF, glycosylated G-CSF, methionyl-G-CSF analogs of G-CSF, enzymatic or chemically modified forms of G-CSF and fused proteins containing G-CSF.

4. The method according to claim 1 or 2, where specified containing G-CSF mixture selected from the group consisting of:
mix, medium or solution, prepared after denaturation, followed by denaturaciei;
the solution or suspension Taurus inclusions under native conditions;
the mixture or solution, prepared from the supernatant after expression in secreting systems or from the culture medium expressing system;
and the eluate, which get previous elution G-CSF from the column for IMAC or any other chromatographic column.

5. The method according to claim 1 or 4, wherein said mixture contains a solution or suspension Taurus inclusions under native conditions.

6. The method according to claim 1 or 2, where the IMAC is performed using chelate compounds, metal ion associated with the carrier for IMAC selected from the group consisting of M(II)-iminodiacetate, M(II)-nitrilotriacetic acid and M(II)-carboxymethylstarch, where M is chosen from the group consisting of Zn, Cu, Co and Ni.

7. The method according to claim 6, where the chelate compound of a metal ion associated with the carrier for IMAC, selected from the group consisting of Zn(II)-iminodiacetate, Ni(II)-iminodiacetate and Ni(II)-nitrilotriacetic acid.

8. The method according to claim 1 or 2, where the biologically active G-CSF obtained by IMAC has a purity of at least 95%.

9. The method according to claim 1 or 2, further comprising a phase(s): cation-exchange chromatography and/or gel filtration chromatography for purification.

10. The method according to claim 9, where the biologically active G-CSF obtained in the chromatographic stages, has a purity of at least 99%.

11. The method according to claim 1, where G-CSF is subjected to chromatographic stage(s), which consists of the IMAC, and optionally at least one of the treatment methods selected from ion-exchange chromatography and gel filtration.

12. The method according to claim 9, where containing G-CSF mixture is essentially free of detergent or solubilizing reagent or contains detergent or solubilizers reagent at a concentration that allows for the presence of G-CSF in native conditions.

13. The method according to claim 11 or 12, where the biologically active G-CSF obtained by chromatographic stages, has a purity of at least 95%, preferably at least 99%.

14. The method according to any one of claims 1, 2, 11 or 12, where the whole process is carried out under native conditions.

15. The method according to any of the preceding paragraphs, further comprising a stage of application of G-CSF obtained for the preparation of medicines.

16. The method according to clause 15, where drugs are used when indicated, selected the C group, consisting of: neutropenia and associated with neutropenia clinical complications, reduction in hospitalization due to febrile neutropenia after chemotherapy, mobilization of hematopoietic progenitor cells, as an alternative to infusion of donor leukocytes, chronic neutropenia, infections in patients with neutropenia and infections that are not associated with neutropenia, recipients of transplants, chronic inflammatory conditions, sepsis and septic shock, reducing the risk of morbidity, mortality, number of days of hospitalization due to infection with neutropenia and infection associated with neutropenia, infection prevention and related infection complications in patients with infection during neutropenia in patients infection associated with neutropenia, prevention of nosocomial infections and lower mortality rates and frequency of nosocomial infections, enteric introduction newborn, strengthening the immune system of newborns, improve clinical outcome in patients in intensive care units and patients in critical condition, healing and the treatment of wounds/skin ulcers/burns, intensification of chemotherapy and/or radiotherapy, pancytopenia, increased levels of inflammatory cytokines, reduction of intervals of high doses of chemotherapy with the use of filgrastim order p is opractice, the potentiation of the antitumor effects of photodynamic therapy, prevention and treatment of diseases caused by various cerebral disorders, the treatment of thrombotic disease and its complications and recovery of erythropoiesis after irradiation.



 

Same patents:

FIELD: biotechnology.

SUBSTANCE: the invention relates to producing new peptides and may be used for treatment and prophylaxis of cytokine-sensitive disorders. Peptides, having a size of 5 to 40 amino acids and arising from cytokines, are used in a vaccine for treatment and prophylaxis of autoimmune diseases, disseminated sclerosis, rheumatoid polyarthritis, psoriasis, autoimmune diabeteses, lupus, allergy, asthma, cancer and AIDS.

EFFECT: allows effective immunization of patients against said diseases while minimizing side effects.

11 cl, 2 dwg, 17 tbl

FIELD: biotechnology, transplantation, in particular, isolation of hematopoietic stem cells.

SUBSTANCE: Lin-negative or weak positive cells derived from bone marrow are cultivated in presence of macrophage colony-stimulating factor (M-CSF) at concentration of approximately 5-20 ng/ml together with cytokine composition including stem cell factor (SCF), interleukin-11 (IL-11) ligand such as fms-like tyrosine kinase (FLT) or thrombocytopoietin (TPO), wherein cytokines are used in the next weight ratio: 1-4 M-CSF:20 SCF:20 IL-11:20 (FLT or TPO).

EFFECT: effective method for targeted propagation of hematopoietic stem cells.

4 cl, 3 dwg, 1 ex

The invention relates to biotechnology, in particular genetic engineering, and can be used to produce secreted modified colony-stimulating factor granulocyte person (hG-CSF)

FIELD: medicine.

SUBSTANCE: claimed invention relates to field of molecular biology, virology and medicine. Claimed is application of adenovirus for manufacturing medication for tumor treatment. Said virus is replication-deficient in cells which do not contain YB-1 in nucleus and encodes oncogenic protein E1A, which transactivates at least one viral gene from group including E1B55kDa, E4orf6, E4orf6 and E3ADP.

EFFECT: invention can be used for treating tumors demonstrating resistance to many cytostatic medications.

19 cl, 19 dwg, 13 ex

FIELD: chemistry, pharmaceutics.

SUBSTANCE: claimed invention relates to method of acidifying of one or several amino groups of peptide which is selected from group including exendin-3, exendin-4, Arg34-GLP-1(7-37), Gly8-GLP-1(7-36)-amide, Gly8-GLP-1(7-37), Val8-GLP-1(7-36)-amide, Val8-GLP-1(7-37), Val8Asp22-GLP-1(7-36)-amide, Val8Asp22-GLP-1(7-37), Val8Glu22-GLP-1(7-36)-amide, Val8Glu22-GLP-1(7-37), Val8Lys22-GLP-1(7-36)-amide, Val8Lys22-GLP-1(7-37), Val8Arg22-GLP-1(7-36)-amide, Val8Arg22-GLP-1(7-37), Val8His22-GLP-1(7-36)-amide, Val8His22-GLP-1(7-37), des(B30)-human insulin and their analogues, in which reaction of acidifying is carried out in water mixture containing less than 10% wt/wt aprotonic polar solvent, and interaction of peptide with acidifying agent of general formula I is carried out, where n is 0-8; R1 represents COOR4; R2 represents lipophilic part of molecule; R3 together with carboxyl group, to which R3 is bound, represents reaction-able ester or reaction-able N-hydroxyimidoesther; and R4 is selected from group including hydrogen, C1-12-alkyl and benzyl; in base conditions in water solution, acidifying agent being added to reaction mixture in form of solution stabilised by adding acid.

EFFECT: obtaining efficient method of peptide acidifying.

17 cl, 2 tbl, 8 ex

FIELD: chemistry, biochemistry.

SUBSTANCE: invention refers to biochemistry and can be used in pharmaceutical industry for mixture purification of natural mixtures containing plasminogen and fibrinogen from plasminogen. Plasminogen is removed from mixture containing plasminogen and fibrinogen using insoluble chromatography matrix that is covalently cross-linked with tranexamic acid through amino group with linker of length exceeding three carbon atoms. Therefore mixture mentioned above is applied on chromatographic column containing specified insoluble matrix. Then column is washed with neutral solution containing salts, while unbound material is collected.

EFFECT: extended technical feasibilities of purification of natural mixtures containing plasminogen and fibrinogen from plasminogen thus keeping original amount of fibrinogen in mixture.

15 cl, 1 dwg, 13 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: method of obtaining dodecapeptide of the formula I: H-Asp-His-Leu-Asp-Lys-Gln-Thr-Gln-Thr-Pro-Lys-Thr-OH and tripeptide of the formula II: X-Asp(Y)-His-Leu-OH is the intermediate compound in its synthesis. Solid-phase synthesis of dodecapeptide I is realised by sequential growth of the peptide chain, beginning with the C-end dipeptidilpolymertill the obtaining of C-end nonapeptidilpolimer, which is condensed with the protected N-end tripeptide of the formula II: X-Asp (Y)-His-Leu-OH where X, Y are protected groups and the obtained dodecapeptidilpolymer is processed with an unblocking agent for removing the protective groups and the polymeric matrix and in 1 stage the end product is given out by means of HELC.

EFFECT: increasing the output of the end product and simplification of the process.

3 cl, 2 ex

FIELD: chemistry.

SUBSTANCE: sunflower seeds are crushed, de-oiled, extracted and separated into phases of centrifugal process. Double precipitation of the obtained lipoprotein complex with an acid at an isoelectric point at a pH=4.6-4.8, processing using an alkaline solution, washing of the fixed residue using water and drying. Extraction of the fat extracted residue is conducted using a trisglycinate buffer at a ratio seeds: buffer 1:16.7, and then held for two hours. Centrifugal process is carried out at 8000 rev/min for 15 minutes, and precipitated 0.1 n amber acid solution. Drying is carried out till a residual humidity of 6-8%.

EFFECT: increase in the protein content, less toxicity while reducing the time interval.

4 cl, 1 tbl, 1 dwg, 4 ex

FIELD: chemistry.

SUBSTANCE: sunflower seeds are crushed, de-oiled, extracted and separated into phases of centrifugal process. Double precipitation of the obtained lipoprotein complex with an acid at an isoelectric point at a pH=4.6-4.8, processing using an alkaline solution, washing of the fixed residue using water and drying. Extraction of the fat extracted residue is conducted using a trisglycinate buffer at a ratio seeds: buffer 1:16.7, and then held for two hours. Centrifugal process is carried out at 8000 rev/min for 15 minutes, and precipitated 0.1 n amber acid solution. Drying is carried out till a residual humidity of 6-8%.

EFFECT: increase in the protein content, less toxicity while reducing the time interval.

4 cl, 1 tbl, 1 dwg, 4 ex

FIELD: biology, biotechnologies.

SUBSTANCE: invention can be used in the food-processing industry. For obtaining of 6-O-αD-(1,6-GPS) isomaltulose would submit to the reactionary solution containing enzyme, possessing ability of catalyzing transformation of isomaltulose in 1,6-GPS from which allocate a target product after an incubation at temperature of 20-40°C. Before or during incubation in a reactionary solution add regenerative equivalents. Allocate enzyme with means of anion exchange and two affine chromatography from a crude extract of a microorganism of sort Gluconobacter containing a gene of enzyme, possessing ability of catalyzing transformation of isomaltulose in 1,6-GPS. The nucleic acid coding enzyme, possessing ability to catalyze transformation of isomaltulose in 1,6-GPS is obtained. The vector containing given nucleic acid, is intended for provision of an expression of this enzyme in a host cell.

EFFECT: invention application allows obtaining 6-O-αD-D-D-sorbite from isomaltulose by means of the unique enzymatic reaction.

26 cl, 2 dwg, 1 tbl, 4 ex

FIELD: chemistry, biotechnology.

SUBSTANCE: invention relates to field of biotechnology and preparation chemistry and can be used in biopharmacology and medicine. Cells of yeast P.pastoris are successively transformed by two different genetic structures, containing gene of human serum albumin (HAS) precursor. Obtained strain-producent is cultivated in nutrient medium. Recombinant HAS is isolated from cultural medium by clarification of said medium, as well as carrying out stages of successive centrifuging at 2000 and 10000 g, ultrafiltration, dialysis and cation-exchanging chromatography on column Source S. Target product represents eluate, including recombinant human serum albumin, 50 mM phosphate buffer, containing 400 mM of sodium chloride, with pH 9. Application of said iclaimed invention allows to extend arsenal of means, directed at production of recombinant HAS, and to obtain recombinant HAS in form of product, which in addition to recombinant HAS contains 50 mM phosphate buffer, containing 400 mM of sodium chloride and has pH 9.

EFFECT: extension of arsenal of means directed at obtaining recombinant HAS.

2 cl, 7 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology. Method versions lie in fact that trisulfide isoform is brought into contact with mercaptocompound, either with chelating agent or with metal salt.

EFFECT: obtaining polypeptide product with reduced amount of trisulfide isoform admixtures.

30 cl, 5 dwg, 7 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology. Method versions lie in fact that trisulfide isoform is brought into contact with mercaptocompound, either with chelating agent or with metal salt.

EFFECT: obtaining polypeptide product with reduced amount of trisulfide isoform admixtures.

30 cl, 5 dwg, 7 ex

FIELD: medicine.

SUBSTANCE: invention relates to medicine and biochemistry and concerns compositions for stabilisation of protein active ingredients in pharmaceutical preparations, which contains two following components: a) surface-active substance, preferably non-ion detergent (tenzid), and b) mixture of four amino acids: glutaminic acid (Glu), glutamine (Gln), aspartic (Asp) and asparagine (Asn).

EFFECT: synergetic effect at combined using of said four concrete amino acids.

10 cl, 11 ex, 6 dwg

FIELD: medicine.

SUBSTANCE: invention refers to medicine and concerns methods of neural disorder treatment with introducing hematopoietic growth factors. Substance of the invention includes G-CSF or its functionally active version 90% identical to human G-CSF sequence presented in SEQ ID NO:28 to be applied in therapy of amyotrophic lateral sclerosis.

EFFECT: benefit of the invention consists in neuroprotection of disease-related nerve cells.

2 cl, 38 ex, 1 tbl, 41 dwg

FIELD: medicine.

SUBSTANCE: medication for treatment and prevention of asthma contains FGF2 (fibroblast growth factor-2) as active ingredient, asthma being caused by overexpression of IL-13 (interleikin-13) or overexpression of IFN-γ (interferon-γ), where FGF2 inhibits IL-13 activity, FGF2 inhibits VEGF activity and FGF2 inhibits TGF-β1 activity (transforming growth factor-β1). Medication for treatment or prevention of chronic obstructive lung disease (COLD), containing FGF2 (fibroblast growth factor-2) as active ingredient, where chronic obstructive lung disease (COLD) is caused by overexpression of IFN-γ (interferon-γ). Inhibitor of IL-13 activity, containing FGF2 (fibroblast growth factor-2) as active ingredient. Inhibitor of VEGF activity, containing FGF2 (fibroblast growth factor-2) as active ingredient. Inhibitor of TGF-β1 activity, containing FGF2 (fibroblast growth factor-2) as active ingredient.

EFFECT: prevention of asthma and COLD.

8 cl, 31 dwg, 7 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, specifically to otorhinolaryngology, and is intended for determination of indications for differential therapy in sickly children (SC) suffering from lymphoepithelial pharynx ring pathology. Method is implemented as follows. Operating or biopsy material of lymphoepithelial pharynx ring is analysed for system and local immunity indices. If system and local immunity indices are below normal, system and local immune-response modulating agents are introduced. If system immunity indices only are below normal, system immune-response modulating agents only are introduced. If local immunity indices only are below normal, local immune-response modulating agents only are introduced. If system and local immunity indices are within normal limits, antiinflammatory therapy is applied.

EFFECT: possibility for estimation of immune system state in SC and selection of pathogenetically valid treatment regimen.

3 tbl, 4 ex

FIELD: medicine; veterinary science.

SUBSTANCE: administer an immunopotentiator to females of mammals into a lactemia initial stage. The immunopotentiator immunifan is administered to lactating females in a dose of mass of a body of 0.0006-0.0008 mg/kg. The immunopotentiator amyxine is administered to lactating females in a dose of mass of a body of 0.0017-0.0018 g/kg.

EFFECT: increase of content of immunoglobulins, leucocytes and mononuclear cells in milk of lactating animals.

FIELD: biotechnology.

SUBSTANCE: the invention relates to producing new peptides and may be used for treatment and prophylaxis of cytokine-sensitive disorders. Peptides, having a size of 5 to 40 amino acids and arising from cytokines, are used in a vaccine for treatment and prophylaxis of autoimmune diseases, disseminated sclerosis, rheumatoid polyarthritis, psoriasis, autoimmune diabeteses, lupus, allergy, asthma, cancer and AIDS.

EFFECT: allows effective immunization of patients against said diseases while minimizing side effects.

11 cl, 2 dwg, 17 tbl

FIELD: technological processess.

SUBSTANCE: oligomeric polypeptide dimmers include ligand-binding leptyne domains. Domains are connected by means of flexible polypeptide linker molecules. Linker molecules might possibly include sites of sensitivity to proteases.

EFFECT: releases biologically active cytokines when injecting to human being or animal.

31 cl, 13 dwg

Up!