Plasmin(ogen) removal from protein solutions

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

 

PREREQUISITES TO the CREATION of INVENTIONS

The technical field

This invention relates to the use of the resin and to provide a method for the specific removal plasmin(ogen)and its derivatives of the protein solution, and the obtained protein solution can be used for intravenous and for local applications, i.e. in the form of a matrix carrier for sustained release and healing of wounds, or in the form of individual active ingredient, or in combination with other relevant pharmaceutical drugs. Remove the plasmin(ogen)and you can maintain the integrity and function of the protein solution during longer periods of incubation. This invention also relates to the production of high-purity the plasmin(ogen)and for therapeutic applications.

Related area

The plasmin(ogen) or its active plasmin molecule (hereinafter plasmin(ogen)) very often pollutes protein solutions, especially solutions extracted from fluids or organs of animals. With the recognition of the plasmin(ogen)and as stable pharmaceutical product of the presence of plasmin(ogen)and in the protein solution represents multiple risk due to the known proteolytic activity of the molecule in relation to various proteins and peptides by peptide St. the relations of arginine and lisala (M.J. Weinstein, Doolittle RF. Differential specificities of the thrombin, plasmin and trypsin with regard to synthetic and natural substrates and inhibitors RF Biochim Biophys Acta. 1972. 258: 577-90 and Ling CM, Summaria L, Robbins KC. Mechanism of formation of bovine plasmin(ogen) activator from human plasmin. J. Biol. Chem. 1965. 240: 4212-B) and basic amino acids, its stimulating activity against various tissues, especially tissues of the Central nervous system, and its role in binding (Chen ZL, Strickland S. Neuronal death in the hippocampus is promoted by plasmincatalyzed degradation of liminin. Cell. 1997. 91: 917-25) and, probably, the maintenance of prions in the blood of mammals (Fischer MB, Roeckl C, 's agenda P, Schwarz HP. Aguzzi A Binding of disease-associated prion protein to plasmin(ogen). Nature. 2000. 408: 479-83).

Developed several chromatographic methods of cleaning the plasmin(ogen)and from protein solutions and, therefore, removal of the plasmin(ogen)and of the protein solution.

The mentioned methods are essentially based on two principles. The first group of methods is based on several successive stages of purification, which use different solubility, isoelectric point or size distribution of the molecules (Alkjaerisig N. The purification and properties of human plasmin(ogen). Biochem. J. 1963, 93: 171-182). Because their main purpose was to clear the plasmin(ogen)referred to ways completely changed the composition of the protein solution. The second group of methods is based on one-step affinity purification. Purification is based on the binding of plasmin(ogen)and with various synthetic ligands ωs karbonovykh acids, who can be contacted at lessinvasive sites on the heavy chain plasmin(ogen). These plots consisting of 5 disulfide bridges triple loop with internal homology sequences, known as Kringle domains of the plasmin(ogen)and localized at NH2-the end of the heavy chain plasmin(ogen)and at a long distance from the catalytic center located at the COOH-end of the light chain, bind fibrin(ogen). Another possible perspective for affinity chromatography is the binding of the plasmin(ogen)and through the catalytic centre, potentially less specific binding, as it can bind many proteins, such as serine protease with similar or lower affinity to the peptide linkages of arginine or lisala and basic amino acids. Briefly it can be concluded that, essentially, plasmin(ogen)-affinity chromatography is performed using a specific ligand, which is chemically and monogenetic reminds ω-aminocarbonyl acid or the substrate of the catalytic center of plasmin. The ligand binds to the resin via a corresponding connecting bridge or linker. But the ideal affinity resin for removal of plasmin(ogen)and essentially is not a resin, which was created as an ideal for cleaning the plasmin(ogen). Such resins must contain the ligand, which Saint who shows the plasmin(ogen) with high affinity and has a very low affinity for other proteins, like other serine proteases, and especially very low affinity to fibrinogen, which is the main protein in fraction I of Cohn (Cohn) plasma or cryoprecipitate. It is also important that the removal of the plasmin(ogen)and using this affinity chromatography can be performed using different buffers and is not limited to a specific buffer, which could compromise the stability and integrity of those proteins in solution, which, unlike plasmino(ogen), and are of interest.

Antifibrinolytic activity (the ability to inhibit high-affinity binding of plasmin(ogen)and fibrinogen) ω-aminocarbonyl acids depends on the presence of free amino and carboxyl groups and the distance between the COOH group and the carbon atoms which is attached to the NH2group (Markwardt 1978), such as a group ε-aminocaproic acid (EAC) and a pair of aminobenzamidine (PAMBA). Comparison of antifibrinolytic activities of the EAC and PAMBA has shown that the latter is about three times more active. Shimura et al. (1984) proposed a resin in which a pair of aminobenzamidine associated with particulate hydrophilic vinylpolymers through binder (linker) the fragment. Using the described resin Shimura et al. managed to split the plasmin and plasmin(ogen) using affinity chromatography high resolution Facts the plasmin(ogen) cannot be eluted using only one 6-aminohexanoic acid and 3 M urea must be entered in lucindy buffer, indicate two-interaction of plasmin with named immobilized ligand, i.e. lessinvasive sites on the heavy chain and the catalytic site in the light chain. This may explain the data collected by other researchers that a couple aminobenzamidine also removes some other proteins. Another resin, lysine-resin, produced and used for affinity purification of the plasmin(ogen). However, the antifibrinolytic activity of lysine is very low, and therefore also his affinity binding. It also binds to other proteins and its specificity depends on the buffer.

Moroz LA. Gilmore NJ in Fibrinolysis in normal plasma and blood: evidence for significant mechanisms independent of the plasminogen-plasmin system, Blood, 1976, 48, 531-45, describe obtaining plasma not containing plasmin(ogen), method of affinity chromatography. On the basis of methods used by the authors made the message data indicating that the processes which contribute to the formation of fibrinolytic enzyme plasmin, play at best a minor role in spontaneous or basal fibrinolytic activity, as measured in normal plasma. Tranexamic acid is used instead of the ones with other protease inhibitors as inhibitors of plasmin for the measurement of fibrinolytic activity. To obtain plasma, not containing plasmin(ogen), use the method of Deutsch and Meltz, Science 170: 1095-1096, 1997.

Iwamoto in Thrombos. Diathes. Heamorrh. (Stuttg.), 1975, 33, 573 describes specific binding of tranexamic acid with plasmin. Although tranexamic acid identified as an effective ligand plasmin, it is shown that the antifibrinolytic effect of tranexamic acid is not only a result of the binding plasmin(ogen), as well as increasing cooperation natural antiplasmin. Therefore it should be concluded that the binding of tranexamic acid with solid media will be removed from the plasma not only plasmin(ogen), but also natural antiplasmin. You should also assume that tranexamic acid can cause the formation of aggregates (conglomerates) with inhibitors of plasmin. This understanding is based on differences, which can be installed when comparing antifibrinolytic activities ε-aminocaproic acid and tranexamic acid, leading to 98% and 91% inhibition of plasma-stimulated urokinase, compared with plasma, which is derived from oral heparinised blood (65 and 39% for ε-aminocaproic acid and tranexamic acid, respectively - see table 2 and 7 in the article Moroz et al.). It should be assumed that due to their high ratio of binding of tranexamic acid is one and the ε -aminocaproic acid are good candidates for high-affinity ligands. However, you should also assume that these ligands will block affine column in the binding of plasmin and plasmin complexes-inhibitor.

Finally, as the closest analogue is claimed in the present invention, a method of removing from a mixture of plasminogen containing fibrinogen and plasminogen, you can specify inventor's certificate SU 1727839 A1 (Chupov CENTURIES, nikolaychik CENTURIES, Bychko GN. et al., from 23.04.1992). In accordance with the specified document plasminogen from plasma was removed by passing the latter through a column of equilibrium swollen copolymer of acrylamide, N,N'-methylenebisacrylamide and methacryloyl-L-lysine. The use of such a sorbent with covalently immobilized L-lysine were 100% cleaning plasma from plasminogen, and loss of lysine was less than 0.3%. Compared with these results proposed in the present invention a method of removing plasminogen allows you to expand the technical capabilities of the cleaning liquid mixtures of natural origin containing plasminogen and fibrinogen, plasminogen while maintaining the original amount of fibrinogen in the mixture.

BRIEF DESCRIPTION of DRAWINGS

The drawing shows a gradient electrophoresis in polyacrylamide gel the presence of sodium dodecyl sulfate (SDS-PAGE) (5-12% polyacrylamide) 7 µg of proteins buervenich two ways (described in Materials and methods), with three different resin - tea-sepharose, Lys-Ceramic Hyper DF and Lys-sepharose 4B. Tracks: 1 - Glu-plasminogen; 2 - fibrinogen; 3 - albumin; 4 - immunoglobulin G; 5 - standard molecular mass; Lucianne peaks: 6 - way using TEM; 7 - method 2 using TEM; 8 - way 1 using lysine-Ceramic Hyper D; 9 - method 1 using lysine-sepharose; 10 - way 1 with lysine-Ceramic Hyper D; 11 - method 1 using lysine-sepharose.

BRIEF description of the INVENTION

The present invention is based on the data that the fixed amino acid, it has been found that is able to specifically bind the plasmin(ogen). In the context of describing the invention, the expression "specifically bind" means that from a mixture containing proteins, such as plasmin(ogen) and fibrinogen, mainly removes the plasmin(ogen), whereas fibrinogen is retained in the mixture almost unchanged. Preferably remove at least 85 to 99% of the plasmin(ogen)and at least 85% of fibrinogen remains in the mixture. More preferably remove at least from 98.0% to 99.9% of plasminogen or retain 95%-99% of fibrinogen.

The amino group of the amino acid and the carboxyl group of the amino acids are located at a distance of about 6-8 angstroms, preferably about 7 angstroms, fixed amino acid Kowal is NTO connected with the carrier via the amino group of amino acids. Especially preferred are tranexamic acid in its transconfiguration and 4-aminomethylbenzoic-[2.2.2.]-octane-1-carboxylic acid (EMBOCA).

Unexpectedly, it was found that, first, ε-aminocaproic acid is not working as tranexamic acid, and that as soon as tranexamic acid bound to a solid surface, it takes all of his increased plasmin(ogen)-binding ability (so-called cooperatively), and the resin removes from plasma or from plasma products only plasmin(ogen). If ε-aminocaproic acid attached to the column, its increased activity against plasmin(ogen)and still remains the same, but nevertheless it binds fibrinogen and other proteins from plasma, whereas according to the invention completely eliminates the increased activity of tranexamic acid in relation to the plasmin(ogen). However, the affinity resin with tranexamic acid to plasmin(ogen)have not changed.

Rigid amino acid attached to the corresponding connecting bridge, in particular, longer than 3 carbon atoms, and media and affine material can delete plasmin(ogen) from a mixture containing proteins, without further changes in the composition of the protein solution. The removal can be carried out in the presence of various buffers. The method of the invention is also suitable for receiving the East fractions of the plasmin(ogen)and after elution from the affinity media.

DETAILED description of the INVENTION

The invention relates to the creation of a specific way of removing or releasing the plasmin(ogen)and in the presence of fibrinogen from a mixture containing plasmin(ogen), by contact of the mixture with a fixed amino acid in which the amino group of the amino acid and the carboxyl group of the amino acids are located at a distance of about 6-8 angstroms, preferably about 7 angstroms, and the fixed amino acid covalently linked to a carrier via the amino group of amino acids.

Preferably in the method according to the invention the mixture is selected from the group consisting of body fluids, such as blood, blood fractions, cryoprecipitate, cell culture, animal tissue extracts, such as the lungs of a bull, the bull intestines, or extracts of animal bones; gelatin, bovine serum albumin, and water-immiscible fats of animal origin such as lanolin (PC-phosphatidylcholine).

The method according to the invention can be used to obtain the high-purity plasmin(ogen)and from the corresponding mixtures. After exposure of the mixture, for example, with chromatographymass substance associated with rigid amino acid, plasmin(ogen) can be eluted from the solid affine substances. As such, in this case, it is also possible to apply known principles of solid-phase extractivism(ogen) can be eluted with a solution, containing ligand which competes with binding sites of rigid amino acids, for example, tranexamic acid, plasmin(ogen)new protein. Such ligands are usually ε-amino acid, preferably lysine. For example, lysine can be used in concentrations of from 0.85 to 0.99 wt.%. You can also use other concentrations, especially when the ionic strength elsinoe environment balanced by the other ingredients such as electrolytes.

The plasmin(ogen), eluruumist with the solid phase, may be exempted from lucynova buffer according to the buffer components are extracted, for example, dialysis. Obtained according to the method of this invention, the plasmin(ogen) is characterized by a very high degree of purity. Exceptional property becomes evident from the following data:

The total table 1

Comparison of the specific activity, purification and output plasmin(ogen)and crioestaminal FFP-plasma in the preferred conditions of application and elution for each of the resins
Used

resin
MethodSpecific activity

(mg plasmin(ogen)and/

mg protein)
Degree

cleanup
Output

(%)
Thea-sefar is for 4B 20,794567to 91.6
Lysine-Ceramic Hyper DF20,44444410,9
Lysine-sepharose 4B10,121101the 11.6
Total table 2: Comparison of removal plasmin(ogen)and crioestaminal FFP-plasma in the preferred conditions of application for each resin
Used resinMethodRemoval (%)
Thea-sepharose 4B1 and 2*99,5
Lysine-Ceramic Hyper DF154,6
Lysine-sepharose 4B158,0
*Both methods are identical up to collect the unbound peak, including this very stage.

As you can see from the summary table, when commercially supplied resin with immobilized lysine ligands and Thea-resin used to optimized the conditions of chromatography, the yield and specific activity of the plasmin(ogen)and were higher in the case of TEM-tar (total tab is Itza 1). Also worthy of note that the resin Thea turned out to be much more effective at removing the plasmin(ogen)and (as demonstrated in the study of Glu-plasmin(ogen) (a) of crioestaminal plasma than lysine-resin (summary table 2).

The purity of the eluates was estimated using SDS-gel electrophoresis. Eluate with three different resins (tea-sepharose, Lys-Ceramic Hyper DF and Lys-sepharose 4B) were subjected to SDS-PAGE using a 5-12% gradient acrylamide and when applied 7 µg of protein per lane. The obtained gel, colored Kumasi blue shown in the drawing.

The drawing shows a gradient gel SDS-PAGE (5-12% polyacrylamide) protein 7 g, buervenich two ways (described in Materials and methods) with three different resin - tea-sepharose, Lys-Ceramic Hyper DF and Lys-sepharose 4B. Tracks: 1 - Glu-plasmin(ogen); 2 - fibrinogen; 3 - albumin; 4 - immunoglobulin G; 5-standard molecular mass; Lucianne peaks: 6 - way using TEM; 7 - method 2 using TEM; 8 - way 1 using lysine-Ceramic Hyper D; 9 - method 1 using lysine-sepharose; 10 - way 1 with lysine-Ceramic Hyper D; 11 - method 1 using lysine-sepharose.

The obtained protein zone and the purity of the product was well correlated with the specific activity of plasmin(ogen)and which are listed in summary table 1. Data show that the application of Thea-sepharose gives highly purified plasmin(the gene) with only minor contamination with albumin. It is obvious that the clinical criteria no further purification is not required for use of this product.

Therefore containing plasmin(ogen) composition is also subject of this invention.

The subject of this invention is a carrier covalently associated with the rigid amino acid and the amino group of the amino acid and the carboxyl group of the amino acids are located at a distance of approximately 6-8 angstroms, preferably about 7 angstroms.

Preferred for implementing the method of the present invention the chromatographic medium is a substance which can bind rigid amino acid in which the amino group of the amino acid and the carboxyl group of the amino acids are located at a distance of about 6-8 angstroms, preferably about 7 angstroms. The distance between the amino group and carboxyl group, essentially, is kept constant by the rigid structure of amino acids. The rigidity of the amino acids can be obtained by using an alicyclic ring, preferably a cyclohexane ring, and the amino - and carboxy groups are in the 1,4-position of the alicyclic ring. Also in the field covered by this invention include aromatic system, for example, substituted benzoic acid or aniline-zameshannaya acid.

According to the invention the carrier is preferably bind with amino acids selected from the group consisting of tranexamic acid and ENGOS.

Chromatographic substance that is used according to the method of the present invention is, for example, a hydrophilic substance, such as agarose, cellulose, glass with adjustable porosity, silica gels, dextrans or synthetic organic polymer, such as polymer-based polyacrylamide, polystyrene. Common substances are supplied commercially under the trade names of sephacryl® (Pharmacia, Sweden), ultragel® (Biosepara, France), TSK-gel Toyopearl® (Toso Corp., Japan), HEMA (Alltech Ass. (Deer-field, Il, USA), Eupergit® (Rohm Pharma, Darmstadt, Germany). You can also use the materials, created on the basis of azlactones (3M, St. Paul, Minn, USA). Particularly preferred is the agarose® or sepharose®. These materials are available commercially, for example, by the company Sigma, St. Louis.

In a preferred aspect of the method according to the invention is carried out with the use of particulate chromatographic material from particulate or monolithic block of material. The particulate substance may be suspended in an appropriate environment, and the resulting suspension can be used, for example, in the chromatographic column. However, the method according to the invention can be implemented portions. Chrome is also the polymers can be used in the form of particulate matter or also in the form of membranes.

Tranexamic acid is associated with a carrier, preferably via a linker, particularly a bifunctional linker between the carrier and tranexamic acid. If you're using a bifunctional linker can be selected from the group consisting of N-hydroxysuccinimide, DAPA, CNBr, epoxy, diaminodiphenylamine (DADPA), 1,6-diaminohexane, succinic acid, 1,3-diamino-2-propanol, ethylene diamine (EDA), TNB, pyridylsulfonyl, iodoacetamide activated maleimido media or combinations thereof.

For implementing the method according to the invention the carrier is preferably modify the fragment that interacts with a primary or secondary amino groups.

In accordance with the method according to the invention the mixture is incubated with media for a sufficient period of time, and after contact of the mixture with the carrier associated with tranexamic acid to elute neutral aqueous solution containing salts of sodium, calcium salts, buffer salts. Subsequently, the plasmin or plasmin(ogen) can be eluted with an aqueous solution containing a sufficient amount of lysine or equivalent, which competes with covalently linked, tranexamic acid.

The subject of the invention is obtained from natural sources CME is ü, which is essentially free of plasmin(ogen)and plasmin.

In particular, the mixture according to the invention is a blood-derived blood or a fraction of blood, cryoprecipitate.

Blood derivatives according to the invention is, in particular, a coagulation factor, obtained from plasma, or a mixture of coagulation factors, such as FVIII, FIX, fibrinogen, fibronectin, α1-antitripsin, antithrombin III, factor von Willebrand's disease, albumin, immunoglobulin.

In addition, the medium containing covalently linked tranexamic acid, is also the subject of the present invention. The media according to the invention is preferably chromatographic substance, more preferably hydrophilic chromatographic substance, such as dextrans or synthetic organic polymer, such as mentioned above. The most preferred carrier is agarose® or sepharose®is associated with tranexamic acid.

The chromatographic material, which forms the carrier may be particulate matter or monolithic block of material. The latter is described in the cited work Hermanson et al., (Hermanson GT, Mallia AK and Smith PK 1992 "Immobilization of Affinity Ligand Techniques" pp. 454 Academic Press, Inc. San Diego, USA).

In another preferred aspect according to the invention tranexamic acid binds with but what ielem through a linker between the carrier and tranexamic acid. Favorably when the media has no functional groups capable of covalently bind tranexamic acid. In this case, the carrier is first activated, and then injected into the reaction with the linker, which is able to bind tranexamic acid. Spacer elements group, or a leash, are low molecular weight molecules, which are used as intermediate linkers between the carrier or matrix and affine ligand, which is in accordance with the invention represents an amino acid having a rigid structure and an amino group at a distance of about 6-7 angstroms from the carboxyl group. Preferably spacer elements groups contain two functional groups at both ends for easy interaction with the ligand and a carrier. Usually spacer elements group is a hydrocarbon compound having at the ends of the two functional groups. One of the two ends covalently attached to the matrix using conventional or known reactions. The second end covalently bound to the ligand using a different method of attachment.

Preferably the linker is a bifunctional linker, such as N-hydroxysuccinimide, DAPA, CNBr, epoxy, diaminodiphenylamine (DADPA), 1,6-diaminohexane, succinic acid, 1,3-diamino-2-propanol, Ethylenediamine (EDA), TNB, pyridylmethyl, iodoacetamide, activated maleimido Sitel or combinations thereof.

As the majority of activated carriers supplied commercially, it may be useful to start with the media, the modified piece, which interacts with a primary or secondary amino groups.

Furthermore, the method according to the invention are described in more detail using as an example the obtain cryoprecipitate essentially free from plasmin(ogen)and that can be the raw material for numerous products derived from blood.

Cryoprecipitate is subjected to affinity chromatography with immobilized ligand, to obtain the adsorbed fraction and readsorbing faction. A substance that can be allerban of the adsorbed fraction is a plasmin(ogen).

Immobilized ligand can be any analogue, which can interact with lessinvasive areas of the plasmin(ogen). The way to obtain immobilized ligands, which are used according to the invention, described below. The following examples illustrate the invention but do not restrict it.

EXAMPLE 1: Immobilization of various ligands ε-aminocarbonyl acids.

Several ligands ε-aminocarbonyl acids in combination with different spacers in several resins were either received or bought (if commercially available)to estimate the IC removal plasmin(ogen)and from solutions obtained from plasma. The following table summarizes all studied combinations (non resins below refer to the section in which the following synthesis for each combination):

Table 1
Ligand

Linker
Pair-benzamidineArginineTranexamic acidε-aminohexanoic acidLysine
N-hydroxyestraSepharose 4V

(1)
DADPAThe agarose

4% (2)
The agarose

4% (3)
The agarose

4% (4)
CNBrSepharose

4B (5)
Sepharose

4B (6)
EpoxySepharose

6B (10)
Sepharose

6B (7)
Sepharose

6B (8)
Ceramic

Hyper DF

(9)

1) Complex N-hydroxysuccinimidyl ether ε-aminohexanoic acid-sepharose 4B was produced by the company Sigma.

2) the Manufacture of a pair of aminobenzamidine-4% agarose.

The following method was used for immobilization diamondpro is ylamine (DADPA) on 4% agarose (Pierce), the reaction occurred at aminoclonazepam binder gel, which is then modified anhydride.

25 ml of DADPA-agarose gel was washed with purified water, and then the gel is suspended in equal volume of purified water. The suspension mixture was slowly stirred for 1 hour at room temperature by adding 2.5 g of succinic anhydride. At the end of the mixing succinylcholine the gel is then washed with purified water, 1 M NaCl and again with purified water to remove excess unreacted succinic acid.

A negative test with TNBS (Sigma) showed that all amines DADPA were successfully blocked succinic acid.

Immobilized succinylcholine DADPA washed with 250 ml of purified water, and then excess water was pumped out to dry to wet sediment, and was transferred into a 500-ml chemical glass. The gel is re-suspended in 25 ml of 0.1 M MES buffer, pH 4.7 and slowly stirred, adding 0.25 g of steam-aminobenzamidine (Sigma) and 0.75 g of EDC (Pierce). The PH of the reaction mixture was maintained at 4.7 in 1 hour, continuously adding 0.5 M NaOH. Then the reaction mixture was left overnight at room temperature with constant slow stirring.

The gel is then washed with 0.5 l of each of the following components: purified water, 0.1 M sodium acetate, pH of 4.7, 0.5 M sodium bicarbonate and purified water.

Immobiliza the ing a pair of aminobenzamidine kept to use in 0.02% sodium azide at 4° C.

3) Arginine-4% agarose

Synthesis of arginine-4% agarose was performed in accordance with the above-described method for pair-aminobenzamidine-4% agarose (see 2).

4) Tranexamic acid (tea)-4% agarose

Synthesis of tranexamic acid (tea)-4% agarose preferably carried out in accordance with the above procedure for pair-aminobenzamidine-4% agarose (see 2).

5) Arginine-sepharose 4B

The following method was used to mobilitat arginine or tranexamic acid on CNBr-sepharose 4B (Pharmacia) as a binder gel. The syntheses of the two ligands with two different concentrations (10 mmol or 0.01 mmol/ml of dry gel) were the same as described in this and the following sections (5-6).

2.5 g of CNBr-activated sepharose 4B suspended in 50 ml of 1 mm HCl. The gel was left to swell for 10 minutes at room temperature followed by a 15-minute washing with 500 ml of 1 mm HCl on a sintered glass filter.

Arginine was dissolved in 12.5 ml of binding buffer, 0.1 M NaHCO3pH of 8.3, containing 0.5 M NaCl. Linking solution containing the ligand was mixed with the gel in a plastic test tube, followed by rotation overnight at 4°C.

At the end of the incubation, the mixture was washed from excess ligand 10 gel volumes of binding buffer through a sintered glass filter. The protein solution was washed for the 3 cycles of 50 ml of the buffer when changing the pH (0.1 M acetate buffer, pH 4, containing 0.5 M NaCl, followed by washing with 0.1 M Tris-HCl-buffer, pH 8, containing 0.5 M NaCl). Immobilized arginine-sepharose 4B kept to use in 0.02% sodium azide at 4°C.

6) Tranexamic acid (tea)-sepharose 4B

This synthesis was carried out according to the above described method for arginine-sepharose 4B (see section 5).

7) Arginine-sepharose 6V

Arginine in two different concentrations (2 mm or 0.2 mmol/ml of dry gel) was added to a commercial immobilized epoxy-sepharose 6B (Pharmacia) as follows:

2.5 g of epoxy-activated sepharose 6V suspended in 200 ml of purified water. The gel was left to swell for approximately 5 minutes at room temperature, then washed for 1 hour with 500 ml of purified water, added to the aliquot through a sintered glass filter.

20 ml of binding buffer (0.1 M NaHCO3pH 9.3 and 0.5 M NaCl) and the swollen gel was poured into two test tubes containing arginine. The mixture was stirred in a plastic tube over night at RT (room temperature).

At the end of the incubation, the mixture was washed from excess ligand 5 gel volumes of binding buffer through a sintered glass filter. The product was washed 3 cycles with changing pH (0.1 M acetate buffer, pH 4.0, and 0.5 M NaCl, followed by washing with 0.1 M Tris-HCl-buffer, pH 8 is 0.5 M NaCl). Immobilized arginine-sepharose 6V kept to use in 0.02% sodium azide at 4°C.

8) Tranexamic acid (tea)-sepharose 6V

This synthesis was carried out according to the above described method for arginine-sepharose 6B (see section 7).

9) L-Lysine epoxyoctadecane Ceramic Hyper DF-hydrogel was acquired by the company Sigma.

10) a pair of Aminobenzamidine covalently attached to sepharose 6V, was acquired by the company Pharmacia.

EXAMPLE 2: Testing different affinity chromatographic resins for removal of plasmin(ogen). United cryoprecipitate of human plasma containing 1 IU/ml plasmin(ogen)and 50 mg/ml of fibrinogen was used for the following studies.

Aliquots frozen cryoprecipitate were subjected to thawing at 37°and were dialyzed against buffer BN1 (0.12 M NaCl, 10 mm Tri-Na-citrate, 1 mm CaCl2at pH 7.0). The obtained protein solution was filtered through a depth filter 5 microns to obtain a clear solution. In the cylinder 5-ml syringe with a diameter of at 8.36 mm made of 1.5 ml (wet volume) of the following affine resin described in example 1: immobilized ε-aminohexanoic acid (Sepharose 4B, used CNBr as spacer)immobilized pair aminobenzamidine/arginine/Thea (4% Agarose, using DADPA as spacer)immobilized arginine/Thea (Sepharose 4V using CNBr in which the quality of the spacer), immobilized arginine/Thea (Separate 6V using epoxy as a spacer) and immobilized L-lysine (Ceramic Hyper DF-hydrogel using epoxy as a spacer). Optimized packing of the gel was as follows: Packed gel was washed with 4 volumes: (i) purified water, ii) 1 M NaCl, (iii) purified water, iv) buffer TLN of 0.1 (0.05 M Tris, 0.02 M lysine, 0.1 M NaCl, pH 9,0), (v) buffer TLN 1 (0.05 M Tris, 0.02 M lysine, 1 M NaCl, pH 9,0) and (vi) of purified water. All samples buffer for applying and rinsing introduced into the cylinder of the syringe after centrifugation at 1000 rpm for 1 minute at 25°C. Balancing spent 4 volumes BN1, and pre-filtered, concentrated cryoprecipitate was applied on the resin (2:1, vol/about., respectively), then the resulting mixture was incubated for 1 hour at room temperature. Aliquots unrelated spin-leaching was collected in plastic tubes after each centrifugation. The resin was washed at least 13 column volumes BN1 buffer up until O.D280(optical density) was not reached 0,02. The elution of the plasmin(ogen)and conducted buffer TLN 1 followed by washing four volumes of layer 3 M NaCl. Mini-column was tightly closed and stored at 4°C.

Resin, which was destroyed during the process or removed less than 50% of the plasmin(ogen)and excluded the school in the beginning of the study. The results are summarized in table 2.

Table 2

Remove the plasmin(ogen)and P

in the unbound material using different immobilized ligands with buffer BN1
Immobilized ligandsRemove the plasmin(ogen)a (%)
ε-Aminohexanoic acid, used CNBr as spacermade 13.36
a pair of Aminobenzamidine used DADPA as spacerNo

(column Packed)
a pair of Aminobenzamidine used epoxy as spacer19,78
Arginine, used DADPA as spacer6,04*
TEM (high concentration), used CNBr as spacer0
Thea (low concentration), used CNBr as spacer0
Arginine (low concentration), used CNBr as spacer23,25
Arginine (low concentration), used epoxy as spacer0
Arginine (high concentration), used epoxy as spacer5,61
*Bring the installed data are average of three experiments.

In the above table shows that the best ligands for removal of plasmin(ogen)and from cryoprecipitate contained affine gels, which were strong enough to withstand the consequences of the application, washing and elution without compression, or gels that hold more than 50% damage of fibrinogen.

In table 3 demonstrated the effectiveness of different types of gels when removing the plasmin(ogen), and their ability to keep the fibrinogen concentrates cryoprecipitate.

Present in cryoprecipitate residual plasmin(ogen) adsorbiroval resins, whereas fibrinogen did not adsorbiroval, which makes the resulting supernatant essentially free of plasmin(ogen).

The content of fibrinogen was determined by clotting time, while the contents of the plasmin(ogen)and was identified in the study with a chromogenic substrate.

The calculated output plasmin(ogen)and fibrinogen with lysine-gels served as the gold standard for all other used gel ligands. The results presented in table 2 show that only TEM-ligands with epoxy-spacer provided good removal plasmin(ogen)and high yield of fibrinogen (see table 3). A high concentration of immobilized TEM showed the best removal plasmin(ogen)and an output fibrinogen is compared with the associated with lysine ligand: yield 89% vs. 92% in the case of fibrinogen and 89% vs. 56% removal plasmin(ogen)and respectively. All other resins was significantly less effective or deleting the plasmin(ogen)and, with respect either to the output of fibrinogen.

Table 3

Summary data on the output of fibrinogen and removal plasmin(ogen)and the unbound material, obtained using different immobilized ligands with buffer BN1
Immobilized ligandsRemove the plasmin(ogen)a (%)The output of fibrinogen (%)
Lysine, when using

epoxy as spacer
56*92*
Thea, when using DADP as spacer4984
Thea (low

concentration), when using epoxy as a spacer
44,3588,62
TEM (high

concentration), when using epoxy as a spacer
8989
Arginine (high concentration), using CNBr as spacer91,2649
*The above data are average values from three studies.

EXAMPLE 3: Effect of phosphate and BN1 buffer on the profile affine chromatographia resin with immobilized lysine and Thea.

Cryoprecipitate was treated with aluminum hydroxide to adsorb coagulation factors dependent on vitamin K, and then incubated with a mixture of detergent solvent (SD-1% Tri (n-butyl)phosphate, 1% Triton X-100) for 4 hours at 30°C to inactivate viruses in the lipid membrane. SD Reagents were removed by extraction of castor oil and hydrophobic interaction chromatography, the product was subsequently pasteurizable (10 hours at 60° (C) in the presence of sucrose and glycine as a stabilizer.

After pasteurization, sucrose and glycine was removed by diafiltration. Tranexamic acid (tea) and arginine hydrochloride was added as stabilizers prior to filtration under sterile conditions. Aliquots of stabilized product was stored until use at -30°C.

Aliquots frozen cryoprecipitate with inactivated viruses were subjected to thawing at 37°and were dialyzed against buffer BN1 (consisting of 0.12 M NaCl, 0.01 M Tri-Na-citrate, 1 mm CaCl2at pH 7.0) or, alternatively, against 25 mm phosphate buffer. The last solution was filtered through a depth filter 5 microns to obtain a clear solution.

In a column of 10 mm in diameter (Biorad, USA), was brought to 6 ml (wet volume) or immobilized TEM (TEM-sepharose 6B), or immobilized lysine (Ceramic Hyper DF/sepharose 4B) and flushing and 4 volumes of each of the following solutions: i) purified water, ii) 1 M NaCl, (iii) purified water, iv) buffer TLN of 0.1 (0.1 M NaCl, lysine 0.02 M, 0.05 M Tris, pH 9,0), (v) the buffer TLN 1 (1 M NaCl, lysine 0.02 M, 0.05 M Tris, pH 9) and (vi) purified water. The equilibration was carried out 4 volumes BN1 or, alternatively, a phosphate buffer. Filtered cryoprecipitate was applied on the column at a speed of current of 100 ál/min

Samples of the unbound material was collected in plastic tubes, and the resin was washed 16 column volumes or BN1 buffer or phosphate buffer. The elution of the plasmin(ogen)and conducted buffer TLN 1 followed by washing four volumes of 3 M NaCl, four volumes of purified water and four volumes of 25% ethanol supplemented with 1 M NaCl.

Table 4 illustrates the removal efficiency of the plasmin(ogen)and various types of resins and the output of fibrinogen. The content of fibrinogen was determined by clotting time (analysis Claus (Clauss)), while the content of the plasmin(ogen)and was determined using the chromogenic substrate.

Comparison of different resins showed that 95,4 to 96.4% of the content of fibrinogen remained in the unbound peak when using buffer BN1. In contrast, the output of fibrinogen was low, if used phosphate buffer. Unexpectedly it was found that only the resin with immobilized Thea provided both a high degree of purification of plasmin(ogen)and high yield of fibrinogen.

Financial p the tats, obtained for lysine-resin, also demonstrated superior efficacy with buffer BN1 compared to phosphate buffer.

Table 4

Summary of data obtained in the determination of the yield of fibrinogen, and plasmin(ogen)and when using immobilized resins or BN1 buffer or phosphate buffer
Immobilized ligandsRemove the plasmin(ogen)a (%)The output of fibrinogen

(%)
TEM (high. conc.)

epoxy+BN1 buffer
77,196,4
Lysine-epoxy+BN1 buffer68,8795,4
Lysine-epoxy+

phosphate buffer
10066,9
Lysine CNBr+ BN1 buffer62,5the level of 121.8
Lysine CNBr+

phosphate buffer
10062,2

EXAMPLE 4

Aliquots frozen cryoprecipitate with inactivated viruses were subjected to thawing at 37°and were dialyzed against buffer BN1 (0.12 M NaCl, 0.01 M Tri-Na-citrate, 1 mm CaCl2at pH 7.0) or, alternatively, against 25 mm phosphate buffer. The last solution was filtered through a depth filter 5 micron to remove undissolved substances.

In column 26 mm in diameter (Pharmacia, Sweden), in which oily 50 ml (wet volume) of immobilized TEM (TEM-sepharose 6B) and washed with 4 volumes of purified water and the same volume of buffer TLN of 0.1 (0.1 M NaCl, lysine 0.02 M, 0.05 M Tris, pH 9,0), buffer TLN 1 (1 M NaCl, lysine 0.02 M, 0.05 M Tris, pH 9) and purified water. The equilibration was carried out by 4 volumes of buffer BN1 (NaCl, Tri-Na-citrate, CaCl2, pH 7.0) and filtered YOU passed through the column at a speed of DC 700 ál/ml

Samples of the unbound material was collected in a plastic Cup, and the resin was washed at least 3 gel volumes of buffer BN1. The elution of the plasmin(ogen)and carried out by the buffer TLN 1 followed by washing 3 M NaCl.

The unbound fraction from the two deletions. were combined and stored at 4°to concentrate. Finally, YOU have concentrated approximately to the original volume by diafiltration, using membrane, cut-off 100, and against buffer B1 (glycine, NaCl, Tri-Na-citrate, CaCl2, pH 7) followed by filtration through a 0.45 µm filter. To the filtrate was added 2% arginine.

To control the stability of the obtained product was filtered under sterile conditions through a 0.2 μm filter.

As shown in table 5, using a larger and more long columns improve the efficiency of TEM-ligand, the output of fibrinogen was 100%, and the destruction of the plasmin(ogen)and was below the detectable level in the study of the plasmin(ogen)and chromogenic method.

When comparing product before and after diafiltration revealed that the content of fibrinogen is reduced by 33%. This phenomenon can in order to explain technical problems, which only occur when handling small quantities. Resin with immobilized Thea certainly provided good removal plasmin(ogen)and good yield of fibrinogen.

Table 5

Summary of data obtained in the determination of the yield of fibrinogen, and plasmin(ogen)and unbound peak. Each data point represents a mean value of the two deletions.
SampleThe output of fibrinogen (%)The output of the plasmin(ogen)a (%)
After applying the1000
After application and

diafiltration
66,20*
*The output of the plasmin(ogen)and after combined sample was concentrated to 3.7 times.

Any remaining Thea was not found in buyowner protein solution and concentrated by ultrafiltration product. Analyses of the residual level of tranexamic acid was performed using HPLC.

Conducted four additional experiment to remove the plasmin(ogen)and, using the same resin and such terms of bandwidth, as described above (example 4). In General the results for all investigated samples was similar to the first results, PR is dostavlenny above.

Any remaining Thea was not found in buyowner protein solution and concentrated by ultrafiltration product. Analyses of the residual level of tranexamic acid was performed using HPLC.

Not watched any adhesion or in the case of strips before removing the plasmin(ogen)nor after it was studied on rat model.

Cryoprecipitate before and after removal of the plasmin(ogen)and explored in relation to degradation during incubation at room temperature. Data, expressed as a percentage of coagulated proteins (by absorption in the UV), for different samples are presented in table 6.

Table 6

The percentage of coagulated proteins after incubation at room temperature, concentrated cryoprecipitate after double inactivation of viruses before and after removal of plasminogen on epoxy-sepharose 6B associated with tranexamic acid
The incubation period (weeks)
0123456
Before processing62,91000000
After processing 73,4470,8068,2465,663,6963,8462,46

Stability studies were performed on strips that were prepared from cryoprecipitate before and after removal of the plasmin(ogen), followed by incubation at 37°in the presence of buffer. These strips were treated with or without adding glycine and/or arginine. The results are summarized in table 7 below.

Table 7

Time degradation on the strips before and after removal of the plasmin(ogen)
CookingTime degradation strips (days)
0123456
Before processing+++
To handle+

2% arginine
+++------
To handle+

2% arginine+

1% glycine
+++------
After processing ++++++++++++++++++-
After processing+2% arginine+1% glycine++++++++++++++++++-
+++ This set of data indicates the presence of strips.

This data set implies a degradation of the strips.

1. The research described in the following examples was carried out on crioestaminal United fresh frozen plasma from normal healthy donors. Due to the high concentration of antiplasmin and small amounts of plasmin from normal healthy donors (1) plasmin cannot be determined using the function (chromogenic) method (Schreiber AD, Kaplan AP, Austen KF, Plasma inhibitors of the components of the fibrinolytic pathway in man. J. Clin. Invest 52: 1394-1401, 1973). Therefore, it is impossible to demonstrate the removal, cleaning and access to TEM-tar plasmin from plasma samples of healthy donors. However, a commercial ELISA-kit for determination of very low amounts of Glu-plasmin(ogen)and, as was established in previous studies, Thea-resin are characterized by the same affinity for both forms of plasmin(ogen), and plasmin (Fredenburgh JP, Nesheim ME. Lys-plasmin(ogen) is a significant intermediate in the activation of Glu-plasmin(ogen) during fibrinolysis in vitro. J. Biol. Chem. 267. 26150-6. 1992 and Miyashita C, Wenzel ., Heiden M. Plasmin(ogen): a brief introduction into ist biochemistry and function. Haemostasis 1:7-13, 1988).

2. Thus, the determination of glu-plasmin(ogen)and can be used as an indicator of the total plasmin(ogen)and in plasma.

EXAMPLE 5

Phase chromatography.

Studies were performed to determine the efficacy of tea, immobilized on sepharose 4FF, when removing the plasmin(ogen)and crioestaminal fresh frozen plasma (FFP), containing 1 IU/ml plasmin(ogen). Aliquots crioestaminal fresh frozen plasma was subjected to thawing at 37°and filtered through a 3 micron depth filter to remove insoluble proteins.

In a column of 10 mm in diameter (Pharmacia, Sweden)was introduced 2 ml (wet volume) of immobilized TEM and washed with 4 volumes of purified water and the same volume of buffer TLN of 0.1 (0.1 M NaCl, 0.02 M lysine, 0.05 M Tris, pH 9,0), buffer TLN 1 (1 M NaCl, lysine 0.02 M, 0.05 M Tris, pH 9,0) and purified water. The equilibration was carried out 4 volumes BN1 buffer (0.12 M NaCl, 0.01 M Tri-Na-citrate, 1 mm CaCl2, pH 7.0)and filtered plasma (˜20 IU the plasmin(ogen) (a) was passed through the column at a speed DC 1 ml/ml

Flowing through the column material was collected and frozen in plastic vial, and the resin was washed at least 3 column volumes of buffer BN1. The elution of the plasmin(ogen)and carried out by the buffer TLN-1 followed by washing approximately three volumes of 3 M solution of NaC, two volumes of purified water and two volumes of 20% ethanol + 1 M NaCl (method 1). With the second method (method 2) was performed with the same procedure used for method 1, but with additional washing 3 M NaCl before elution of the plasmin(ogen).

Analytical research.

The method of determination of Glu-plasmin(ogen): ELISA-kit for Glu-plasmin(ogen)and, Kunkle, (Imunclone®) (American Diagnostica, Greenwich, CT, USA), used in the described experiments, is a sandwich enzyme-linked immunosorbent analysis specific to determine levels of native plasmin(ogen)and man. Quantitative threshold analysis (in accordance with the lowest standard on the calibration curve) in the plasma or plasma derivatives is 0,063 µg/ml.

The activity of plasmin: fibrinolytic the study was performed by semi-quantitative determination of the activity of plasmin in the eluates. Briefly, free from the plasmin fibrinogen(ogen) (Enzyme Research) were incubated with different concentrations of normal United plasma (Unicalibrator, Stago) or purified plasminogen lirovannomu with affinity columns, in the presence of an excess of streptokinase. The time during which culminated in the degradation of the clot was recorded and compared with the complete degradation of the clot in the sample.

Determination of protein: Total protein was analyzed using the method of Bradford (Bradford MM. A rapid and sensitive mthod for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-54, 1976). In tables 8A and 8b presents the results of chromatographic removal and cleaning Glu-plasmin(ogen)and human plasma using TEM-ligand. In table 8A summarizes the data obtained by purification of plasmin(ogen). The table shows that method 2 gives some of the best cleaning of the plasmin(ogen)rather than the method 1 cleanup in 567-time and yield of 91.6%. In table 8b shows that, on average, 99.5% of the plasmin(ogen)and was removed from the unbound fraction, mostly composed by the number obtained in the eluate (table 8A).

Table 8A

The effect of an additional washing TEM-tar 3 M sodium chloride (method 1 compared to method 2) on the specific activity, purification and output plasmin(ogen)and from plasma
Method 1Method 1Method 2Method 2
FactionSource materialThe eluateSource materialThe eluate
Volume (ml)4013,24020
Protein (mg/ml)46,960,291 55,410,180
Glu-plasmin(ogen)

(ág/ml)
65,418,278,0142,9
The plasmin(ogen)

(IU/ml)
Did not˜0,5˜1
Specific activity

(mg plasmin(ogen)and/

mg protein)
0,0014of 0.6250,00140,794
The degree of purification446567
The output of the plasmin(ogen)and,

%
91,8to 91.6
Table 8b

Remove the plasmin(ogen)and plasma (mean results for the two methods)*
MethodChromatogra-

specific faction
Glu-

The plasmin(ogen) (average,

µg/ml)
Volume

(average, ml)
Remove the plasmin(ogen)

(%)
1 & 2*Plasma71,740,0 99,5
The unbound fraction is0,18to 83.5
*Methods 1 and 2 are identical and includes a collection of disjoint (flowing) fractions.

EXAMPLE 6: Effect of conditions chromatography on efficiency of lysine-immobilized ligand in affinity purification of plasmin(ogen).

Phase chromatography.

Studied affinity purification using resin with immobilized lysine ligand, using two commercial lysine-resin. Used chromatography method presented in the literature (see Robbins KC, L. Summaria Plasmin(ogen) and Plasmin. Methods Enzymol. 45: 257-73, 1976., for method 2, described below), and the method developed in the laboratory of the authors of the invention (method 1 below).

Each of the two resins with immobilized lysine (Ceramic Hyper DF obtained using Biosepra and sepharose 4B manufactured by Pharmacia) was made in a column with a diameter of 10 mm (Pharmacia, Sweden). Each column contained 2 ml (wet volume) of resin.

Aliquots crioestaminal fresh frozen plasma was subjected to thawing at 37°and filtered through a depth filter of 3 μm, to obtain a clear solution.

Phase chromatography was performed using one of the following methods:

Method 1

The column was washed with 4 volumes of each of the following process is s: 1) purified water, 2) TLN-0,1, 3) TLN-1 and 4) purified water. The equilibration was carried out 4 volumes BN1. The filtered plasma (40 ml) was applied on the column at a speed of current of 1 ml/min Sample flowing through the column material was collected in plastic bottles, and the resin was washed with buffer BN1. The elution of the plasmin(ogen)and conducted buffer TLN-1 followed by washing 3 M NaCl and 2 volumes of purified water and 2 volumes of 20% ethanol+1 M NaCl.

Method 2: (reference 5)

Aliquots crioestaminal fresh frozen plasma (40 ml) was filtered through a filter depth of 3 μm. To 40 ml of the filtered plasma was added 4 ml of 0.5 M Tris, 0.2 M lysine, 1 M NaCl buffer, pH 9.

The column was washed with 4 column volumes of purified water and balanced 0.1 M phosphate buffer, pH 7.4. Diluted plasma was passed through the resin at a rate of 1 ml/min Samples of the unbound material was collected in plastic bottles, and the resin was washed with 0.1 M phosphate buffer, pH 7.4 until such time as the optical absorption of the effluent at 280 nm reached baseline values. Then the plasmin(ogen) was suirable 0.2 M ε-aminocaproic acid, dissolved in 0.1 M phosphate buffer, pH 7.4, and collected in a plastic Cup. For the elution was followed by washing with about 2 volumes of 3 M NaCl and purified water.

In tables 9a and 9b compare the removal and cleaning of the plasmin(ogen)and using two commercial buildings, the ski resins with immobilized lysine, using each of the resins of two different cleaning method. Although there are relatively small difference in the output of the plasmin(ogen)and eluate (PL. 9a), it can be seen that when using method 2 and the resin Ceramic Hyper DF is higher purification of plasminogen.

The results showed that the application of resin Lys-Ceramic Hyper DF and chromatographic method 2 was able to achieve 444-fold purification. In addition, most of the plasmin(ogen)and the source material was obtained in the fractions of peak (76,5% in the unbound fraction + 10.9% in the eluate, only about 10% of the applied plasmin(ogen)and remains unaccounted for). However, removing the plasmin(ogen)and the unbound fraction was only 23.5%.

1. Method specific removal of plasminogen from a mixture containing the substance and fibrinogen by applying this mixture to the chromatographic column containing insoluble matrix for chromatography, covalently cross-linked to tranexamic acid, washing the column neutral aqueous solution containing salt, and collecting the unbound material, and tranexamic acid is linked to the matrix via the amino group via the linker, whose length is more than 3 carbon atoms.

2. The method according to claim 1, wherein the mixture is selected from the group which ostoja of body fluids, such as blood, blood fractions, cryoprecipitate, cell culture, animal tissue extracts, such as the lungs of a bull, the bull intestines, or extracts of animal bones, as well as water-immiscible animal fats.

3. The method according to claim 1, characterized in that the matrix for chromatography is a chromatographic material that is hydrophilic substance, such as agarose, cellulose, glass with adjustable pores, silica gel, dextran or synthetic organic polymer, such as polymer-based polyacrylamide-polystyrene.

4. The method according to claim 1, characterized in that the matrix for chromatography is a chromatographic material that is agarose or separate.

5. The method according to claim 1, characterized in that the matrix for chromatography is a chromatographic material that is particulate matter or monolithic block of material.

6. The method according to claim 1, wherein the linker is a bifunctional linker.

7. The method according to claim 6, wherein the bifunctional linker selected from the group consisting of N-hydroxysuccinimide, DAPA, 1,6-diaminohexane, succinic acid, TNB, pyridyldithio or combinations thereof.

8. The method according to claim 1, characterized in that the mixture after application to the chromatographic column incubated with indicated the second matrix over a period of time, sufficient to bind plasminogen.

9. Insoluble matrix for the purification of mixtures containing plasminogen and fibrinogen, plasminogen using affinity chromatography, which represents an insoluble matrix for chromatography, which covalently cross-linked to tranexamic acid through the amino group of this amino acid via the linker, whose length is more than three carbon atoms.

10. Insoluble matrix according to claim 9, characterized in that the insoluble matrix for chromatography is a chromatographic material.

11. Insoluble matrix of claim 10, wherein the chromatographic material is a hydrophilic substance, such as agarose, cellulose, glass with adjustable pores, silica gel, dextran or synthetic organic polymer, such as polymer-based polyacrylamide-polystyrene.

12. Insoluble matrix of claim 10, wherein the chromatographic material is agarose or separate.

13. Insoluble matrix of claim 10, wherein the chromatographic material is a particulate substance or monolithic block of material.

14. Insoluble matrix according to claim 9, wherein the linker is a bifunctional linker.

15. Insoluble matrix according to claim 9, characterized in that myfunc the optional linker selected from the group consisting of N-hydroxysuccinimide, DAPA, 1,6-diaminohexane, succinic acid, TNB, pyridylsulfonyl or combinations thereof.



 

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SUBSTANCE: method involves treating sunflower oilcake with catholyte; removing treated solution; extracting protein; filtering; drying sediment; grinding; simultaneously with treatment of sunflower oilcake with catholyte, providing treatment of soya with anolyte, with catholyte and anolyte circulating at equal velocities. Apparatus has two chambers connected with each other through semi-permeable partition. Each of said chambers is equipped with electrodes and dc source. Apparatus is further equipped with collecting chamber and pumps with pipelines.

EFFECT: increased efficiency of method and apparatus, reduced production time and decreased costs of additives.

1 dwg, 1 tbl

FIELD: biotechnology.

SUBSTANCE: method involves purification of protein raw, addition of water and ice, milling a mixture followed by hydrolysis. After hydrolysis collagen-containing solution is homogenized and collagen is separated. Hydrolysis is carried out for two stages. The first stage is carried out by treatment of the reaction mixture with lipase from fungus Rhizopus oryzae, and at the second stage a proteolytic enzyme as neutral protease is used. Invention provides preparing collagen approaching to natural collagen by physicochemical and structural-mechanical properties. Invention can be used in food processing industry, cosmetic, medicinal and other branched of industry.

EFFECT: improved preparing method.

4 cl, 3 ex

FIELD: biotechnology, preparative biochemistry.

SUBSTANCE: invention proposes a method for preparing the recombinant human tumor necrosis factor-aplha (TNF-alpha). Method involves culturing the strain-producer E. coli SG20050/pTNF311Δ, disruption of cells by ultrasonic oscillation, extraction of the end protein and 3-step chromatography purification procedure on DEAE-cellulose column in the linear gradient concentrations of NaCl at pH 8.0, on hydroxyapatite in the linear gradient concentrations of potassium phosphate at pH 8.0 and on hydroxyapatite in the linear gradient concentrations of potassium phosphate at pH 6.7. The recombinant TNF-alpha prepared by the proposed method shows the reduced content of impurity proteins, nucleic acids and lipopolysaccharides especially that provides its direct medicinal using. Invention can be used in medico-biological industry.

EFFECT: improved preparing method, enhanced quality of polypeptide.

2 tbl, 5 dwg, 2 ex

FIELD: biotechnology, preparative biochemistry.

SUBSTANCE: invention proposes a method for preparing the recombinant human tumor necrosis factor-aplha (TNF-alpha). Method involves culturing the strain-producer E. coli SG20050/pTNF311Δ, disruption of cells by ultrasonic oscillation, extraction of the end protein and 3-step chromatography purification procedure on DEAE-cellulose column in the linear gradient concentrations of NaCl at pH 8.0, on hydroxyapatite in the linear gradient concentrations of potassium phosphate at pH 8.0 and on hydroxyapatite in the linear gradient concentrations of potassium phosphate at pH 6.7. The recombinant TNF-alpha prepared by the proposed method shows the reduced content of impurity proteins, nucleic acids and lipopolysaccharides especially that provides its direct medicinal using. Invention can be used in medico-biological industry.

EFFECT: improved preparing method, enhanced quality of polypeptide.

2 tbl, 5 dwg, 2 ex

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