Method of cleaning thrombin solution from infectious particles
SUBSTANCE: invention relates to biochemistry. A method of cleaning thrombin solution from infectious particles is provided. Macromolecules are added to the starting thrombin solution. The obtained solution is then passed through a nanofilter to obtain a thrombin solution free from infectious particles. Said macromolecule is not a nonionic surfactant, is different from thrombin and can be selected from a polymer containing at least 3 monomers of sugar, amino acids, glycols, alcohols, lipids or phospholipids. A thrombin-containing solution obtained using said method is also provided.
EFFECT: invention increases efficiency of removing infectious particles from thrombin solution; thrombin output reaches 93% using said method.
20 cl, 2 dwg, 13 tbl
The scope of the invention
This invention relates to the purification of protein solutions. In particular, the invention relates to the purification of solutions of proteins from infectious particles by nanofiltration.
Human blood is the source of a wide range of medical products used in the treatment of acquired and/or inherited hematologic disorders and life-threatening diseases. Examples of such products are immunoglobulin, factor VIII, albumin, α1-antitripsin, antithrombin III, factor IX, factor XI, factor concentrates prothrombinase complex, fibrinogen and thrombin.
Because human blood is the source of raw materials for these products may contain infectious agents that are responsible for the transmission of serious diseases, sterility of the products is the main requirement. Making big efforts to inactivate and/or remove infectious agents such as hepatitis, human immunodeficiency virus (HIV), the causative agent of transmissible spongiform encephalopathies (TSES), which may be present in such preparations.
Viruses with lipid membranes can be effectively inactivated by exposure to the material being processed organic solvents and surfactants, as described for example in the R 0131740 and US 4481189. However, because the processing of such solvents and detergents no significant impact on bezobolochnye the virus or pathogen TSES should apply other methods of inactivation, such as pasteurization, gamma or ultraviolet (<280 nm) radiation or nanofiltration.
Among these methods of inactivation nanofiltration is the most mild. There are two process nanofiltration: a dead-end flow and tangential flow.
(1) dead-end flow - the fluid is directed to the membrane under pressure, particles with dimensions greater than the pore size of the membrane accumulate on the membrane surface, whereas the molecules of smaller size to pass through it.
(2) a Tangential flow (nanofiltration with tangential flow, tangential nanofiltration, FBL) - fluid is pumped tangentially to the membrane surface, which creates a pressure difference across the membrane, allowing particles smaller than the pore size to pass through the membrane, while larger particles continue to flow along the membrane tangential flow. Tangential flow does not allow detainees molecules to accumulate on the membrane surface.
DiLeo A.J. et al.(1992) Biotechnology 10:182-188 describes the removal from solution of viral particles of small diameter (<30 nm), such as phage φ174 using membra the s Viresolve/70™. To determine the extent to which the adsorption of the particles contributes to the destruction of viral particles to the solution of viral particles were added to human serum albumin (CSA) at a concentration of 2.5 mg/ml, due to competition reduce adsorption of viral particles on the membrane. It was found that in the presence of the protein CSA delay viral particles increases by 0.5 to 0.7 log. The authors argue that these results indicate that the ability of a membrane to hold the particles depends primarily on the screening properties of the membrane and increase delay viral particles, most likely, is the result of protein concentration polarization on the membrane surface.
DiLeo AJ. et al. (1993) Biologicals 21:275-286 describes further experiments with membrane Viresolve/70™. It was again shown that the presence of the protein increases the degree of delay of the model viruses in accordance with the concentration polarization, where the charged protein introduces additional resistance to the passage of viruses through the membrane.
Hoffer L. et al. (1995) J. Chromatography 669:187-196 describes the purification of factor IX from human plasma and virus removal using membrane Viresolve/70™. The smallest of the measured viruses was canine parvovirus (16-18 nm in diameter), which was removed with efficiency, expressed as the logarithm of the concentration reduction (LRV). logreduction value) equal to 5.2. The output of factor IX averaged 83±9%.
In the publication WO 96/00237 (Winge) describes how filtering of viral particles from a solution containing macromolecules, mainly proteins, polysaccharides and polypeptides, by increasing the salt content in the solution is from 0.2 mol to saturation of the solution of the corresponding salt. The method reduces the processing time and the degree of dilution of the solution by filtering viruses and optimizes the output of the filter virus. The described method provides virus filtering by using the so-called technique of "dead-end stream, which gives several operational and economic advantages compared to the commonly used technique of tangential filtration of viruses.
In the publication US 6,096,872 (Van Holten et al.) the described method nanofiltration anti-D immunoglobulin in buffer solution with high ionic strength and in such a filler as Polysorbate 80™. Additional steps include diafiltration to concentrate anti-D protein and to reduce the concentration present filler. In particular, the method comprises the following steps: (a) fractionation of human plasma in alcohol; b) re-suspension of the obtained Precipitate II; C) mixing resuspending Precipitate II with the buffer solution with high ionic strength, containing a filler; d) conducted the e nanofiltration immunoglobulin.
In the publication US 6,773,600 (Rosenblatt et al.) the described method of purification protein material, such as immunoglobulin, to remove impurities, such as pathogenic viruses, consisting of the following stages:
a) mixing the protein material with:
1) buffer solution with a low pH, low conductivity, designed to reduce the pH between 5.0 and 6.0 and to achieve the ionic strength of less than 30 mS/cm;
2) non-ionic surface-active agent;
3) clathrate modifier and
b) conducting nanofiltration protein material for the production of purified material, essentially free of viral particles.
Preferably, clathrate modifier represented a polyol or sugar alcohol having 4 to 8 hydroxyl groups.
In one aspect of the invention, a method of purification of the protein solution from infectious particles. The method includes:
(a) the addition of macromolecules to the solution of protein and
(b) passing the solution through nanofilter,
thus obtaining a solution of proteins, essentially cleared of infectious particles.
In another aspect of the invention, a method for increasing the efficiency of removal of infectious particles from a solution of the protein with NF, including the addition of macromolecules to the solution before and/or during nanofiltration.
The term "registered the ion particle" refers to a microscopic particle, such as a virus or a prion, which can infect the cells of a biological organism and multiply in them. In one embodiment of the infectious particle is a virus particle. Infectious particles that must be removed, have a size of from 15 to 80 nm. In another embodiment of the infectious particles are larger than 80 nm.
The term "protein solution" refers to a homogeneous mixture composed of one or more proteins, dissolved in another substance. In one embodiment, the solution containing the protein originates from whole blood. In another embodiment, the solution is a plasma. Other possible sources of proteins can be animal proteins and recombinant proteins produced in cell culture or transgenic animals.
Proteins that can be purified by the method according to the present invention, include all proteins used for therapeutic purposes, which may contain infectious particles and which is filtered through a filter that removes infectious particles. In one embodiment of the proteins have a molecular weight of less than 180 kDa. In another embodiment, the proteins have a molecular weight of about 160 kDa. In the following embodiment of the proteins have a molecular weight less than 150 kDa, or less than 140 kDa.
These proteins include:
coagulation factors such as factor IX/IXa, factor VIII, prothrombin/thrombin, factor VII/VIIa, factor X/XA, factor XI/Ia, factor XII/XIIa, prekallikrein, high kininogen, plasminogen, urokinase, an inhibitor of protein C;
anticoagulation factors or subunit, such as protein C, protein S, antithrombin III, heparin cofactor II, A2-doesn? t, protein Z;
growth factors such as insulin-like growth factor 1 (IGF-1), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF);
neurotropic factor, such as nerve growth factor tissue (NGF), glial-derived neurotrophic factor (SPF);
hormones, such as erythropoietin, growth hormone
interferons and interleukins, such as interferon alpha, beta and gamma, interleukin 1, 2, 3, and 7;
recombinant proteins, for example; recombinant coagulation factors or protivosvertyvayushchei factors, recombinant growth hormone
but not limited to the above.
In one embodiment the protein is a charged protein. Charged protein refers to hydrophilic molecules.
The protein yield using the method of the present invention is high and may be at least 59, 65, 71, 72, 80, 81, 84, 85, 87, 93, 94, 96 or 100%. The average yield of protein using the method of this invention was found to be within 75-93%.
The term "essentially purified" refers to the removal efficiency infectious the particles. In one embodiment, the removal efficiency of infectious particles is more than 4 LRV (log reduction in concentration) or about 5, 6, 7 or 8 LRV.
The definition of the term "macromolecule" means generally accepted polymers and biopolymers, as well as polimernye molecules with large molecular mass. Macromolecule that can be used in the method of this invention should possess in one embodiment at least one of the following properties, in another embodiment, two properties, and in the next incarnation - all of the following properties:
- biocompatibility - toxicity;
- no vzaimodeistvie with protein, exposed to treatment;
- solubility in water.
The macromolecule according to the invention may be a polymer of at least 3 monomers of sugars, amino acids, glycols, alcohols, lipids or phospholipids. In one embodiment, the macromolecule is a polymer of at least 6 monomers. In another embodiment, the macromolecule is a polymer of at least 9 of the monomers. In the following embodiment, the macromolecule is a polymer of at least 12 monomers. Examples of macromolecules according to the invention include proteins (such as albumin, gelatin), polysaccharides (e.g. dextran, chitosan, carrageen), polymers, sugar alcohols (e.g. the measures the polymer mannitol), phospholipids, polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), cellulose derivatives, vinyl polymers and polyglycols. The macromolecule does not contain a polyol or sugar alcohol having 4 to 8 hydroxyl groups or non-ionic surface-active agent.
In one embodiment of the invention, the macromolecule is an albumin. In another embodiment of the invention, the macromolecule is a dextran. The dextran molecules can have different molecular weight, for example, in the range of 5-80 kDa, or about 5, 25 and 80 kDa.
In one embodiment, the macromolecule can be added in concentrations from values greater than 0.01 mg/ml to values less than or equal to 2 mg/ml In another embodiment, the macromolecule is added in concentration range from values higher than 0.15 μm and up to values less than or equal to 31 μm. In the following embodiment, the macromolecule is added in a concentration less than the concentration of the protein solution.
Nanofiltration refers to the process of separation under pressure, which may occur on the selective separating layer is formed by a semi-permeable membrane. The driving force of the separation process is the pressure difference between the party, which is part of the feed stream (retentate) and the side of the filtrate (permeate) on the separating layer of the membrane.
In one embodiment of the invented what I nanofilter, which can be used in the method of the invention may exclude particles larger than 104Daltons. In another embodiment, this maximum size can be more than 105Daltons. In another example, the cutoff occurs in the region of 104Dalton and 3×105Daltons. In one embodiment of nanofilter is a membrane Viresolve/70™ or Viresolve/180™.
The solution is passed through nanofilter in accordance with standard filtration methods. For example, the solution can pass through nanofilter or according to the method of tangential nanofiltration or method stub thread. Solution protein can also add stabilizers such as mannitol and salt.
Another aspect of the invention is a solution of the protein, purified from infectious particles by the method of the invention.
Brief description of drawings
For the understanding of the invention and how it may be embodied in practice, the following examples of embodiments that are possible embodiments are not limited to, with reference to the below drawings, where:
Figure 1 is a schematic diagram illustrating the Assembly of the membrane system Viresolve/70™
Figure 2 is a graph showing the effect of concentration of CSA (human serum albumin) on the removal efficiency of the parvovirus MVM(p) (expressed in LRV) when Phi is Tracii through the Viresolve/70.
Descriptions of the applications, patents and publications mentioned above or below, are included here in their entirety by reference.
These examples are illustrative, but not exhaustive.
Detailed description of exemplary embodiments of the
Materials and methods
Viruses and cells
A9 (ATSS-CCL1.4), the cell line of mouse fibroblasts were maintained in the environment. Needle in the modification Dulbecco (DMEM) supplemented with 2 mm L-glutamine, 1% solution of penicillin, streptomycin and amphotericin b In 0.1% solution of gentamicin sulfate and 5% fetal serum of calves (FCS) (Biological Industnes).
FrhK-4 (R.E. Wallace et al. In vitro 8:333-341, 1973), a permanent cell line from kidney rhesus monkeys, were grown in DMEM with 4.5 g/l glucose, 110 mg/l sodium pyruvate and 1.1 g/l sodium bicarbonate with the addition of 4 mm glutamine, 1% nonessential amino acids and 10% FCS.
The mouse parvovirus MVM(p) was kindly provided by Dr. Jacob tal (Jacov Tal, University of Ben-Gurion) and were cultured in cell A9. Production of virus was produced as described previously (Tattersall R, Bratton J. (1983)). The A9 cells were infected with MVMp multiplicity of infection (MOI)of 0.1-1. Upon reaching the cytopathic effect, for example, when the surface was separated about 30% of the cells, the cells and medium were collected and centrifuged at 1000 g for 10 min at 4°C. the Cell precipitate is then resuspendable in the morning the e TNE (Tris-HCl 50 mm pH 7,2, NaCl 150 mm, EDTA 50 mm) and again centrifuged at 1000 g, as described above. The procedure was repeated 3 times. At the end of the last stage centrifugation of cell sediment resuspendable in TNE buffer (Tris-HCl 50 mm pH of 8.7, 0.5 mm EDTA), and then subjected to 3 cycles of freezing in liquid nitrogen and thawing at room temperature. For isolation of the virus destroyed the cells were subjected to centrifugation at 20000 g for 10 min at 4°C. the Supernatant containing the virus was poured into the aliquot and stored at -70°C. Before use the original preparation of the virus was passed through a filter with pores of 0.2 μm (Minisart, Sartonus), and then through a filter with pores of 0.1 μm (Durapore, Millipore).
Hepatitis a virus (HAV) (NM) (Cooper P, et al, 1978) was produced in the supernatant of infected cells FrhK-4. After clarification at 3500 rpm for 15 min (Sigma 3K2) cells were collected and resuspendable in phosphate-buffered saline (PBS), and treated with ultrasound and osvetleni at 2500 rpm for 10 minutes and Then the supernatant was poured into the aliquot and stored at < -70°C. the Supernatant after the first centrifugation was concentrated by ultracentrifugation at 19000 rpm for 7 hours at 4°C (Beckman R19). Sediment resuspendable in PBS, was poured into the aliquot and stored at -70°C.
Titration of virus and determination of the logarithm of the concentration reduction LRV
The titer of the original drug virus MVM(p)and all of the samples were determined using test TCID 50(50% tissue culture infective dose, dose infecting 50% tissue culture). Cultivation of virus/samples was done in a series of 2-fold dilution in 96-well-plate with a nutrient media A9 as a diluent (final volume 50 μl) To each well was added 100 μl of A9 cells (5×103) and the plates were incubated at 37°C for 7-10 days. Samples containing the virus MVM(p) in high titer, pre-diluted for the quantitative determination in the above test. Each well was tested for infectivity and titer expressed as the dose infecting 50% tissue culture per milliliter (TCID50/ml) according to the formula of Spearman-Karber. Titers of HAV in the samples was determined as described above, with minor changes; cells FrhK-4 was used at a concentration of 1×104on the hole and the infectivity was determined approximately 10-12 days after infection.
If in the sample it was not possible to determine the infectivity in determining took either a larger volume of sample in the minimum netforensics.com breeding, or were subjected to ultracentrifugation large number of sample (Beckman with the rotor R19 at 19000 rpm for 15 hours at 4°C) and the infectivity was determined in the whole volume.
LRV was calculated by the following formula:
LRV = log (titer of the virus in the first sample × sample volume) - log (titer of the virus in the second sample × the volume of the sample)
In the samples used in the experiments with filtering using Viresolve/70™, made MVM(p) or the CAA to reach the final concentration above 1×105TCID50units/ml Fraction of deposited material does not exceed 10%. Loaded with virus solutions to filter through the Viresolve/70™ was filtered through a filter with pores of 0.2 μm (Millipak 40, Millipore).
Determination of thrombin
The determination of the activity of thrombin was carried out according to the Protocol of the European Pharmacopoeia Test (0903/1997) with slight modifications. The calibration curve (4-10 IU/ml) was obtained when mixing standard thrombin with a solution of fibrinogen (Enzyme Research Laboratories, Ltd) containing fibrinogen of 0.1%. The concentration of thrombin in the sample was calculated by the standard curve and multiplied by the dilution factor.
Determining the concentration of total protein
Protein concentration was determined using the Biuret method (Doumas et al, 1981).
Before adding biuret reagent, the samples were subjected to precipitation with acetone. Samples and standard were diluted 2.5 times with acetone, incubated for 5 min, and then centrifuged for 5 min at 20000 g. Sediment resuspendable in 0.1 ml of saline solution, mixing thoroughly on a vortex, and to each sample was added to 0.9 ml of total protein reagent (Sigma). After incubation for 15 min at room the temperature was measured spectrophotometrically the absorbance of samples and standards at 540 nm. The protein concentration was calculated by comparison of the results in the samples and the standard.
Thrombin in the processed material
Thrombin is usually made from plasma without cryoprecipitate the company Omrix (tel Aviv, Israel). During production the product is processed detergent solution (1% tri-n-butylphosphate / 1% Triton-X100) for removal of enveloped viruses. The solvent and the detergent is then removed by chromatography, and the product is subsequently extracted by elution. To increase the stability of the product add mannitol at a final concentration of 2%. Human serum albumin (CSA) is added to a final concentration of 0.2%. The product is then filtered through the Viresolve/70™ at room temperature with a flow rate of retentate 1400-1600 ml/min and permeate 65-70 ml/min
Samples were stored frozen until filtration. Before filtration, the samples were thawed at 37°C.
Raw materials and buffers to filter
To determine the effect of macromolecules on the nanofiltration were prepared with different source materials and appropriately selected buffers to filter (see table 1).
Tangential nanofiltration (FBL) with the application module Viresolve/70™
Module Viresolve/70™ production size is the nominal size of the membrane is 0.1 m2. In d nom study used a smaller modules with membranes the size of 1/3 or 1/6 from the place of production of the membrane and the conditions were changed respectively Tangential nanofiltration was carried out according to the recommendations the manufacturer In the process of filtering through the Viresolve/70™ used 2 peristaltic pump: one (retentate) used for the circulation of the product module, and the other (permeate) was used for extraction of the filtrate and his collection (Figure 1), When reducing the size 1/6 pumps were set up to achieve the desired speed current 250±10 ml/min pump, pumping over retentate, and 11-12 ml/min for pump, Sipper permeate. When reducing to 1/3 of the speed of the current is doubled. Before filtration the product was washed with purified water, and then buffer for filtering. The composition of the buffer to filter selected in accordance with the source material (see Table 1).
For a module, reduced 1/6, for filtering used 1-1 .5 liters of source material, and for a module, the reduced 1/3, used 2-3 liters. Before filtering through the Viresolve/70™ samples were filtered through a filter with pores of 0.2 μm (Millipak 40, Millipore). The filtering process was carried out at room temperature using the above mentioned speed of the current. After filtration of the source material module Viresolve/70™ were washed 3 times the corresponding buffer for filtration, followed by the purification procedure, including the washing of the filtrate and retentate purified water. Collected during the technological process samples used to determine the titer of MVM(p) or HAV and conc is the protein and thrombin (Table 2).
To assess the effect of addition of macromolecules to remove small bezobolochnym viruses and the product yield in filtering through the Viresolve/70™ conducted a series of experiments with tangential nanofiltration. As a model explored the purification of thrombin using module Viresolve/70™. The first part of the experiment was designed to optimize the operating conditions of the system Viresolve/70™ (1/3 ft2or 0.03 m2with application of the Protocol of the manufacturer to change the speed of the current. The experiment was carried out as follows: the day of the examination the thrombin solution was thawed at 37°C and then filtered through polyethersulfone filter with pore size 0.2 μm and an area of 500 cm2(CH5925PPZK, PALL). The feed container was then filled with 300 ml of the filtered solution of thrombin. The flow rate of the pump, pumping over retentate, was set at 500 ml/min, and the product was recycled through the membrane for 30 minutes and Then the pump is pumping over permeate, configured to obtain speed current 3,5, 5, 10, 15, 20, 25 and 30 ml/min and worked on each speed current so that the permeate is returned to the channel retentate for 30 minutes (full recirculation). Sample retentate (R) and permeate (P) were collected at each speed of the current and stored at -70°C.
Then membra who have purified according to the instructions Millipore.
Frozen samples were thawed and measured the protein concentration and the activity of thrombin.
The experiment was repeated twice, the first time after the cleansing of the membrane, and the second time using the new membrane. Each time the cycle is repeated with a new vial of thrombin.
The optimal flow rate was chosen for the highest values of protein yield and activity of thrombin.
The integrity test was performed on the module Viresolve 70 according to the manufacturer's instructions.
The results showed (see Table 3 and 4)that the best current velocity was in the range of 20-25 ml/min. At such speeds, the current average yield of thrombin and protein yield was approximately 93% and 75%, respectively.
After determining the optimal speed of the current was possible to evaluate filtering thrombin in the conditions of production on a smaller scale. For these experiments the feed container was filled with 3000 ml of a solution of thrombin and samples were taken (T0).
The recirculation pump was configured on the flow rate 500 ml/min and the product was recycled within 30 minutes, the Volume of the whole party passed at optimal speed current determined in experiments with a velocity change of current, until then, until reaching the final volume. Collected samples from the feed container (C1and PE is meat (P 1). The solution was diafiltrate the corresponding buffer in the amount of 3-fold greater than the final volume to obtain the greatest possible weight of a protein without significant dilution of the product, and then collected the following sample of the permeate (P2).
We measured the protein concentration and the activity of thrombin in the samples before and after diafiltration to determine product yield when diafiltration without it. Module Viresolve 70 kept at 4°C and to assess the lack of damage to the membrane spent the integrity test according to the manufacturer's instructions.
The obtained results (Tables 5 and 6) showed that the average yield of thrombin was 96%, and the average protein yield was approximately 77%. In addition, the stage of diafiltration (W) led to an additional increase in activity of thrombin by 3-5%.
After you have established conditions for the tangential filtration of thrombin using Viresolve 70™, it has become possible to assess the effectiveness of the system for virus removal.
In the first experiment, described below, used the optimal velocity change of current. The nominal size of the membrane modules used was 1/3 ft (0.03 m2or 1/6 ft (0,015 m2). Due to the volume of product to be filtered and the speed of the current was changed accordingly.
what about all the experiments carried out in the samples of thrombin, to filter, were made MVM(p) or the CAA to achieve a final concentration of over 1×105TCID50units/ml (see details in Methods section). Loaded with virus solutions were filtered through a filter with pores of 0.2 μm and the first samples to the filter (L1 and L2) were selected for titration of the virus. The remaining material is loaded with a virus, filtered through a pre-equilibrated membrane Viresolve 70™ with the appropriate buffer. The filtering process continued as long as the volume of retentate not reached 100-250 ml Whole filtrate was collected and selected the sample for titration of the virus (F). The filtering process continued with repeated washing of the filter corresponding buffer for filtering. The filtrate from the washing step were either taken separately and for titration of virus selected an additional sample (W), or together with the previous filtrate (titres of virus in these samples were expected, respectively). At this stage, collected retentate whole process and selected a sample of (R) for titration of the virus. After filtering, all modules were washed and tested for damage in the test of integrity offered by the manufacturer of the module (Millipore).
The results (shown in Table 7) showed that under optimal conditions the output of thrombin and protein, UDA is giving parvovirus using this system highly efficient. In addition, the results unexpectedly turned out to be better than described in previous studies (Hoffer et al 1995, DiLeo et al 1992, DiLeo et al 1993, Jemberg et al 1996, Adamson 1998).
To confirm these results and clarify the mechanism for obtaining the best indicators of concentration reduction was conducted a number of experiments.
The first series of experiments was designed to evaluate the effect of albumin concentration on removing the virus. Used 4 of albumin concentration: 2 mg/ml, 1 mg/ml, 0.2 mg/ml and 0.01 mg/ml with the addition of 20 mg/ml of mannitol. These solutions were loaded with either the CAA or MVM(p), in accordance with the conditions and procedures described previously. However, in the experiment with MVM the flow rate was maintained at the level recorded at the beginning of the filter (the worst conditions; the increase in pressure during filtration). On the contrary, to HAV the flow rate recorded at the beginning of the filtration, and then filtration was performed without increasing pressure. The results showed that the reduction in the concentration obtained previously for MVM(p) at the level of approximately 6.0 log reduction (LRV) were reproducible (see Table 8 and Figure 2). It is noteworthy that the addition of albumin human serum at concentrations of 0.2-2 mg/ml had a comparable effect on the performance of the removal MVM(p). However, the addition of albumin human serum at a concentration 20 times lower (0.01 mg/ml) or nedbalek the e CSA in the feedstock has led to considerably less destruction of MVM(p) (the logarithm of the concentration reduction of approximately 5.0 or 4,7, respectively), indicating a role of molecules such as albumin in improving the rate of decrease in the concentration of the virus. It should be noted that both molarity of thrombin in various solutions of albumin amounted to 5.4 μm. These results suggest that in order to improve removal of MVM(p), it is enough to use CSA in a lower molar concentration than the molar concentration of thrombin.
Values of the rate of decrease in concentration for HAV were generally higher (>7,0 LRV), since particles of hepatitis a virus (27-32 nm) is larger compared with MVM(p) (18-26 nm). As shown for MVM, albumin human serum at concentrations of 0.2-2 mg/ml had a similar effect on the ratio of removal of the CAA. In addition, increasing pressure to maintain the initial speed of the current in the loops, which included MVM did not reduce the destruction of viral particles.
To further study the involvement of albumin in the virus removal process was carried out the following series of experiments. Studied the effect of the following solutions to remove viral particles: thrombin solution containing 0.2 mg/ml albumin and 20 mg/ml mannitol solution, mannitol and medium DMEM. Conditions in the process and procedures load viruses have been described above.
The obtained results (see Table 10) showed that a further decline in virus removal was obtained, when the source material is not added, no albumin, no thrombin (2,84 LRV), and the lowest values of the rate of decrease in concentration were obtained using medium DMEM (Biological Industries) (1,72 LRV).
To evaluate the effect of adding other macromolecules to delete the virus and the possible influence of the molecular size on the removal of the virus, used a solution of dextran with different size molecules and the same both molarity. These molecules were added to the treated sample, as described above. Procedure load viruses and filtration were carried out in accordance with the conditions set forth above.
The results obtained (Table) showed that in the first experiment using 80 kDa dextran at a final concentration of 0.23 mg/ml log reduction in titer MVM(p) was 6.2. A similar effect on the performance of the removal of the virus was discovered and when using dextran molecules of smaller size (5 kDa, 25 kDa) in the same molar ratio, and the titer of MVM(p) after filtration was also decreased by 6.1 to 6.2 log.
To determine whether the increase in the rate of current to influence the level of removal of the virus, the flow rate of the permeate was increased to 25 ml/min and maintained during the entire filtration process. The results showed that the increase in the rate of current had only a borderline effect is the index of the removal MVM(p) (Tabl), lower concentrations were recorded at the level of 5.7 log.
In addition, when the measured average speed of the current of the different cycles of filtration (see Table 13), it was found that the flow rate sharply declined in solutions containing macromolecules (e.g., albumin or dextran) in addition to thrombin. The lowest flow rate of 5.4 ml/min, approximately 46% of the initial speed of the current was observed when added to a solution of thrombin solution of human albumin at a final concentration of 2.0 mg/ml At a final concentration of bovine serum albumin (0.2 mg/ml or when using equimolar concentrations of dextran with different sizes of molecules was achieved by the flow rate from 63 to 74% from the initial one. Such differences in average speed current does not affect the performance of removal of viral particles, as described in the previous experiments. However, when the solution of thrombin was not made macromolecule or when used only buffer solutions (for example, DMEM, buffer + mannitol), the average speed of the current was close to the initial velocity of the current.
1. The method of purification of a solution of thrombin from infectious particles
(a) in the above solution add macromolecule; and
(b) pass this solution through nanofilter, thus obtaining a solution of thrombin, beings who, cleared of infectious particles, and this macromolecule is not a non-ionic surfactant and non-thrombin.
2. The method according to claim 1, where these infectious particles are viral particles.
3. The method according to claim 1, where the specified solution comes from whole blood or plasma.
4. The method according to claim 1, where the yield of purified thrombin after step (b) is in the range 59-100% or within 75-93%.
5. The method according to claim 1, where the logarithm of the concentration reduction (LRV) of these particles after step (b) is about 4-8.
6. The method according to claim 1, where these particles have a size of from 15 to 80 nm 80 nm or more.
7. The method according to claim 1, where the specified macromolecule selected from the group consisting of a polymer consisting of at least 3 monomer sugars; amino acid; glycols; alcohols; lipids; phospholipids.
8. The method according to claim 1, where the specified macromolecule selected from the group consisting of proteins; polysaccharides; sugar alcohols; phospholipids; polyvinylpyrrolidone; polyethylene glycol; cellulose derivatives; vinyl polymers; and glycols.
9. The method according to claim 1, where the specified macromolecule is a dextran.
10. The method according to claim 1, where the specified macromolecule is a dextran with a molecular weight in the range of 5-80 kDa.
11. The method according to claim 1, where the specified macromolecule is an albumin.
12. The method according to claim 1, g is e specified macromolecule add in the concentration range from values greater than 0.01 mg/ml to values less than or equal to 2 mg/ml
13. The method according to claim 1, where the specified macromolecule is added in the concentration range from values higher than 0.15 μm to less than or equal to 31 mm.
14. The method according to claim 1. where specified macromolecule type at a concentration lower than the concentration specified solution of thrombin.
15. The method according to claim 1, where the specified nanofilter has a border pass particle size above 104Yes.
16. The method according to claim 1, where the specified nanofilter has a border pass particle size lying approximately in the range of 104-3×105Yes.
17. The method according to claim 1, where the specified nanofilter is a membrane Viresolve/70™ or membrane Viresolve/180™.
18. The method according to claim 1, where the specified solution is passed through nanofilter method nanofiltration with tangential flow.
19. The method according to claim 1, where the specified solution is passed through nanofilter method nanofiltration with a dead-end thread.
20. A solution containing thrombin obtained according to the method according to claim 1.
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: gene engineering, in particular method for treatment of viral infections.
SUBSTANCE: protein ZCYTO21 has amino acid sequence which is nearly similar to amino acid sequence of interferon-α. Protein and antibodies thereto have antiviral activity and are useful in treatment of hepatitis B and C as well as other diseases.
EFFECT: new protein with antiviral activity.
71 cl, 1 dwg, 6 tbl, 7 ex
FIELD: preparative biochemistry, medicine, pharmacology.
SUBSTANCE: method for purification of interferon proteins is based on using cation-exchange chromatography on a solid matrix. Method is realized at more basic pH value, i. e. at relatively higher pH value corresponding to the isoelectric proteins point, pI, designated for purification. However, at this pH value indicated proteins are remained to be absorbed and therefore method involves using buffer solutions of organic or inorganic salts able to modify the solution ionic strength. Invention provides a simple method for industrial realization of the method and economy availability.
EFFECT: improved purifying method.
8 cl, 1 tbl, 6 ex
SUBSTANCE: disclosed is an isolated thymic stromal lymphopoietin protein (TSLP) or antigenic fragment thereof for inducing immune response, as well as a nucleic acid molecule coding it.
EFFECT: invention can find further use in therapy of atopic diseases.
7 cl, 8 dwg, 2 tbl, 7 ex
SUBSTANCE: compound contains IL-15 indirectly bound by covalent links with a polypeptide containing a sushi domain of an extra-cellular region of the alpha IL-15R subunit.
EFFECT: invention allows inducing and stimulating activation and proliferation of said cells, and effectively treating the conditions and diseases which requires higher activity of IL-15.
44 cl, 42 dwg, 4 tbl, 1 ex
SUBSTANCE: what is described is a composition containing an ordered antigen pattern where antigen represents IL-1, mutein IL-1 or fragment IL-1. There is also offered a based vaccine. The compositions offered in the invention can be applied for producing vaccines for inflammatory diseases and chronic autoimmune diseases, transmittable diseases and cardiovascular diseases.
EFFECT: compositions effectively induce immune responses, particularly humoral immune responses; compositions are the most suitable for effective induction of autogenic immune responses.
46 cl, 2 dwg, 3 tbl, 14 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: group of inventions relates to medicine, namely to immunology, and can be used for treatment of immune-mediated disease in mammal which needs it. For this purpose efficient amount of one or more antagonists capable of inhibiting both IL-17A (SEQ ID NO: 3), and IL-17F (SEQ ID NO: 4) in polypeptide IL-17A/F is introduced.
EFFECT: inventions ensure higher efficiency of treatment of immune-mediated diseases, due to novel polypeptide of interleukin17 (IL-17) family.
25 cl, 20 dwg, 9 tbl, 15 ex
SUBSTANCE: recombinant technique is used to obtain a fused polypeptide with activity of interleukin-7, containing a modified human IL-7 molecule in which the T-cell epitope is modified to reduce T-cell response against IL-7, and the Fc part of an immunoglobulin molecule which is fused through its C-end with the N-end of said modified IL-7 molecule. The obtained polypeptide is used in a pharmaceutical composition to stimulate immune response in a patient.
EFFECT: invention enables to obtain a polypeptide with interleukin-7 activity, having low immunising capacity.
8 cl, 43 dwg, 14 tbl, 12 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to biotechnology and medicine. What is offered is ProThrThrLysThrTyrPheProHisPhe peptide, a based pharmaceutical composition which is used for antitumour immune response stimulation, and also to methods of treating a mammal and immune response modulation.
EFFECT: range of products for cancer treatment is extended.
19 cl, 49 tbl, 3 ex
SUBSTANCE: invention relates to novel versions of peptides IL-21, where amino acids are delegated and/or replaced in region, consisting of amino acids No 83-96.
EFFECT: possibility of manufacturing medication for cancer treatment.
8 cl, 2 dwg, 1 ex
SUBSTANCE: invention relates to molecular pharmacology and specifically to a peptide which is part of an interleukine-15 (IL-15) sequence which can inhibit biological activity of the said molecule.
EFFECT: obtaining a peptide which inhibits T cell proliferation induced by IL-15, and apoptosis caused by tumour necrosis factor when bonding with the alpha subunit of the (IL-15R) receptor.
8 cl, 4 dwg, 5 ex
SUBSTANCE: in modified molecule IL-4RA, which inhibits mediated IL-4 and IL-13 activity, amino-acid remains 37, 38 or 104 represent cysteine. Polynucleotide, which codes specified antagonist, in composition of expression vector, is used to transform host cell and produce IL-4RA. Produced molecule IL-4RA is PEGylated and used to eliminate abnormalities that are related to high activity of IL-4 and IL-13.
EFFECT: invention makes it possible to produce antagonist with longer period of half-decay compared to non-modified IL-4RA.
17 cl, 1 dwg, 7 tbl, 7 ex
SUBSTANCE: invention relates to biotechnology, particularly to genetic engineering and it can be used in the biomedical industry to produce active medications of interleukin-29 (IL-29). Mutant forms of IL-29 (SEQ ID NO: 27, 29, 40, 41, 149 and 159) were offered with substitution of the cysteine residue in the position in accordance with position 171 of the aminoacid sequence of a mature protein of a wild type that are characterised by a correct formation of intramolecular disulfide bonds and accordingly they provide production of polypeptides with an antiviral activity as homogeneous medications at expression in the heterologous system.
EFFECT: vector structures and the host cells transformed by these structures for the expression of new versions of IL-29 are described.
15 cl, 37 tbl, 45 ex
SUBSTANCE: peptide is described by general formula I: X1-X2-X3-X4-X5 (I) where XI represents Q or V; X2 represents P; X3 represents G or (β-A); X4 represents R, or X4 represents K with X1-Q or with X1-V and X3-(p-A); X5 represents G, where G is glycine, (β-A) is β-alanine, P is proline, V is valine, Q is glutamine, K is lysine, R is arginine.
EFFECT: invention presents extended range of effective therapeutic agents involved in liver regeneration.
4 dwg, 1 tbl, 3 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: present invention relates to molecular biology and can be used in designing agent and methods of modulating body functions associated with HGF/c-met signalling pathway. The invention discloses HGF/c-met polypeptide-antagonists which are mutant forms of HGF which contain a mutation in the N-terminal part of the β-chain and/or in its dimerisation part. The disclosed polypeptides have lower biological activity compared to wild type HGG and can be used in modulating activity of c-met, cell proliferation, cell migration and angiogenic cell activity.
EFFECT: invention describes a method of obtaining HGF muteins using DNA recombinant technology and agents which are necessary for its existence.
22 cl, 8 dwg, 1 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: invention pertains to biotechnology. In particular, the invention relates to an Escherichia coli BL21 (pVEGF-A165) strain and can be used to produce a vascular endothelial growth factor - GST-VEGF-A165 protein. A novel Escherichia coli BL21 (pVEGF-A165) cell strain is obtained, which is transformed by the pGEX-VEGF-A165 plasmid. This strain produces a recombinant GST-VEGF-A165 protein.
EFFECT: invention enables to obtain a Escherichia coli BL21 (pVEGF-A165) strain which is stably transformed by plasmid which codes VEGF, and which secrete this factor in extracellular space when cultured in vitro.
3 dwg, 4 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: invention relates to molecular biology, specifically to proteins which regulate cell differentiation through inhibition of the TGF-beta cell superfamily. The invention is aimed at treating and preventing diseases related to human VgI orthologs. Noggin2 protein which contains VgI protein is injected into tissue in amount which is efficient for inhibiting VgI activity.
EFFECT: invention enables to solve the task of blocking the signal path activated by the TGF-beta factor of VgI in aniamal cells.
2 dwg, 4 ex
SUBSTANCE: invention refers to biotechnology, specifically to Noggin2 proteins and can be used in medicine. The signalling cascade activin is blocked by introducing Noggin2 in an organism, tissue or cell in amount effective to inhibit or decrease activin activity.
EFFECT: invention allows blocking effectively activity of protein activin.
2 dwg, 8 ex
SUBSTANCE: invention refers to medicine and concerns angiogenesis-preventing immunotherapy. Invention substance includes immunogenic compositions for treatment of disorders associated with angiogenesis intensification, containing oligonucleotides, coding polypeptides VEGFR2, introduced as a part of plasmid or viral vectors, as well as polypeptides VEGFR2, oligonucleotides, coding autologous VEGF with damaged function of receptor activation, polypeptides VEGF and their combinations. Immunogenic compositions can be used for treatment of malignant neoplasms and metastasises, at benign neoplasm and chronic inflammatory and autoimmune diseases. Advantage of invention lies in humoral and cellular immunity induction by means of specified compositions.
EFFECT: development of effective method angiogenesis preventive immunotherapy.
19 cl, 11 ex, 7 tbl
FIELD: medicine; pharmacology.
SUBSTANCE: releasing peptides of growth hormone are described with formula (I): R1-А1-А2-А3-А4-А5-R2, where:А1 designates Aib, Apc or Inp; А2 designates D-Bal, D-Bip, D-Bpa, D-Dip, D-1Nal, D-2Nal, D-Ser(Bzl) or D-Тrp; А3 designates D-Bal, D-Bip, D-Bpa, D-Dip, D-1Nal, D-2Nal, D-2Ser(Bzl) or D-Trp; А4 designates 2Fua, Orn, 2Pal, 3Pal, 4Pal, Pff, Phe, Pim, Taz, 2Thi, 3Thi, Thr(Bzl); А5 designates Apc, Dab, Dap, Lys, Orn or deleted; R1 designates hydrogen; and R2 designates NH2; and their pharmaceutically acceptable salts.
EFFECT: pharmaceutical compositions and the methods of their application are presented.
25 cl, 1 tbl, 2 ex
FIELD: biotechnology, immunology.
SUBSTANCE: invention reports about preparing and characterizing two forms of Nogo protein bound with the natural myelin with respect to the presence of immunogenic properties in their. These two forms correspond to natural products of alternative splicing, their fragments and derivatives, in particular, derivatives comprising deletions in amino acid residues, and chimeric protein also and comprising novel immunogenic polypeptides and their fragments. Invention can be used in medicine for diagnostic and curative aims.
EFFECT: valuable medicinal properties of protein.
18 cl, 79 dwg, 3 tbl, 8 ex