RU2180233C1 - Method of recombinant protein liquid medicinal form preparing - Google Patents

Abstract

FIELD: biotechnology, medicine, pharmaceutical technology. SUBSTANCE: invention relates to technology of preparing medicinal forms of recombinant proteins. In the process of preparing liquid form of corresponding recombinant protein an effective amount of the latter is dissolved in biocompatible buffer solution containing nonionic detergent and, in some cases, stabilizing agent additionally. For preparing the medicinal form buffer solution with pH value distinguishing by more 1 unit from isoelectric point of protein is used. This provides stable state of protein at low concentrations (1- 100 mcg/ml) in solutions without aggregation and oligomerization. Tween-20, Tween-80, Tween-60, Nonidet P-40 and others can be used as detergent. Isotonic buffer solutions based on citrate, acetate, phosphate and others can be used as biocompatible buffer systems. In some cases for correction of osmomolarity solutions can contain polyatomic alcohols, dextrins, polyvinylpyrrolidones and others biocompatible polymeric components as stabilizing agent. EFFECT: decreased cost, safety of preparation. 4 cl, 4 tbl, 3 dwg

Description

 The present invention relates to biotechnology, and in particular to a technology for producing dosage forms of recombinant proteins.

 Therapeutically significant proteins derived from recombinant DNA technology have been widely used in recent years to treat a wide range of diseases. The most significant and common drugs of this class include drugs created on the basis of recombinant erythropoietin, interferon-alpha, granulocyte colony stimulating factor and some other human cytokines.

 Common to this class of substances is the practical impossibility and inappropriateness of isolating them from natural sources and, as a result, obtaining them in artificially created prokaryotic (usually E. Coli) or eukaryotic (usually CHO) systems.

 Proteins of this class have a high specific biological activity, as a result of which their single therapeutic dose is in the microgram range. Some of the proteins of this class used in medical practice (usually obtained in prokaryotic systems) are in a partially denatured state and are prone to aggregation and precipitation. This primarily relates to recombinant interferons-alpha. In the process of preparing dosage forms based on proteins of this class, the most common approach currently is to introduce serum albumin as a stabilizer and / or freeze-drying as a stabilizer. However, both of these approaches are not free from disadvantages. The introduction of human serum albumin as a stabilizer increases the risk of viral contamination of the drug and limits its use by patients of certain religious denominations. Freeze-drying not only reduces the activity of the drug, but also causes partial denaturation of albumin, which in turn sometimes leads to allergic reactions in patients. Previously, it was described and implemented the preparation of a dosage form of a recombinant human interferon-alpha drug free of human serum albumin. For this, dextrans (REALDIRON, BIOTECHNA, Lithuania) were introduced into the composition for drying as a filler. Such an approach allows the preparation to be dried, but the stability in solution remains low due to losses on the vessel walls and aggregation.

 Another approach to solving the stability problem of recombinant proteins in finished dosage forms is the introduction of urea, a complex of amphoteric electrolytes (amino acids) and detergents, followed by freeze drying (RU 2043118), where it was possible to obtain a stable dosage form of recombinant human erythropoietin. At the same time, there are significant restrictions on the shelf life of the drug after its dissolution.

 The closest set of essential features to the claimed method is a method for preparing finished forms of recombinant proteins [US 5,656,730, 1996, IPC A 61 K 035/14; A 61 K 38/00; C 07 To 014/505; C 07 K 014/535], according to which dosage forms of recombinant proteins were prepared using complex compositions consisting of a complex of amino acids, chlorobutanol, benzyl alcohol, benzalkonium chloride and inorganic components of buffer solutions. According to the prototype, to obtain a finished dosage form containing 10-2000 μg / ml of recombinant protein, the corresponding amount of substance is dissolved in the specified solution and the resulting solution is used equally for the preparation of a liquid or lyophilized form. A significant drawback of this approach is the need to use an extremely complex composition containing components such as 1,1,1-trichloro-2-methyl-2-propanol, benzyl alcohol and benzalkonium chloride.

 The objective of the invention is to develop a simpler and cheaper way to obtain dosage forms of recombinant proteins with stable biological activity during storage.

 The technical result consists in the fact that as a result of the developed method receive a cheaper and safer drug that does not include toxic components.

 The problem is solved by the fact that when preparing a liquid dosage form of the corresponding recombinant protein, an effective amount of the latter is dissolved in a biocompatible buffer solution containing a non-ionic detergent and, in some cases, an additional stabilizer. To prepare the dosage form, take a buffer solution with a pH value that differs by more than 1 unit from the isoelectric point of the protein. This ensures a stable presence of protein at low concentrations (1-100 μg / ml) in solutions without aggregation and oligomerization.

 As a detergent, Tween-20, Tween-80, Tween-60, Nonidet P-40 and others can be used. Isotonic buffer solutions based on citrates, acetates, phosphates, etc. can be used as biocompatible buffer systems. In some cases, for adjustment osmolarity solutions may contain biocompatible polymer components as a stabilizer - polyhydric alcohols, dextrins, polyvinylpyrrolidones, etc.

 The proposed method is illustrated by the following examples.

Example 1. Obtaining a liquid dosage form of recombinant human interferon-alpha
To obtain a liquid dosage form of recombinant interferon-alpha, a highly purified preparation was used, isolated from inclusion bodies of E. coli strain IF212S, which did not contain noticeable impurities of oligomeric forms, proteolysis products, and impurities of producer cell proteins. The specific biological activity of the preparation was 2 • 10 ⁸ IU / ml, the apparent molecular weight was 18.1 kD, the isoelectric point was pl = 5.96.

Biological activity was monitored by the method of inhibiting the cytopathogenic effect of the vesicular stomatitis virus on human embryonic lung cells of the L-68 line. In the wells of 96-cell microplates, 0.1 ml of a suspension of cells in a nutrient medium Eagle DMEM was added with the addition of 80-160 μg / ml gentamicin and 2% serum of cow fruits at a concentration of 200-300 thousand cells / ml. To determine the activity of interferon, two dilutions (above and below the expected titer) of the studied preparations and the standard of activity of interferon in the Medium Needle DMEM containing 2% serum of cow fetuses and antibiotics (penicillin 100 Units / ml, gentamicin 80 μg / ml) are prepared. At least 4 wells with cell culture are used for each dilution of the preparation. 0.1 ml of prepared interferon samples were added to each cell culture well. Four culture wells in each microplate were left as controls. In addition, 16 holes were left to control the dose of the indicator virus. 0.1 ml of culture medium was added to these wells. Inoculated and control cell cultures were incubated for 1 day at (37.0 ± 1.0) ^o C in an atmosphere with (5.0 ± 0.5)% CO ₂ , after which a predetermined dose was introduced into each well with test materials vesicular stomatitis virus corresponding to 100 TCD ₅₀ in 0.1 ml. At the same time, the taken dose of the virus was monitored in 16 culture wells intended for this operation. After the introduction of the indicator virus and titration of its dose, the cell culture was incubated for 1-2 days at a temperature of (37.0 ± 1.0) ^o С in an atmosphere with (5.0 ± 0.5)% СО ₂ under the control of the virus dose after which the interferon activity was counted. For the interferon titer, the reciprocal of the preparation was taken, in which the cell culture in 50% of the wells was completely protected from the cytopathic effect of the virus.

 The physical content of interferon was determined by enzyme immunoassay using ProCon IF2 test systems (Protein circuit, Russia). To control the oligomeric forms, an enzyme-linked immunosorbent assay with PC / IF1 antibodies (Protein circuit, Russia) was used as a sorbing and opening reagent.

A series of 0.05 M ammonium acetate buffer solutions containing 0.1 M sodium chloride and 0.02% Tween-20 was prepared in the pH range from 4 to 8. Recombinant interferon-alpha was added to each buffer solution at a rate of 3 • 10 ⁶ and 6 • 10 ⁶ IU / ml, the solutions were subjected to sterilizing filtration and 1 ml were sterilized aliquotted into glass ampoules. The ampoules were sealed and in equal quantities were stored at temperatures of -70, +4 and +37 ^o C. With a frequency of 7 days, the ampoules were removed from thermostats and subjected to analysis for the total content of interferon and the content of oligomeric forms in relation to the frozen preparation. At the beginning and at the end of the experiment, additional measurements of biological activity were carried out. The resulting data on the storage of the drug with an initial activity of 3 • 10 ⁶ IU / ml are presented in FIG. 1 and table 1. Completely similar data were obtained for the drug with an activity of 6 • 10 ⁶ IU / ml.

Example 2. Preparation of a liquid dosage form of recombinant human erythropoietin
To obtain a liquid dosage form of recombinant human erythropoietin, a highly purified preparation was used that was isolated from conditioned culture liquid of CHO SP / M cells pZip NeoEPO SV (x) DFR in accordance with the method described in the patent [RU 2145610, Mcl ⁶ C 12 N 11 / 00] and not containing noticeable impurities of oligomeric forms, proteolysis products, and impurities of producer cell proteins. The specific biological activity of the preparation was 1.4 • 10 ⁵ IU / ml, the apparent molecular weight was 35.2 kD, pl = 3.5-5.0.

Control of biological activity was carried out in vitro according to the method of [Krystal G., 1983], based on the evaluation of stimulation of proliferation of blood cells of polycythemic mice in the presence of erythropoietin. Anemic condition in F1 CDF • C57Bl hybrid mice was caused by a double, after 30 hours, intraperitoneal administration of phenylhydrazine at a dose of 60 μg / kg. Splenocytes were isolated 3 days after the last injection of phenylhydrazine and were cultured in cells of 96-well plates in DMEM medium with 5% calf embryonic serum, 2 mM L-glutamine and 80 μg / ml gentamicin and the presence of cultivated samples of standard and test preparations in CO ₂ - incubator for 24 hours. Two hours before the end of the cultivation, 40 μBq ³ H-thymidine were introduced into the cells. Cell cultures were transferred to filters, washed, and ³ H-thymidine incorporation was counted using a RackBeta 1217 scintillation counter, Pharmacy. According to the graph of the level of ³ H-thymidine incorporation for a standard sample, the biological activity of the studied preparations was determined.

The physical content of erythropoietin was determined by enzyme-linked immunosorbent assay using ProCon EPO test systems (Protein circuit, Russia). To control the oligomeric forms, an enzyme-linked immunosorbent assay with antibodies PC / ED7 (Protein circuit, Russia) was used as a sorbing and opening reagent. A series of 0.05 M sodium citrate buffer solutions containing 0.12 M sodium chloride and 0.02% Tween-20 was prepared in the pH range from 4 to 8. Recombinant human erythropoietin was added to each buffer solution at a rate of 2 • 10 ³ and 4 • 10 ³ IU / ml, the solutions were subjected to sterilizing filtration and 1 ml were sterilized aliquotted into glass ampoules. The ampoules were sealed and in equal quantities were stored at temperatures of -70, +4 and +37 ^o C. With a frequency of 7 days, the ampoules were removed from thermostats and subjected to analysis for the total content of erythropoietin and the content of oligomeric forms in relation to the frozen preparation. At the beginning and at the end of the experiment, additional measurements of biological activity were carried out. The resulting data on the storage of the drug with an initial activity of 4 • 10 ³ IU / ml are presented in FIG. 4 and table 2. Completely similar data were obtained for the drug with activity of 2 • 10 ³ IU / ml.

Example 3. Obtaining a liquid dosage form of a recombinant granulocyte colony stimulating factor (G-CSF) person
To obtain a liquid dosage form of recombinant human G-CSF, a highly purified preparation was used, isolated from conditioned culture liquid of CHO cells pZip NeoGCSF CMV (x) DFR, not containing noticeable impurities of oligomeric forms, proteolysis products, and protein cell impurities of producer cells (Fig. 5) . The specific biological activity of the preparation was 1.1 • 10 ⁸ IU / ml, the apparent molecular weight of 20 kD, pl = 5.7-6.3.

The control of biological activity was carried out by the method of evaluating the stimulation of proliferation of bone marrow cells in mice [Okabe M., et al., 1990]. The bone marrow cells of F1 CBA • C57Bl hybrid mice were isolated by the standard method and cultured at a concentration of 5 × 10 ⁵ cells / ml in cells of 96-well plates in DMEM medium with 10% calf fetal serum, 2 mM L-glutamine and 80 μg / ml gentamicin in the presence of propagated samples of standard and test drugs in a CO ₂ incubator for 72 hours. 20 hours before the end of the cultivation, 40 μBq of ³ N-thymidine were introduced into the cells. Cell cultures were transferred to filters, washed, and ³ H-thymidine incorporation was counted using a RackBeta 1217 scintillation counter, Pharmacy. According to the graph of the level of incorporation of ³ N-thymidine for a standard sample, the biological activity of the studied drugs was determined.

 The physical content of G-CSF was determined by enzyme-linked immunosorbent assay using ProCon G-CSF test systems (Protein circuit, Russia). To control the oligomeric forms, an enzyme-linked immunosorbent assay with antibodies PC / G36F11 (Protein circuit, Russia) was used as a sorbing and opening reagent.

A series of 0.02 M sodium phosphate buffer solutions containing 0.15 M sodium chloride and 0.02% Tween-80 was prepared in the pH range from 4 to 8. Recombinant human G-CSF was added to each of the buffer solutions 3 • 10 ⁷ IU / ml, the solutions were sterilized by filtration and 1 ml was sterilized aliquotted into glass ampoules. The ampoules were sealed and in equal quantities were stored at temperatures of -70, +4 and +37 ^o C. With a frequency of 7 days, the ampoules were removed from thermostats and analyzed for the total content of G-CSF and the content of oligomeric forms with respect to the frozen preparation . At the beginning and at the end of the experiment, additional measurements of biological activity were carried out. The resulting drug storage data in FIG. 6 and table 3.

 From the data of tables 1-3 and FIG. 1-3 it is seen that the most stable compositions of protein solutions are in areas remote from the isoelectric points of the objects in question by more than 1 unit. The reduced stability of interferon-alpha in the acidic region is apparently determined simultaneously by a combination of factors — hydrolytic cleavage of the protein at elevated temperature, oligomerization due to thiol disulfide exchange with the participation of free sulfhydryl groups of the incompletely renatured protein, and increased sorption of the positively charged protein on surface silanol groups of glass. At the same time, the transition to the main region beyond the isoelectric point leads to stabilization of this protein. The apparent dependence of the stability of erythropoietin preparations on pH is clearly visible. In the region close to the isoelectric point, stability is reduced due to the formation of aggregates, and as they move away from pl, electrostatic interactions are suppressed. The same trend can be noted in the case of G-CSF.

Example 4. Obtaining a liquid dosage form of a recombinant granulocyte colony stimulating factor (G-CSF) of a person containing lactose
A series of solutions of lactose, mannitol, polyvinylpyrrolidone 8000 (PVP) and dextran 10000 in 0.02 M sodium phosphate buffer solution containing 0.15 M sodium chloride, 0.02% Tween- was prepared to study the effect of the stabilizing properties of some components of non-protein nature. 20, pH = 7.0. Recombinant human G-CSF, as in example 3, was introduced at the rate of 3 • 10 ⁷ IU / ml in each of the solutions. The solutions were aliquoted and stored as in Example 3, subjected to weekly analysis for the activity and content of oligomeric forms. The results are presented in table 4.

 In the processes of preparing finished forms of preparations of genetically engineered origin, the components of monomeric and polymeric carbohydrate nature, polyhydric alcohols, and some biocompatible polymers are often used as stabilizing fillers. From table 4 it is seen that the introduction of such components in the finished form of preparations of proteins of genetically engineered origin, located at pH, significantly different from this protein does not have a significant effect on the stability of the composition.

Claims (4)

 1. A method of producing liquid dosage forms of recombinant proteins (genetically engineered proteins), comprising dissolving these proteins in a biocompatible buffer solution, characterized in that a non-ionic detergent is added to the biocompatible buffer solution, after which a recombinant protein of effective concentration is added, and a buffer is used a solution having a value of the hydrogen potential index of more than one unit different from the isoelectric point of this protein.  2. The method according to p. 1, characterized in that the detergent is introduced into the buffer solution in an amount of 0.01-0.05 wt. %  3. The method according to p. 1, characterized in that the biocompatible buffer solution is additionally injected with a stabilizer of carbohydrate nature in an amount of 2-5 weight. %  4. The method according to p. 1, characterized in that the biocompatible buffer solution contains polyhydric alcohols and / or a water-soluble polymer as stabilizer.

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