EA027609B1 - Method of producing interferon beta 1-alfa and pharmaceutical composition comprising interferon beta 1-alfa - Google Patents

Abstract

Provided is a method of producing a pharmaceutical composition comprising interferon beta-1α as an active ingredient in the form of a solution for injection. Preferably the solution for injection is used as a pre-filled syringe. The proposed method provides producing a pharmaceutical composition with high content of stability with regard to biological activity.

Description

The invention relates to the field of biotechnology and pharmaceutical industry, in particular, to a method for producing a pharmaceutical composition in the form of a solution for injection containing interferon beta 1-alpha as an active agent, and a pharmaceutical composition obtained in accordance with the claimed method.

State of the art

Interferons are divided into three classes, designated from type I to type III according to the corresponding receptor complexes. Interferon-alpha together with interferon-beta are type 1 interferons. Interferon-gamma is the only type II interferon in humans. The relatively new subclass of type III interferons, which is designated as interferon lambda, includes interleukins G-28A and (I-29 (ΖίΙ / tapp e! A1., 2006).

Although interferon alpha and interferon beta use the same ΣΕΝΑΚΣ receptor complex, ίη νίνο and ίη νίΐτο, they exhibit different effects due to different binding affinities of these proteins (1 e! A1., 2006). Several proteins induced by interferon type I have been identified that affect the reproduction of the virus. Activation of RNAase of endonuclease L and the resulting degradation of viral mRNAs are triggered by the formation of the oligonucleotide ppr ppr (A2'p) ηΑ (2-5A), catalyzed by synthetase 2-5A. The polyribosomal translation of viral mRNAs is inhibited by an RKK called protein kinase and inactivation of the eGR2 peptide initiation factor. These two mechanisms are involved in the replication of RNA-containing small viruses, such as EMC, picornaviruses, and are also involved in the replication of other groups of viruses. In addition, it has been described that Mx proteins specifically inhibit the replication of other virus groups (Meadeg, 2002).

Recombinant interferon beta-ία (ΓΒ1α) is a glycosylated cytokine used in the treatment of recurrent forms of multiple sclerosis (M8), a chronic progressive disease that affects the central nervous system.

Recombinant interferon beta-1α is an analogue of interferon produced by human diploid fibroblasts with respect to amino acid sequence (166 amino acids), disulfide bonds and a similar glycosylation mechanism. This means that both types of interferon have similar structural and immunological characteristics. Recombinant interferon beta-1a has antiviral, antiproliferative and immunomodulating properties.

SUMMARY OF THE INVENTION

The invention describes a method for producing a pharmaceutical composition containing, as an active ingredient, interferon beta-1a, in the form of an injection. Preferably, the injection is used as a pre-filled syringe. The proposed method provides a pharmaceutical composition with a high degree of stability relative to biological activity. Interferon beta-1a is a glycoprotein used as a subcutaneous injection and is indicated for the treatment of recurrent forms of multiple sclerosis.

A method for producing a pharmaceutical composition in the form of an injection solution includes the steps of obtaining a concentrated solution of the active ingredient and mixing the concentrated solution and buffer, and characterized in that each stage is performed at a temperature of 4.0 ° C ± 0.5 ° C; the buffer is added to a concentrated solution of interferon beta-1 α at a rate of 1 l per minute, without stirring; wherein the buffer is prepared for a time not exceeding 5 minutes

Another aspect of the invention relates to a pharmaceutical composition in the form of a solution for injection, obtained by the proposed method, containing as an active ingredient interferon beta-1a and excipients, including solvents, stabilizing substances, buffers and diluents.

List of figures

The present invention is illustrated by the following figures:

FIG. 1C and 1Ό - dose-response curves of standard samples, analysis 1 (Fig. 1C) and analysis 2 (Fig. 1Ό);

FIG. 2ES - presents dose-response curves of samples obtained by the claimed method, analysis 1;

FIG. 2Ι-2Ν - presents dose-response curves of samples obtained by the claimed method, analysis 2;

FIG. 3 - molecular weight data of interferon beta-1a; FIG. 4 and 5 - distribution of glycoform interferon beta-1 α;

FIG. 6-8 - peptide mapping of Interferon beta-1a, used in the pharmaceutical composition according to the invention, by splitting with trypsin and endolysin (molar mass distribution);

FIG. 9 - UV absorption spectra, Extinction coefficient 1.574 (mg / ml) ^-1 cm ^-1 ; FIG. 10 - spectra of fluorescence emission; FIG. 11 - far UV CO spectrum; FIG. 12 - near UV SE spectrum;

- 1,027,609 in FIG. 13 is a mass spectrum corresponding to a trypsinized unreduced sample; FIG. 14 is a mass spectrum corresponding to a trypsinized, ΌΤΤ reduced sample.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes a method for producing a pharmaceutical composition containing, as an active ingredient, interferon beta-1a in the form of an injection solution (Genfaxon preparation). Preferably, the injection is used as a pre-filled syringe. The proposed method provides a pharmaceutical composition with a high degree of stability relative to biological activity.

The method of obtaining a pharmaceutical composition in the form of an injection solution includes the steps of obtaining a concentrated solution of the active ingredient and mixing the concentrated solution and buffer, and characterized in that each stage is performed at a temperature of 4.0 ± 0.5 ° C; moreover, the buffer is added to the concentrated solution of interferon beta-1a at a speed of 1 l per minute, without stirring; wherein the buffer is prepared for a time not exceeding 5 minutes

In a preferred embodiment, when preparing the buffer solution, mannitol and sodium acetate are dissolved in water for injection (90% of the final volume of the prepared solution) at the indicated temperature for five minutes. Then the resulting solution is slowly, at a speed of 1 l per minute, added to the concentrated solution of interferon beta-1a, without stirring.

After 5 minutes, the pH is adjusted and immediately diluted with water for injection to the desired final volume. The solution is filtered and then proceed to fill the syringes.

Significant distinguishing features of the claimed method, such as the indicated temperature values, the order of mixing of the buffer and a concentrated solution of the active ingredient, the time of mixing the buffer with a concentrated solution of interferon beta-1a and the absence of mixing when adding buffer to a concentrated solution of interferon beta-1a, ensure the stability of the protein structure, associated with the side chains of residues important for the biological activity of interferon beta-1a, which provides, in turn, by comparison iju to known methods, a high degree of reproducibility of the end product and its increased stability. The proposed method provides a uniform biological activity of the final product of various batches (high degree of compatibility) during the shelf life. The product can be stored at a temperature of 2 to 25 ° C for a maximum of one month; and then at a regular storage temperature of 2 to 8 ° C until the expiration date, and the biological activity remains stable.

Another aspect of the invention relates to a pharmaceutical composition in the form of a solution for injection, obtained by the proposed method, containing as an active ingredient interferon beta-1a and excipients, including solvents, stabilizing substances, buffers and diluents. The product of the claimed method has a high stability of biological activity during the period of storage with a high degree of correspondence of biological activity in various batches.

Preferably, human serum albumin and mannitol are used as stabilizing substances, sodium acetate and acetic acid are used to prepare the buffer, and water for injection is used as a diluent.

Human serum albumin, the main plasma protein, has good binding properties for water, Ca ² '. Να '. K ', fatty acids, hormones, bilirubin and medicines. Its main function is to regulate the colloidal osmotic pressure of the blood. The main plasma zinc transporter typically binds approximately 80% of all plasma zinc. Upon receipt of the final product of interferon beta-1a of the proposed method, this protein plays an important role in stabilizing the solution (stabilization of the molecular structure of interferon beta-1a). Albumin, in addition, reduces the level of protein adsorption on the surfaces of containers, is a cryoprotectant and prevents the formation of aggregates.

Mannitol provides an isotonic environment by stabilizing the solution. Sodium acetate and acetic acid act as a buffer and regulate the pH of the solution.

Physical instability, which disrupts the activity and effectiveness of interferon beta-1a in pharmaceutical formulations, includes the denaturation and formation of soluble and insoluble aggregates, which, as is known, leads to the loss of biological activity of interferon beta-1a or to a reduction in its shelf life.

The formation of degradation products leads to undesirable side effects. High or unstable temperature during the manufacturing process of interferon beta-1a causes the instability of its molecules. Even at a low, but not constant temperature during the manufacturing process, some molecules can also undergo denaturation or form aggregates, which makes the injection solution unstable and reduces biological activity. Subject to strict temperature control from 3.5 to 4.5 ° C during the entire manufacturing process provides high stability of the claimed pharmaceutical composition. Chemical instabilities include hydrolysis, imide formation, oxidation, racemization and deamidation. Reason

- 2 027609 oxidation can be mixing the solution, quick and vigorous mixing of the buffer and concentrated solution of the active ingredient and a long period of time for the manufacture of the final product. The optimization of the time of the process of obtaining the pharmaceutical composition and the absence of mixing when adding buffer to the concentrated solution of the active ingredient ensures the preservation of interferon beta-1a molecules and, therefore, the stability of the solution. This stability is demonstrated by the reproducibility of biological activity during the shelf life of the product under storage conditions.

Example 1. Characteristics of interferon beta-1a as an active ingredient in the claimed pharmaceutical composition (pre-filled syringes).

Molecular mass

Recombinant human interferon beta-1a is a single chain glycoprotein with a molecular weight of approximately 22,500 Da. This is confirmed by analysis of 8-8-ΡΆΘΕ (Fig. 3), where lane 1 corresponds to pre-stained low range standards, and lane 2 corresponds to interferon beta-1a according to the invention.

Profile (distribution) of glycoform (Fig. 4 and 5).

Mature interferon beta-1a is a 166 amino acid protein containing one Ν-glycosylation site in Άδπ-80. Natural glycoforms occur during differentiated glycosylation of the Ν-glycosylation site, leading to differences in the branching profile and different degrees of sialylation in glycan. The glycoforms of interferon beta-1a are confirmed by mass spectrometry (MS). According to the European Pharmacopoeia, there are 6 glycoforms of interferon beta-1a in the analysis of MS (Table 1).

Table 1

Peak MS Glycoforms * Expected M Level sialylation BUT 2A281P 22375 Disialylation IN 2A281P 22084 Monosialization FROM ZA281R and / or 2A281R + 1 NehShsNekh repeat 22739 Disialylation ϋ 3Α381Ρ 23031 Trisialization E 4A381P and / or 3Α381Ρ + 1 NehIasNeh repeat 23400 Trisialization G 2A081R 21793 Without sialing * 2A-oligosaccharide with a double branching profile; ZA-oligosaccharide with a triple branching profile; 4A-oligosaccharide with a four-complex branching profile; 08- without sialylation; 18-monosialylation; 28-disialylation; 38-trisialylation; 1P-fucosylation

To confirm the distribution of the glycoform of interferon beta-1a in accordance with the invention, a comparative analysis of a standard batch of CK8 1 interferon beta-1a and interferon beta-1a, used as an active ingredient in the claimed pharmaceutical composition according to the invention, by ΜΆΕΌΙ-спект spectrometry was performed.

The results presented in FIG. 5 and 6 show that the structure of Interferon beta-1a glycoform used as an active ingredient in the claimed pharmaceutical composition according to the invention is comparable to the structure of glycoform of a standard batch of CK8 1 and meets the requirements of the European Pharmacopoeia. Peaks AE in the batch of interferon beta-1a according to the invention correspond to glycoforms AE of the standard batch of CK8 1 interferon beta-1a (Table 2).

Table 2. Distribution of glycoforms in interferon beta-1a and standard batch of SC8 1 interferon beta-1a

Glyco forms Expected pier weight according to European Pharmacopoeia Peak pier. masses, interferon beta-1a according to the invention Peak pier. mass, standard batch of SK8 1 interferon beta-1a Dalton % deviations Dalton % deviations BUT 22,375 22 359 0,07 22 365 0.04 IN 22,084 22,061 0.10 22 094 0.05 FROM 22,739 22,736 0.01 22,731 0.04 Ό 23 031 23 028 0.01 23 016 0,07 E 23,400 23,392 0.03 23,384 0,07 R 21 793 21,804 0.04 21,813 0.09

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The results obtained by the ΜΑΡΌΙ-ΤΟΕ method indicate that the glycoforms existing in the Interferon beta-ία invention correspond to the glycoforms Α-P in the standard lot of SIZ 1 interferon beta-ία, and are similar to the structure of isoforms according to the requirements of the European Pharmacopoeia (01/2009: 1639) .

The primary structure of Interferon beta-ία, used as an active ingredient in the claimed pharmaceutical composition according to the invention (data obtained using MAMITOR mass spectrometry, Tables 3 and 4 and Figs. 6-8).

Table 3. Peptide mapping of interferon beta-ία upon trypsin cleavage (molar mass distribution)

Observed I'm supposed. weight Theoretical cheskaya pier weight Peptide Position Modifications 973,691 973,489 ΝΡΥΡΙΝΚ 153-159 1235,611 1235,598 ΜΝΡϋΙΡΕΕΙΚ 36-45 1341,681 1341,698 ΜδΥΝΙΤ, ΟΡΕΟΚ 1-11 1421,751 1421,663 ΕΥ3ΝΟΑ \ νΤΐνΚ 137-147 (1xSUZ CAM) 2258,165 2258,153 ΕϋΛΆΙΤΙΥΕΜΕρΝΙΡΑΙΡΚ 53-71

Table 4. Peptide mapping of Interferon beta-ία upon cleavage by endolysin C (molar mass distribution)

Watching Teoreti Peptide Polo- Modifika- given -cha zhenie tions pier pier weight weight 1481,587 1481,838 KUUOMNUK 124-134 1506,543 1506,726 ϋΚΜΝΡϋΙΡΕΕΙΚ 34-45 1805,733 1805,973 JACECOCTEUSEC 20-33 (1xSUZ CAM) 2320,887 2321,117 Μ8ΥΝΕΕΟΡΕ0Κ88ΝΡ0ε0Κ 1-19 (1xSUZ CAM)

The data presented in table. 3 and 4, demonstrate that nine peptides have sequences corresponding to the interferon beta-ία sequence. Peptides obtained by trypsin and endolysin cleavage cover approximately 60% of the Interferon beta-ία sequence, position 17 (Cous) is on.

Secondary and tertiary structure (circular dichroism spectroscopy and fluorescence spectroscopy)

In FIG. Figure 9 shows the UV absorption spectra of the sample. The extinction coefficient is 1.574 (mg / ml) ^-1 cm ^-1 .

In FIG. ί0 presents the fluorescence emission spectra. The maximum emission wavelength is 343 nm.

Spectroscopy of circular dichroism was carried out in the far and near UV spectra in order to analyze the secondary and tertiary structures of Interferon beta-ία, used as an active ingredient in the claimed pharmaceutical composition according to the invention.

The data presented in FIG. 11 (far UV UV spectrum), demonstrate that the protein has an alpha helix structure.

The analysis of disulfide bonds of Suz 31-Suz 141 was carried out by mass spectrometry (Ma1FTOR). Prior to trypsin digestion, the native protein was treated with iodoacetamide (ΙΑΑ) to alkylate all free cysteine residues (Cous). Then the sample was divided into two parts: one part was treated with dithiothreitol (ΌΤΤ) to restore disulfide bonds between cysteine residues, and the second part was not processed in order to detect dipeptides with disulfide bonds, if any (data on the distribution of molar masses of peptides obtained without and with processing ΌΤΤ are presented in tables 5 and 6 and in Fig. 13 and 14, respectively).

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1. Trypsin digestion after treatment ΙΑΑ, but without restoration of disulfide bonds ΌΤΤ. Table 5

Received mol. weight Expected molar mass Peptide Position Famous modifications 714,436 714.3682 ΚΥΥΟΚ 124-128 722,517 722,4195 Bastard 160-165 786,542 786.4872 ΙΙΉΥΙ, Κ 129-134 919,608 919,4996 (widoc 6-52 928,653 928.5284 Bm88bn 116-123 973,679 973,489 ΝΡΥΡΙΝΚ. 153-159 998,637 998,436 88ΝΡ () Ο (2Κ 12-19 (Souz SAM: 17) 999,795 99,584 LIVE 20-27 1341,997 1341,698 ΜδΥΝΙΧΟΡΙΛϊΚ 1-11

The peak corresponding to the KEUSKK-EU8PSASh'PUK dipeptide (theoretical molar mass 2130.9 Da) is detected in the sample with unreduced disulfide bonds, which indicates the correct formation of the disulfide bond Suz 31-Suz 141 in the molecule of interferon beta-1a used in the claimed method (Fig. 13). On the other hand, this peak is not detected in the recovered sample (Fig. 14).

2. Trypsin digestion after treatment with ΙΑΑ and subsequent reduction of disulfide bonds ΌΤΤ.

After the disulfide bonds were restored using ΌΤΤ, a peptide was found containing Suz 141 (molecular weight 1364.6 Da) (Fig. 14). Its alkylated analogue (molar mass 1421.6 Da) was not detected, which indicates that Souz in this position is not free in the native protein. Peptide mol. mass 1364.6 Yes are absent in the unreduced sample (Fig. 14). As a BEUSBC peptide with a theoretical mole. weighing 768.39 Da, and its alkylated analogue (mol. mass 825.4 Da) was not detected. All these data indicate that the disulfide bond Suz31-Suz141 is correctly formed in the native protein.

Table 6

Received mol. weight Expected molar mass Peptide Position Famous modifications 999.46 999,573 ΙΑ \ ν () ΕΝΟΚ 20-27 998,335 998,436 δδΝΡΟΟΟΚ 12-19 (Souz SAM: 17 1235.53 1235,598 ΜΝΡϋΙΡΕΕΙΚ 36-45 1341,623 1341,698 ΜδΥΝΙΧΟΡϋΟΚ 1-11 1364,639 1364,642 ΕΥδΗΑΑΊΤνΚ. 137-147 2257,994 2258,153 ΕϋΑΑΕΤΓΥΕΜϋΟΝΙΡΑΙΡΚ 53-71

The data presented in table. 5 and 6 also demonstrate the presence of peptides having sequences corresponding to the interferon beta-1a sequence and covering approximately 85% of its sequence, position 17 (Cs) is included.

Example 2. Biological activity of interferon beta-1a. The activity of interferon beta-1a used in the claimed method was evaluated by analyzing its ability to protect cells from viral cytopathic effect compared with human recombinant interferon beta1α international standard (IEVZS 00/572). Using a standard culture of the A549 cell line (ATCC No. SSB-185), sensitive to the cytopathic effect of the encephalomyocarditis virus (VEMK, ATCC No. UK-129B) and sensitive to interferon, the biological activity was analyzed in accordance with the requirements of the European Pharmacopoeia (01/2005: 50600600 ) The activity was evaluated using statistical software for the analysis of parallel samples (REA 2.0, 8Re2shapp 8uzResz).

In the table. 7 shows the results of the biological activity of 6 different batches of interferon beta-1a used in the preparation of the pharmaceutical composition according to the invention.

- 5,027,609

Table 7

The results show that interferon beta-ία used to obtain the pharmaceutical composition according to the invention (drug Genfaxon) is stable and identical to human interferon beta 1-α in structure and biological activity.

Example 3. Obtaining interferon beta-ία.

The human interferon beta-ία gene (which is present on chromosome 9, locus 9ς2ί) was isolated from the human embryo liver cDNA library by polymerase chain reaction (PCR), and cloned into the expression vector using restriction enzymes апοί I apb Xa I. The resulting vector was introduced into the strain IN5a Excepsois for amplification and further transfection of CHO cells. Stable transfectants were selected for further cloning and amplification.

The cell bank was obtained from the selected amplified clone after analysis of its productivity and the quality of recombinant interferon beta-ία. The bank was stored in the vapor phase from liquid nitrogen at -150 ° C; Further, the bank was analyzed for purity, viability, identity and stability.

A concentrated solution of interferon beta-ία was obtained using continuous fermentation of a suspended culture. The supernatant was obtained, clarified and purified using pseudo-affinity chromatography, hydrophobic and gel filtration chromatography.

Sterile interferon beta-ία was stored at a temperature below -20 ° C in a sterile, non-pyrogenic PET bottle (polyethylene terephthalate copolymer modified with glycol), with a threaded cap made of high density polyethylene (NIRE) in a dark place.

Example 4. Obtaining a pharmaceutical composition in the form of prefilled syringes according to the invention.

Equipment used: magnetic stirrer;

glass containers - sterile and non-pyrogenic - of various volumes;

a stainless steel reactor with a collecting tube and a rubber tube, alternatively with a metal submersible device used to filter the solution supplied by a peristaltic pump;

distiller providing water for injection; peristaltic pump system for filtration; filter: 0.22 microns;

industrial steam sterilizer; syringe filler and locking machine; cabinet with laminar air flow.

Mannitol and sodium acetate are dissolved by gentle stirring in a glass container, sterilized and depyrogenated, containing 80% of the final volume of water for injection.

Interferon beta-ία is added to this solution with gentle stirring, avoiding the formation of foam, then diluted with water for injection to the required volume. Prior to that, adjust the pH. Next, the solution is filtered and syringes are filled. An analysis of the quality of the batch.

In a preferred embodiment of the pharmaceutical composition according to the invention, mannitol and sodium acetate are dissolved in water for injection (90% of the final volume of the prepared solution) at a temperature of 4 ° C ± 0.5 ° C for 5 min, then added to a concentrated solution of interferon beta ία without stirring at a speed of ί l per minute. After 5 minutes, adjust the pH and immediately dilute with water for injection at 4 ° C ± 0.5 ° C to the desired final volume. After filtration, syringes are filled.

In a preferred embodiment, the product obtained in accordance with the claimed method, is a solution for injection in pre-filled syringes with the following composition (tables. 8 and 9):

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Table 8

Component Interferon Beta-1A 44 g (12 MMU) Human albumin 4.0 mg Mannitol 27.3 mg Sodium Acetate 0.4 mg Acetic acid 1.85 mg Water for injection 0.5 ml

Table 9

Component Interferon beta-1 a 22 g (6 MMU) Human albumin 2.0 mg Mannitol 27.3 mg Sodium Acetate 0.4 mg Acetic acid 1.85 mg Water for injection 0.5 ml

Example 4. The stability of the pharmaceutical composition obtained by the claimed method. The pharmaceutical composition according to the invention is more stable with respect to biological activity than the composition obtained in a known manner.

In the table. A list of batches of product (samples) with the results of the analysis of biological activity during the shelf life of 6, 12, 18 and 24 months, obtained according to the claimed method and the known method.

Table a

Biological Activity (%)

The consignment Concentration (mcg / syringe) 0 6 months 12 months eighteen months 24 of the month The method according to the invention ΙΝ221Α 44 (12 million IU) 99 98 one hundred one hundred 99 ΙΝ222Α 44 one hundred one hundred one hundred 99 99 ΙΝ223Α 44 98 98 99 99 98 ΙΝ224Α 44 96 97 98 99 97 AC002588 44 95 95 95 95 95 ΙΝ441Β 88 101 102 101 101 102 ΙΝ442Β 88 103 102 102 102 102 ΙΝ443Β 88 104 105 104 106 104 ΙΝ444Β 88 one hundred 101 one hundred 101 101 AC002498 88 102 102 102 102 102 Standard way ΙΟ221Α 44 99 80 93 82 79 ΙΟ222Α 44 89 106 82 101 84 ΙΟ441Β 88 80 88 75 91 80 ΙΟ442Β 88 one hundred 111 90 81 84

The data presented demonstrate that in batches produced in accordance with the usual production process, there is a large variability of biological activity at different time periods, and the variability of the biological activity of two batches exceeds the limit allowed by the requirements of the European Pharmacopoeia (80-125%). Parties made

- 7 027609 in accordance with the proposed method, demonstrate stable high biological activity at different periods of storage time. Data on the biological activity of the samples according to the invention obtained at 30 months of storage, essentially, did not differ from data obtained at 24 months of storage.

In order to study the dose-response relationship, two different analyzes of biological activity were performed. The first analysis included the following samples: ΙΝ221Α, ΙΝ222Α, ΙΝ223Α, ΙΝ224Α and AI002588. The second analysis included samples ΙΝ441Β, ΙΝ442Β, ΙΝ443Β, ΙΝ444Β and Αυ002498.

The dose-response curves (dose-response ratio) of standard samples (control) are shown in FIG. 1C (analysis 1) and FIG. 1Ό (analysis 2).

Dose-effect (dose-response) curves of samples obtained by the claimed method are shown in FIG. 2Ε-2Ι (analysis 1) and FIG. 2Ι-2Ν (analysis 2).

The results obtained indicate that the addition of buffer to a concentrated solution of interferon beta-1a at a rate of 1 liter per minute, without stirring, at a strictly defined temperature regime contributes to the stability of the final product, minimizing the incorporation of air with subsequent oxidation and degradation of molecules.

Thus, the present invention provides a pharmaceutical composition with a high degree of stability with respect to biological activity during the entire shelf life, and the shelf life of the composition obtained by the claimed method is 30 months instead of 24 months in accordance with the standard method.

Claims (4)

CLAIM 1. A method of obtaining a pharmaceutical composition in the form of an injectable solution containing interferon beta-1a as an active ingredient, which provides a concentrated solution of the active ingredient from a culture of CHO cells transformed with an expression vector containing the interferon beta-1a gene by purification using chromatography and mixing of the concentrated solution and buffer, characterized in that at a temperature of 4.0 ± 0.5 ° C, mannitol and sodium acetate are dissolved in water for injection for 5 minutes, then poured to a concentrated solution of interferon beta-1a at a speed of 1 l / min without stirring; after 5 min, adjust the pH and immediately dilute with water for injection at a temperature of 4.0 ± 0.5 ° C to the desired final volume. 2. The pharmaceutical composition obtained by the method according to claim 1. 3. The pharmaceutical composition according to claim 2, containing auxiliary substances, including sodium acetate and acetic acid as a buffer, mannitol and human serum albumin as a stabilizer. 4. The pharmaceutical composition according to claim 2, containing interferon beta-1a in an amount of 44 μg (12 MMU) in a pre-filled syringe.