RU2804622C2 - Method of industrial purification of romiplostim - Google Patents

Abstract

FIELD: pharmaceutical biotechnology.

SUBSTANCE: invention relates to a method of purifying recombinant proteins, namely to obtaining romiplostim, a highly purified medicinal product. The invention is a method of purification of recombinant romiplostim obtained in E. coli including isolation and washing of inclusion bodies, protein refolding and chromatographic purification, while the main removal of contaminating impurities is carried out at the stage of isolation of inclusion bodies by sequential washing, and additional removal of impurities includes carrying out 2 consecutive stages of chromatography on the same cation-exchange sorbent, where the first stage is separating, and the second is polishing and concentrating.

EFFECT: development of a method of industrial purification of romiplostim.

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Description

The invention relates to the field of pharmaceutical biotechnology, namely to methods for the production of therapeutic drugs, isolation and purification of recombinant proteins, in particular to the production of highly purified drug romiplostim. The invention relates to a method for producing correctly folded proteins and to a chromatography method for separating correctly folded and incorrectly folded conformations of a given protein. The method is highly effective for separating correctly folded romiplostim from misfolded romiplostim and contaminants with high yields of high purity product and with a good ratio of correctly folded romiplostim to misfolded romiplostim. The main area of application of the therapeutic recombinant protein romiplostim obtained by the proposed method is the treatment of thrombocytopenia in patients one year of age and older, resistant to other types of treatment (for example, glucocorticosteroids, immunoglobulins) with chronic immune (idiopathic) thrombocytopenic purpura (ITP).

The invention solves the problem of creating a method for producing the therapeutic drug romiplostim in industrial quantities and contributes to the fulfillment of the goal of the state policy of the Russian Federation for the development of the national pharmaceutical industry in the field of providing the population with vital and essential drugs of domestic production.

DESCRIPTION OF DRAWINGS

SEQ ID NO 1: Primary sequence of the DNA fragment containing the romiplostim protein gene.

SEQ ID NO 2: Amino acid sequence of TMP peptide (from the English TPO Mimetic Peptid, a peptide that imitates TPO).

SEQ ID NO 3: Amino acid sequence of a chimeric protein monomer with a C-terminal chimerization of the IgG heavy chain arm, composed of two peptide sequences of 14 amino acids (SEQ ID NO: 1) with polyglycine linker inserts, 269 amino acid residues long.

Fig. 1 Mechanism of action of romiplostim and eltrombopag.

Activation of the thrombopoietin receptor by thrombopoietin (TPO) or receptor agonists. TPO or romiplostim binds to the thrombopoietin receptor on the surface of platelets through the CRH-2 domain, converting the inactive receptor into an active dimer, triggering an intracellular cascade through phosphorylation of the tyrosine kinase JAK2 and subsequent activation of the transcription factor STAT, which leads to the proliferation of these cells. Eltrombopag binds to the transmembrane region of the TPO receptor and also triggers an intracellular cascade leading to platelet proliferation.

Fig. 2 Prevalence of rare diseases from the group “24 nosologies” by nosology (80 constituent entities of the Russian Federation, 93.0% of the country’s population). The prevalence of idiopathic thrombocytopenic purpura is highlighted in gray.

Fig. 3 Structure of romiplostim

Romiplostim consists of part IgG1 (Fc domain) with two peptides - thrombopoietin mimetics (Thrombopoietin receptor binding domain, 14 a.a.), which are connected to each other and the C-terminal part of the T heavy chain of IgG by glycine linkers (Polyglycidine linker 5 a.a. . and Polyglycidine linker 8 aa).

Interchain disulfide bonds (amino acid cysteine residues at positions C7 and C10) and intrachain disulfide bonds CH2 and CH3 (amino acid cysteine residues at positions C42-C102, C148-C206).

Fig. 4 Structure of romiplostim with the dimeric sequence of the TMP mimetic. The sequence of the TMP mimetic is indicated using a one-letter code.

Fig. 5 Electropherogram of samples of washing solutions and solution of inclusion bodies. Samples 1, 2, 3 are washing solutions, sample 4 is a solution of inclusion bodies.

Fig. 6 Determination of the content of the monomeric unit of romiplostim using the GP HPLC method. Curve 1 - placebo buffer + dithiothrietol (DTT), 2 - buffer for dissolving inclusion bodies, 3 - solution of the reconstituted drug (LP) Enplate, 4 - solution of romiplostim inclusion bodies.

Fig. 7 Electropherogram of samples of the refolded mixture and LP Enplate under reducing and non-reducing conditions. Sample 1 - refoldable mixture under non-reducing conditions, Sample 2 - under reducing conditions,

Sample 3 - LP Enplate in non-reducing conditions,

Sample 4 - LP Enplate under reducing conditions.

Fig. 8 Determination of the chromatographic purity of the refolded mixture by RP HPLC.

Fig. 9 Electropherogram of fractions from chromatography on Q Sepharose FF and MabSelect SuRe under reducing and non-reducing conditions.

Sample 1 - breakthrough fraction with MabSelect SuRe; Sample 2 - wash fraction with MabSelect SuRe; Sample 3 - eluate with MabSelect SuRe; Sample 4 - eluate with Q Sepharose FF;

Fig. 10A Chromatography profile with MabSelect SuRe.

Peak 1 - washout; Peak 2 - eluate 1, fractions containing the main form of romiplostim no less than 60%, form 1 no more than 2%, form 4 no more than 10% according to the results of RP HPLC analysis are highlighted in gray; Peak 3 - eluate 2; Peak 4 - regeneration;

Fig. 10B Determination of the chromatographic purity of fractions from chromatography on MabSelect SuRe using RP HPLC.

Fig. 11 Chromatography profile with YMC S30-1.

Peak 1 - eluate 1, fractions containing the main form of romiplostim no less than 87%, form 3 no more than 8%, form 4 no more than 4% according to the results of RP HPLC analysis are highlighted in gray; Peak 2 - eluate 2; Peak 3 - regeneration;

Fig. 12A Chromatography profile with YMC S30-2.

Peak 1 - eluate 1, fractions with a content of the main form of romiplostim of at least 90% according to the results of RP HPLC analysis and at least 87% according to the results of capillary zone electrophoresis are highlighted in gray; Peak 2 - regeneration;

Fig. 12B Determination of the chromatographic purity of fractions from chromatography on YMC S 30 -2 by RP HPLC.

Fig. 12C Determination of chromatographic purity of fractions from chromatography on YMC S 30 -2 by capillary zone electrophoresis.

Fig. 13A Determination of the chromatographic purity of the finished form of romiplostim by RP HPLC.

Fig. 13B Determination of the chromatographic purity of the finished form of romiplostim by GP HPLC.

Fig. 13C Determination of the chromatographic purity of the finished form of romiplostim by capillary zone electrophoresis.

Fig. 14A Inhibition of thrombopoietin binding to the TPO receptor by various series of romiplostim GF.

Fig. 14B Graphs of the dependence of the concentration of phosphorylated tyrosine kinase JAK2 on the logarithm of the concentration of romiplostim in dilutions of the GF series of romiplostim: A-donor No. 1, B-donor No. 2, C-donor No. 3.

Immune thrombocytopenia (ITP) (formerly known as idiopathic thrombocytopenic purpura) is a disease characterized by immune-mediated thrombocytopenia. Antibodies directed against a narrow spectrum of glycoproteins on the surface of platelets and megakaryocytes play a leading role in the development of ITP. Most often, ITP occurs in women of childbearing age and during pregnancy (Abir Zainal et al., Immune thrombocytopenic purpura // Journal of community hospital internal medicine perspectives - 2019 - V. 9. - N. 1. - p. 59-61). However, it occurs in both sexes at any age. Given the relatively reduced levels of thrombopoietin in the blood of patients with ITP, as well as the ability of antiplatelet antibodies to induce apoptosis of megakaryocytes and impair their platelet production, it has been suggested that activation of c-Mpl receptors (thrombopoietin receptor) may lead to increased platelet counts in patients with chronic ITP. AND SO ON.

The demonstration of the effects of thrombopoietin (TPO) on megakaryocyte growth and platelet production, and confirmation of its role as a major growth factor regulating platelet production, was quickly followed by the development of recombinant human rHuTPO. A glycosylated full-length molecule with an amino acid sequence identical to that of native TPO was obtained in mammalian cell culture. PEG-rHuMGDF (megakaryocyte growth and development factor), a PEGylated product containing part of the native TPO sequence, was produced in E. coli and subsequently modified by the covalent attachment of a 20 kDa polyethylene glycol moiety (Archimbaud E. et al., A randomized, doubleblind, placebo- controlled study with pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) as an adjunct to chemotherapy for adults with de novo acute myeloid leukemia // Blood. - 1999. - 94. - p. - 3694-3701). However, the development of both MGDF and rHuTPO was stopped after it was discovered that some patients and healthy volunteers developed antibodies to PEG-rHuMGDF upon repeated administration. These antibodies cross-reacted with native TPO, which led to long-term resistance to treatment of thrombocytopenia (Basser R.L. et al., Development of pancytopenia with neutralizing antibodies to thrombopoietin after multicycle chemotherapy supported by megakaryocyte growth and development factor//Blood. - 2002. - 99 . - p. 2599-2602). Research continued, leading to the development of the next generation of thrombopoiesis-stimulating agents—TPO peptides and non-peptide mimetics with little structural homology to natural TPO. Two families of small peptides, structurally unrelated to each other, that bind to the human TPO receptor and compete for binding to natural TPO were identified from recombinant peptide libraries. Mutagenized libraries were then generated to further optimize proteins with the best binding ability, resulting in the isolation of a highly active peptide. The specified peptide, consisting of 14 amino acid residues (SEQ ID NO: 2), designated as TMP (from the English TPO Mimetic Peptid, a peptide that imitates TPO), did not show homology in amino acid sequence with TPO (S.E. Cwirla et al, Peptide agonist of the thrombopoietin receptor as potent as the natural cytokine // Science. - 1997. - 276. - 5319. - p.1696-1699).

Amgen scientists constructed a recombinant protein consisting of an Fc carrier domain associated with several c-Mpl binding domains and named AMG531 or AMP-2 (Amgen Megakaryopoiesis Protein-2) (David J. Kuter, The biology of thrombopoietin and thrombopoietin receptor agonists // Int J Hematol. - 2013. - 98. - p. 10-23), original name romiplostim (Virginia C. Broudy, Nancy L. Lin, AMG531 stimulates megakaryopoiesis in vitro by binding to Mpl // Cytokine - 2004. - 25. - p. 52-60). AMG 531, AMP-2 (romiplostim) was a biological TPO stimulator designed to overcome the problem of antibody cross-reaction to TPO by using a random peptide sequence to activate the c-Mpl receptor. (Wojciech Krzyzansk et al, Pharmacokinetic and Pharmacodynamic Modeling of Romiplostim in Animals // Pharm Res. - 2013. - 30. - p. 655-669).

Subsequently, pharmacodynamic, pharmacokinetic and metabolic studies were carried out in mice, rats, rabbits, dogs and monkeys. Romiplostim was well tolerated and caused a dose-dependent increase in platelet levels in all animal species with a wide range of interspecies differences. (Hartley, C, The novel thrombopoietic agent AMG 531 is effective in pre-clinical models of chemo/radiotherapy induced thrombocytopenia//Proceedings of the American Association for Cancer Research. - 2005. - 46. - (abstract 1233)).

The effectiveness of romiplostim was demonstrated in phase I - II clinical studies of romiplostim (Bussel et al, AMG 531, a Thrombopoiesis-Stimulating Protein, for Chronic ITP1 // The New England Journal of Medicine, - 2006. - 355. - pp. 1672-1681 ) and phase III in patients with ITP (Kuter D.J. et al,. Efficacy of romiplostim in patients with chronic immune thrombocytopenic purpura: a double-blind randomized controlled trial // Lancet. - 2008.-371. - p. 395-403. ). As a result, romiplostim was approved for the treatment of ITP in adults in the United States and Australia in 2008.

Romiplostim was first licensed in the EU in 2009 for use in splenectomized and non-splenectomized patients with chronic ITP that is refractory to other treatments (eg corticosteroids, intravenous immunoglobulins, (http://www.ema.europa.eu/ docs/en_GB/document_librarv/EPAR_Product_Information/human/000942/WC500039537.pdf).Nplate Summary of Product Characteristics 2018. Accessed May 4, 2018).

In 2014, the indications were expanded to include non-splenectomized patients in whom surgery is contraindicated. Indications were limited to adult patients at the time of this study, but romiplostim is now approved for patients over 1 year of age. Romiplostim was first recommended by the National Institute for Health and Care Excellence (NICE) in 2011. NICE currently recommends romiplostim as a treatment option for adults with chronic ITP if their condition is refractory to standard active treatment and rescue therapy, or they have severe disease and a high risk of bleeding that requires frequent courses of rescue therapy (https://www. nice.org.uk/guidance/ta221/ chapter/1 - Guidance. Romiplostim for the treatment of chronic immune (idiopathic) thrombocytopenic purpura. National Institute for Health and Care Excellence 2014; Technology appraisal guidance (TA221)).

Romiplostim is currently manufactured by Amgen Inc. under the trade name NPLATE®.

Patients with chronic ITP experience decreased Health-Related Quality of Life (HRQoL) associated with fatigue, concerns about appearance due to bruising, and impaired ability to perform routine daily activities. Indeed, it has recently been reported that such patients have worse HRQoL than patients with hypertension, arthritis, or cancer (McMillan et al, Self-reported health-related quality of life in adults with chronic immune thrombocytopenic purpur // American Journal of Hematology. - 2008 . - 83. - p. 150-154).

Clinical studies of thrombopoiesis stimulators such as romiplostim (Enplate®, Amgen) and eltrombopag (Revolade®, Glaxo Smith Klein) have shown a response rate of more than 80% of patients with low toxicity. Therefore, these drugs are now the mainstay in the treatment of patients with severe chronic ITP (Indraraj Umesh Doobaree et al., Primary immune thrombocytopenia (ITP) treated with romiplostim in routine clinical practice: retrospective study from the United Kingdom ITP Registry // Eur J Haematol - 2019. - 102. - p.416-423, Drew Provan, Adrian C. Newland, Current Management of Primary Immune Thrombocytopenia//Adv. Ther. - 2015. - 32. - p.875-887). In FIG. 1. The mechanism of action of romiplostim and eltrombopag has been demonstrated.

Romiplostim belongs to a group of drugs intended for the pathogenetic therapy of rare (orphan) diseases. As a rule, these are expensive drugs that have no analogue substitutes and are vital. An important feature of these drugs is their “targeting”, high efficiency (with a response to therapy approaching 100%), good safety and tolerability profile in most cases, which is important given long-term lifelong therapy. The use of just such drugs reliably and significantly reduces the number of irreversible, disabling lesions of vital organs and mortality, improves the quality of life of patients compared to previously used methods of best supportive (symptomatic) therapy, leads to a significant improvement in the medical and social rehabilitation of patients and improved long-term survival rates (up to a level comparable in gender and age to a healthy population).

To solve the problem of expanding access to life-saving therapy for patients with rare diseases, the authors of the invention propose a new method for producing the recombinant highly purified drug romiplostim in industrial quantities. Romiplostim is a peptide antibody consisting of a disulfide-linked human IgG1 constant heavy chain and a kappa light chain (Fc fragment) with two identical peptide sequences covalently linked at residue 228 of the heavy chain using polyglycine (James B. Bussel et al., AMG 531, a Thrombopoiesis-Stimulating Protein, for Chronic ITP // The new England journal of medicine - 2006. - 355. - p. 1672-81) (Fig. 3).

To enhance the binding ability, a strategy of dimerization of the C-terminal regions with respect to TPO mimetic peptide (TMP) was applied. The C-terminal bound dimer was found to have improved binding affinity and significantly increased activity in vitro in cell proliferation studies (William Dower, Steven Cwirla et al., Peptide Agonists of the Thrombopoietin Receptor, Thrornbopoietin: From Molecule to Medicine // Stem Cells. - 1998. - 16. - p.21-29-). The structure of romiplostim with the dimeric sequence of the TMP mimetic is presented in Fig. 4.

Peptides are a unique class of pharmaceutical compounds, molecularly balanced between small molecules and proteins, yet biochemically and therapeutically distinct from both. As intrinsic signaling molecules for many physiological functions, peptides provide an opportunity for therapeutic intervention that closely mimics natural pathways. However, enthusiasm for peptide therapy is tempered by several limitations of native peptides, such as short plasma half-life and negligible oral bioavailability. The short half-life is explained by the presence of numerous peptidases and excretory mechanisms that inactivate peptides (Jolene L. Lau, Michael K. Dunn, Therapeutic peptides: Historical perspectives, current development trends, and future directions // Bioorganic & Medicinal Chemistry. - 2018. - 26 . - p. 2700-2707). Various strategies have been developed to expand the stability and improve the pharmacokinetic properties of peptides, one of these strategies has been to cross-link peptides to the Fc region of immunoglobulin G (IgG), thereby extending the plasma half-life of bioactive peptides. The presence of the Fc moiety results in an increase in the hydrodynamic radius of the molecule above the threshold for glomerular filtration by the kidney, due to increased molecular weight (about 60 kDa), the possibility of using the neonatal Fc receptor and protection from lysosomal degradation (Grant Shimamoto, Colin Gegg, Tom Boone and Christophe Queva, Peptibodies A flexible alternative format to antibodies // mAbs. - 2012. - V. 4. - I.5. - p. 586-591).

Fc containing polypeptides can be produced using recombinant technologies in cell systems of yeast, bacteria, insects, plants and animals (EP 2 752 427 A1, Antibodies containing therapeutic peptides).

Romiplostim is currently manufactured by Amgen Inc, Boulder, CO, USA. using recombinant DNA technology in Escherichia coli (E. coli). The commercial manufacturing process uses E. coli fermentation and cell processing to produce an intermediate product (suspension). The purification process begins with thawing of the suspension followed by solubilization. Romiplostim is purified using a series of chromatography, concentration and diafiltration steps to obtain the purified base material (Australian Public Assessment Report (AUSPAR) For Romiplostim//Sample Auspar. - 2008. - p. 1-31).

There are three main advantages of the production of biologically active substances, the biosynthesis of which is carried out in E. coli, compared with biosynthesis in mammalian or plant cells: short cultivation time, high level of expression, absence of post-translational modifications.

The literature describes methods for the production of E. coli-expressed peptide antibodies, starting with the creation of a genetic construct of a recombinant protein-producing strain, fermentation unit operations, which include cell cultivation, cell collection, disintegration of cell biomass under high pressure or ultrasound, followed by production and by washing the insoluble inclusion bodies that contain the denatured recombinant protein. Inclusion bodies are formed as a result of the inability of the bacterial host cell to correctly fold recombinant proteins at high expression levels and, as a consequence, the proteins become insoluble. This is especially true for large, complex proteins of eukaryotic origin.

The formation of misfolded recombinant proteins has to some extent limited the commercial utility of bacterial fermentation for producing recombinant large, complex proteins with high efficiency.

Since the advent of recombinant expression of proteins at commercially viable levels in non-mammalian expression systems such as bacteria, various methods have been developed to obtain correctly folded proteins from inclusion bodies. These methods typically follow the procedure of expressing the protein, which is typically precipitated into inclusion bodies, lysing the cells, collecting the inclusion bodies, and then solubilizing them in a solubilization buffer including a denaturing agent or surfactant and, optionally, a reducing agent that opens the proteins and disassembles inclusion bodies into separate protein chains with virtually no structure. The protein chains are then diluted or washed with a refolding buffer, which supports renaturation to the biologically active form. When cysteine residues are present in the primary amino acid sequence of a protein, it is often necessary to perform refolding in an environment that allows proper disulfide bond formation (for example, using a redox couple (Grant Shimamoto, Colin Gegg, Tom Boone and Christophe Queva, Peptibodies A flexible alternative format to antibodies // mAbs. - 2012. - V. 4, - I.5, - p. 586-591, US 6,835,809 B1 Liu et al. Thrombopoietic compounds, WO 00/24770, Liu Chuan -Fa, Thrombopoietic compounds, AU 2004200687 B2, Feige U. et al, Modified peptides as therapeutic agents, Shultz et al. Refolding proteins using a chemically controlled redox state // US 8,952,138 B2) The refolded protein is subjected to a chromatographic purification process that is optimized for each peptide antibody to ensure the best yield product with optimal purification from impurities such as degradation products, misfolds, aberrant isomers, aggregate, oxidized, deamidated and other forms of the target protein, impurities of cellular proteins, nucleic acids, endotoxins and lipids. Then the purified product is concentrated and transferred into a buffer of the finished form by diafiltration in a tangential flow on membranes and a pharmaceutical substance (PS) is obtained. To obtain the drug, the PS is lyophilized.

The commercial manufacturing process for romiplostim is addressed in the reports (Report on the Deliberation Results. - 2010. - p. - 102, https://www.pmda.go.jp/files/000236304.pdf, Australian Public Assessment Report (AUSPAR) For Romiplostim//Sample Auspar. - 2008. - p. 1-31), but neither the process conditions nor the materials used are indicated.

In Amgen publications: US6,835,809 B1, Liu et al. Trombopoietic compounds, US2006/0234307 A1, Feige et al., Modified peptides as therapeutic agents, EP1144454 B2, Feige et al., Modified peptides as therapeutic agents, US7,169,905 B2, Feige U., Modified peptides as therapeutic agents, US2004/ 0053845, Feige et al., Modified peptides as therapeutic agents, US2006/0234307, Feige et al., Modified peptides as therapeutic agents, US8044174 B2, Liu et al., Trombopoietic compounds, US9,534,032 B2, Liu et al., Trombopoietic compounds, US2006/0234307 Al, Modified peptides as therapeutic agents, US7,169,905 B2, Feige, Modified peptides as therapeutic agents, describes a method for obtaining variants of Fc containing antibodies with TMP peptides at the N or C end or TMPi-(L)n-TMP2 (Ln are amino acid residues of glycine (Gly), where n is from 1 to 20, and when n is greater than 1, up to half of the Gly residues can be replaced by another amino acid selected from the remaining 19 natural amino acids or their stereoisomers), using standard methods for obtaining a genetic construct in the pAMG21 Fc plasmid with expression in E. coli GM221. One of the variants of the above compounds, described in patent US6,835,809 B1 Liu et al. Thrombopoietic compounds, and is romiplostim. The genetic construct contained in the paMG21 plasmid (pAMG21-Fc-TMP-TMP), which in turn is contained in the host strain GM221, is registered with the ATCC under accession number 98957, with a deposit date of October 22, 1998.

Cultures of E. coli GM221 with pAMG21-Fc-TMP-TMP or Fc-TMP-(L)n-TMP are grown in LB medium containing 50 μg/ml kanamycin and incubated for 3 hours at 37°C before induction. Induction of Fc-TMP-TMP gene products with the LuxPR promoter is carried out by adding the synthetic autoinducer N-(3-oxohexanoyl)-Sb homoserine lactone. Fermentation was carried out under standard conditions at 10L scale, resulting in Fc-TMP-TMP expression levels similar to those obtained at laboratory scale. The expressed product accumulated in the cells in the form of inclusion bodies. To obtain inclusion bodies, the cellular biomass is dispersed in water at a ratio of (1/10) and disintegrated by high pressure homogenization (2 passes at 14,000 psi (965.3 bar)). Inclusion bodies are collected by centrifugation at 4200 rpm for 60 minutes and solubilized using 6 M guanidine hydrochloride as a chaotropic agent under reducing conditions with 8 mM DTT. The protein is renatured by diluting it 20-25 times in a 50 mM Tris-HCl buffer solution with 2 M urea, 160 mM arginine and 3 mM cysteine at pH 8.5. The mixture is stirred for 16-48 hours at 4°C and then concentrated approximately 10 times using ultrafiltration. The concentrate is diluted with a buffer solution of 10 mM Tris with 1.5 M urea, pH 9.0, the pH of this mixture is adjusted to 5.0 using acetic acid. The resulting precipitate is removed by centrifugation. The target protein from the supernatant is purified by two sequential chromatographies on SP Sepharose Fast Flow using a 20 column volume (CV) gradient in a sodium acetate buffer system, pH 5.0.

In patents US2006/0140934 A1, Gegg et al., Modified Fc molecules, US2007/0269369 A1, Gegg et al, Modified Fc molecules, US7,442,778, Gegg et al, Modified Fc molecules, US7,750,128 B2, Gegg et al. , Modified Fc molecules, describes the method closest to the claimed invention for producing peptide antibodies, in which at least one biologically active peptide is included as an internal sequence in the Fc domain. In another embodiment of this invention, a linear, free peptide, AMP 2 (AMP-2, IEGPTLROWLAARA (SEQ ID NO: 2) was inserted into the Fc-loop domain of human IgG1. The AMP2 Fc-loop construct is designated Amgen clone #6875. Clone Fc- loop AMP 2 (#6875) was transformed into E. coli by conventional methods with expression at high levels in the insoluble inclusion body fraction.The isolated inclusion body fraction was solubilized at a ratio of 1/10 in 6 M guanidine-HCl, 50 mM Tris, 8 mM DTT , pH 9 at room temperature with stirring for 1 hour. Refolding was carried out by dilution 1:25 in 2 M urea, 50 mM Tris, 4 mM cysteine, 1 mM cystamine, pH 8.5 at 4°C. with stirring for 48 hours, then aliquots were evaluated by SDS-PAGE and reverse phase HPLC.

The peptide antibody was purified using 2 sequential chromatographies. The first stage of purification was carried out on a protein A column equilibrated in 2 M urea, 50 mM Tris buffer with pH 8.5. The filtered reaction mixture was applied to the column and then the column was washed with 2 volumes of equilibration buffer followed by 2 volumes of PBS. The peptide antibody was eluted with 50 mM sodium acetate buffer (NaOAc), pH 3, and quickly neutralized with a 1:4 dilution of 10 mM NaOAc, 50 mM NaCl, pH 5.0. The diluted eluate was again filtered and applied to an SP Sepharose HP cation exchange column (Pharmacia) equilibrated with 10 mM NaOAc, 50 mM. NaCl, pH 5. The peptide antibody fractions were then eluted with a linear gradient of 50-500 mM NaCl. Fractions were combined and concentrated to approximately 2 mg/ml.

To solve the problem of obtaining a highly purified and biologically active drug romiplostim in industrial quantities, the authors of the invention propose an improved method for its isolation and purification. The recombinant protein was produced in E. coli as insoluble inclusion bodies. Next, inclusion bodies were isolated and washed from E. coli, followed by their solubilization and protein refolding to obtain a dimeric form due to the formation of 4 intrachain and 2 interchain disulfide bonds both within the molecule of each monomer and between different monomer molecules in solution . Next, it is proposed to carry out chromatographic purification of the protein from the refolded mixture, where the first stage is carried out on a strong anion-exchange sorbent, which is a highly cross-linked granular agarose, in the breakthrough mode, followed by sorption of the target protein on an affinity sorbent, the matrix of which is cross-linked agarose, modified with recombinant protein A ; followed by effective washing and mild elution conditions, and two cation exchange chromatographies, the first of which is separating, the second polishing and concentrating, and subsequent transfer of the finished form into a buffer by gel filtration to obtain the finished form of the highly purified drug romiplostim, not less than 0.9 g with liter of bacterial culture.

The main technical result of the invention is the production on an industrial scale of a highly purified and biologically active drug romiplostim.

Since the product is synthesized in the bacterial cytoplasm predominantly in an undissolved state in the form of inclusion bodies during the cultivation of E. coli VKPM B-13251 with a biosynthesis level of at least 900 mg/l of culture liquid, refolding is required.

Highly efficient extraction of inclusion bodies from cells is achieved by bacterial cell lysis using an ultrasonic homogenizer or by scaling a high-pressure homogenizer.

The claimed invention demonstrates the production of the pharmaceutical substance of the highly purified drug romiplostim thanks to the developed scheme. As a result of effective washings, cellular proteins of inclusion bodies were obtained, purified from impurities, after optimal refolding and subsequent chromatographic purification, which, in combination with the use of some standard techniques for chromatographic purification of proteins and the introduction of two stages of cation exchange chromatography, the first of which is separating, and the second is polishing and concentrating, followed by transfer to the buffer of the finished form by gel filtration, is effective for separating correctly folded romiplostim and incorrectly folded romiplostim and contaminating impurities, such as degradation products, misfolds, aberrant isomers, aggregate, oxidized, deamidated and other forms of the target protein, impurities cellular proteins, nucleic acids, endotoxins and lipids. The resulting product yield is capable of providing a high purity romiplostim preparation in relation to correctly folded romiplostim compared to incorrectly folded romiplostim and obtaining a finished form in an amount of at least 0.9 g per liter of bacterial culture.

The implementation of the invention is illustrated by the example of obtaining a genetically engineered construct (GEC) pGNR69-001 and expression of the target protein by cultivating E. Coli VKPM B-13251 in a Biostat Bplus Twin 5L reactor. The process of culturing the VKPM B-13251 cell bank was aimed at obtaining a high amount of biomass with targeted localization of the romiplostim protein in inclusion bodies.

The example of protein purification presented in the invention illustrates the lysis of cellular biomass and the production of inclusion bodies, the use of washes of inclusion bodies to remove contaminating impurities of cellular components already at the first stage with buffer solutions containing 2% Triton X-100, 2M urea, 2M NaCl and 5 mM EDTA. The washes were effective and removed most of the contaminating cellular proteins, nucleic acids, lipids and endotoxins. In FIG. Figure 5 shows an electropherogram of samples of washing solutions and a solution of inclusion bodies obtained using washing solutions, samples 1, 2, 3 demonstrate the presence of impurity proteins in washing solutions and the absence of the target protein, sample 4, a solution of inclusion bodies, the presence of denatured cell proteins practically free from impurities of cellular proteins chimeric protein monomer.

Dissolution of the resulting inclusion bodies can be accomplished in an appropriate solution of guanidine hydrochloride, usually at a level equivalent to 7-8 M guanidine hydrochloride, which completely denatures the protein. The protein was reduced with DTT (dithiothreitol), 5-20 mM, at pH 8.5, preferably 10 mM, and incubated at room temperature for approximately 1.5-2 hours. The refolding example provided herein illustrates the identified and evaluated refolding conditions that optimize the yield of the target protein and minimize the formation of aberrant isomers, aggregates, and misfolds. First of all, the concentration of the refolded protein was selected. Typical refolding concentrations for complex molecules, such as molecules containing two or more disulfides, are less than 2.0 g/L and more typically 0.001-0.5 g/L (US20100292447, Pitner WR, et al. Method and agent for refolding proteins, US7732183, Tomasselli A. et al., Methods for refolding enzymes, Krishnanand Tiwari et al., Refolding of recombinant human granulocyte colony stimulating factor: Effect of cysteine/cystine redox system// Indian Journal of Biochemistry & Biophysics. - 2012. - V. 49, - p. 285-288). Thus, refolding large masses of a complex protein such as an antibody, peptide antibody, or other Fc protein on an industrial scale presents significant limitations due to the large volumes required to refold proteins at these typical concentrations and is a common industry problem.

Due to the large volumes of material and pool sizes involved in industrial scale protein production, significant time and resources can be saved by eliminating or simplifying one or more process steps, for example by increasing the protein concentration in the refolded mixture. Although protein refolding had previously been demonstrated at higher concentrations, the refolded proteins were either significantly smaller in molecular weight or less complex molecules containing only one or two disulfide bonds (see, for example, Creighton T., Kinetic Role of a Meta-stable Native-like Two-disulphide Species in the Folding Transition of Bovine Pancreatic Trypsin Inhibitor // J. Mol.Biol. - 1984. - 179. - p. 497-526, Creighton T., Renaturation of the Reduced Bovine Pancreatic Trypsin Inhibitor, // J. Mol. Biol. - 1974. - 87. - p. 563-577). In addition, the refolding processes for such proteins used detergents (US 2011/0077384 A1, Yumioka et al., Protein refolding method) or used a high pressure renaturation strategy (US 7,767,795 B2, Randolph et al., High pressure refolding of protein aggregates and inclusion bodies). More complex molecules such as antibodies, peptide antibodies and other large proteins, often having more than two disulfide bonds, from 8 to 24, as a result of the conditions described above, either do not dissolve completely when detergents are used, or may form homo- or hetero-dimers . US patent 20100324269 A1, Shultz et al., Refolding proteins using a chemically controlled redox state, describes a method for refolding Fc-containing proteins with a molecular weight above 57 kDa and containing 8 disulfide bonds at a concentration of 6 g/l, when present in a buffer for refolding additives that prevent aggregation and promote stability of the refolded protein, such as 4 M urea, 20.9% glycerol, 150 mM arginine and, in the presence of a redox couple, 2.03 M cysteine/2.75 mM cystamine. The diluted reaction mixture was titrated to an alkaline pH (between pH 8 and pH 10) and incubated at 5°C under non-aerobic conditions for 12-72 hours. The described process demonstrated stable scalability from 1 L to 2000 L with desired product yields of approximately 27-35% at both scales.

The above method has its drawbacks, the high content of urea and glycerol interferes with further chromatographic purification and requires either dilution or diafiltration, as well as the presence of additional equipment to create non-aerobic conditions, which creates certain difficulties when scaling the process.

The present invention proposes a scalable method for refolding romiplostim using a 21-fold dilution method at a concentration of 0.7 to 1 g/l and established stabilizing concentrations of urea, arginine, pH, redox couple, temperature and refolding time. The best results, determined by RP HPLC and HFHPLC, were obtained using a refolding buffer containing the denaturing agent urea at a concentration of 2.1 M, the aggregation suppressor arginine at a concentration of 0.168 M, and the redox components 3 mM cystine / 4 mM cysteine in buffer 50 mM Tris-HCl, pH-9.0, with constant stirring at 4-8°C under aerobic conditions for 60-72 hours. The described process demonstrated stable scalability from 3 L to 30 L, with desired product yields of approximately 40-50% at both scales.

The described technical result is one of the decisive values for obtaining high-quality target protein and methods for further purification, since it is at this stage that the structural assembly of the target protein molecule, its dimeric form, occurs due to the formation of disulfide bonds both within the molecule of each monomer and between those located in solution with molecules of monomers, each of which is a fusion of amino acid sequences of the Fc part of a human antibody of isotype 1 with a synthetic thrombopoietin mimetic peptide at the C-terminus. A properly refolded protein should have four intrachain and two interchain disulfide bonds.

From those described in the romiplostim report Kyowa Hakko Kirin Co., Ltd. Australian Public Assessment Report (AUSPAR) for Romiplostim. - 2010. - p.102 and Amgen patents (US 6,835,809 B1, Liu et al. Thrombopoietic compounds, US 6,660,843 B1, Feige et al., Modified peptides as therapeutic agents, EP 1 144 454 B2, Feige et al., Modified peptides as therapeutic agents, AU 2004200687, Feige Ulrich Modified peptides as therapeutic agents, US 457169905 B2, Feige Modified peptides as therapeutic agents, US 2004/0053845 Modified peptides as therapeutic agents, US 2006/0234307 Modified peptides as therapeutic agents, US 6,835,809 B1 , Liu et al. Thrombopoietic compounds, US 8044174 B2. Liu et al., Thrombopoietic compounds, US 9,534,032 B2, Liu et al. Thrombopoietic compounds, WO 00/24770, Thrombopoietic compounds, US 2006/0234307 A1, Modified peptides as therapeutic agents , 2006. US 7,169,905 B2 Modified peptides as therapeutic agents, 6,117,655 A 9/2000 Capon et al.) acid treatment of refolded protein followed by concentration and diafiltration was abandoned due to high losses of the target protein.

Chromatographic purification consisted of anion exchange chromatography on a strong anion exchange sorbent in breakthrough mode, affinity chromatography on a sorbent for monoclonal antibodies MabSelect SuRe with effective washes and mild elution conditions, two cation exchange chromatography on YMC S30, the first of which is separating, the second polishing and concentrating, and subsequent transfer to the buffer of the finished form by gel filtration on Superdex G25. The specific conditions for each stage of chromatography and the brands of sorbents used are disclosed in the description, but can vary within limits known to those skilled in the art. For the first stage of purification, based on the physicochemical properties of the target protein and according to literature sources (Fayaz S. et al. Expression, purification and biological activity assessment of romiplostim biosimilar peptibody // DARU J. Pharm. Sci. - 2016. - V. 24. - p. 1-5), in particular, the conditions for affinity chromatography on MabSelect SuRe, a specially created affinity sorbent based on an agarose matrix and a ligand of a protein A derivative, which specifically binds to the Fc part of antibodies, have been developed. The use of such a sorbent allows all variants of romiplostim molecules with a formed Fc part to be adsorbed onto a chromatographic column directly from the reaction mixture after refolding. Other sorbents with protein A ligand attached to a wide variety of matrices are available on the market, such as cross-linked agarose, surface-modified porous glass, coated polystyrene, ceramic shell-filled hydrogel, and other organic polymer-based materials. For example, the MAbSelect sorbent is based on a highly cross-linked agarose gel matrix to accommodate higher flow rates, the MAbselect Xtra sorbent uses the same backbone chemistry as MAbSelect but has a wider pore size for improved mass transfer and, hence, dynamic binding capabilities. The decrease in surface area due to larger pores is compensated by an increase in ligand density. EMD Millipore's controlled pore glass-based Protein A resin (ProSep) has better flow and diffusion properties than agarose-based Protein A resins. However, it suffers from the disadvantage of reduced surface area for binding due to the larger pore diameter. The MabSelect SuRe sorbent consists of an engineered homotetramer-Z domain protein ligand in which a number of asparagine residues have been replaced to eliminate interaction with the variable region, thereby reducing binding heterogeneity between antibodies and allowing the resin to withstand stronger alkaline conditions. Alkali resistance is an important advantage as it allows for repeated use of 0.1-0.5M NaOH for cleaning and disinfection. Although the basic matrices and ligand densities for MabSelect and MabSelect SuRe are identical, the latter provides greater stability under alkaline conditions used in CIP protocols (Anurag S. Rathore et al., Re-use of Protein A Resin: Fouling and Economics // BioPharm International. - 2015. - V. 3. - , I. 28. - p.28-33).

To optimize chromatography on MabSelect SuRe and increase the yield of the target protein, it was necessary to solve several problems: to increase the lifetime of the expensive sorbent, the completeness of placement and the protein yield in elution, taking into account that the target product is stable only in a narrow pH range from 4.5 to 5 ,5.

The cost of sorbents with protein A ranges from 8,000 to 15,000 US dollars per liter, however, in addition to this aspect, another problem is contamination of the sorbent due to nonspecific adsorption, as well as loss of ligand as a result of proteolysis due to proteases present in cell lysates, causing a decrease in adsorption capacity and a shorter service life of the resin (Ana Mayela Ramos-de-la-Pena et. al, Protein A chromatography: Challenges and progress in the purification of monoclonal antibodies // J. Separation Science. - 2009. - 42. - p. 1816-1827, Anurag S. Rathore et al., Re-use of Protein A Resin: Fouling and Economics // BioPharm International. - 2015. - V. 3. - , I. 28. - p. 28- 33). To solve this problem, the authors used anion exchange chromatography on Q Sepharose FF in breakthrough mode before affinity chromatography on MabSelect SuRe. Chromatography conditions were selected under which lipopolysaccharides, nucleic acids and some cellular proteins were adsorbed onto Q Sepharose FF, while the target protein passed through the column without loss and was adsorbed onto MabSelect SuRe. In order to avoid aggregation and ensure complete loading of the target protein at pH 7.2 and high arginine content, which reduces the specific binding of the protein to the ligand, the refolded protein solution was diluted 3 times with equilibration buffer for MabSelect SuRe 20 mM sodium phosphate, pH - 7.2 containing 1.5 M urea. To solve problems with the complete elution of the target protein and conformational changes in antibodies that cause degradation and aggregation at the low pH (2.5 - 3.5) necessary for the elution of antibodies from the protein-A column, milder elution conditions were developed. It is known from literary sources that arginine facilitates the elution of antibodies from a column with protein-A without disturbing the structure and maintaining the monomeric state (Arakawa T. et al. 6 Elution of antibodies from a Protein-A column by aqueous arginine solutions // Protein Expr. Purif. - 2004. - V. 36. - 2. - P. 244-248). By varying the pH and concentration of arginine in the elution buffer, washing conditions were established with a buffer solution of 30 mM sodium citrate containing 0.2 M arginine, pH-4.4 (W1), which removed related impurities that are difficult to remove during further purification, and elution conditions - linear gradient over 10 column volumes: from W1 to 100% 30 mM sodium citrate buffer containing 0.5 M arginine, pH 4.0. The eluate was collected in fractions; 1M Tris was added to each fraction immediately after obtaining the fraction and the pH was adjusted to 5.0-5.05 with a 5M NaOH solution. Fractions were selected with a content of the main substance of at least 60%, and with a content of impurity form 1 of no more than 2%, determined by the RP HPLC method (Table 1, Fig. 10. A., 10. B.). The use of optimized chromatography on MabSelect SuRe made it possible to obtain from the first stage of purification a product with a high content of structurally correctly folded romiplostim monomer (more than 98.8% by GP HPLC and more than 70% by RP HPLC). For subsequent purification stages, it was necessary to select such sorbents and purification conditions in order to separate closely related misfolds, aberrant isomers, oxidized, deamidated and other modified forms from the target protein. It was necessary to take into account that, as stated above, the protein is stable only in a narrow pH range from 4.5 to 5.5. Cation exchange and hydrophobic chromatography and, accordingly, sorbents such as SP Sepharose FF, Capto S, CM Sepharose FF, Butyl Sepharose, Capto Butyl were considered the most suitable for this purpose. A number of experiments were carried out under various sorption and elution conditions. All these methods did not lead to a significant reduction in the amount of closely related impurities in the product.

Unexpected progress was achieved by performing 2 sequential cation exchange chromatographies on two columns with different volumes of sorbent, which is a hydrophilic polymer matrix with particle sizes from 30 μm to 75 μm, and a pore size of approximately 100 nm. In particular, the YMC S30 sorbent was used under conditions of sorption and elution with a long-term (40-42 column volumes) linear gradient to 0.3 - 0.38 M NaCl (Fig. 11A, Tables 2, 3). The first of the two stages of cation exchange chromatography is separating, the second is polishing and concentrating.

For both stages of cation exchange chromatography, a single equilibration buffer is acceptable, the pH of which varies in the range of 4.9-5.2, containing 0.1 M NaCl. Elution on the first cation exchange chromatography is carried out with a linear gradient of 0.1 M to 0.30-0.38 M NaCl in sodium acetate buffer having a pH in the range of 4.9-5.1 over 40 column volumes. In the second cation exchange chromatography, which is both polishing and concentrating for the next process - gel filtration, the target protein is eluted with a linear gradient of 0.1 M to 0.30 - 0.3 5 M NaCl in sodium acetate buffer having a pH in the range of 4.9-5 ,1 for 41-42 column volumes.

To obtain the final finished form of romiplostim, two methods were developed and studied: concentration and diafiltration into a buffer of the finished form in a tangential flow system using a Pellicon XL Cassette 30 kDa membrane cassette: PES (50 cm2) and transfer of the finished form into a buffer by gel filtration chromatography on Superdex G25. The most preferred and convenient method for commercial production turned out to be gel filtration on a column with Superdex G25 sorbent, since with this method the target protein is gently and without loss transferred into a buffer that contains all the necessary additives to maintain structural homogeneity, physicochemical properties and biological activity the resulting finished form. The final stage of gel filtration chromatography allows you to obtain a homogeneous drug in a ready-made buffer.

As a result of purification using the claimed method, it was possible to obtain the target protein with a main substance content of at least 88%, determined by the RP HPLC method (Fig. 13A), with a monomer content of at least 98%, determined by the GP HPLC method (Fig. 13B) and the content of the main charged form not less than 70%, determined by capillary zone electrophoresis (Fig. 13C) and having specific biological activity (Fig. 14A, Fig. 14B).

Materials and methods

Protein synthesis can be carried out using various genetic constructs; to implement the invention, the authors created a strain of the genetically modified microorganism E. coli VKPM B-13251: recipient host strain BL21(DE3) with plasmid pGNR-069-001, carrying the protein gene Fc- peptidylated protein romiplostim. For translation into E. coli, the optimized DNA sequence encoding the romiplostim protein, the nucleotide sequence of which is given in SEQ ID NO1, was introduced into the expression plasmid vector encoding a monomer of a chimeric protein with a C-terminal chimerization of the IgG heavy chain arm, composed of two peptide sequences from 14 amino acids (SEQ ID NO: 2) with polyglycine linker inserts, a length of 269 amino acid residues and a molecular weight of 29.5 kDa (the amino acid sequence is shown in SEQ ID NO: 3, (the nucleotide sequence of romiplostim is presented in US patent 6,835,809 B1, Liu et al. , Thrombopoietic compondus)). The romiplostim gene sequence consists of the Fc fragment of human immunoglobulin IgG1, in which each single-chain subunit is linked by a covalent bond at the C-terminus to a doubly repeating synthetic peptide that binds to the thrombopoietin receptor, with polyglycine linker inserts, consisting of 269 amino acid residues with a calculated molecular weight 29.543 kDa (Fig. 3).

To express Romiplostim, a genetically engineered construct (GEC) pGNR69-001 is obtained. To obtain an expression vector, one optimized for expression in E. coli was synthesized. Nucleotide sequence (SEQ ID NO 1) containing the romiplostim coding open reading frame together with the ATG start codon and the TAA stop signal, flanked at the 5' and 3' ends by NcoI and BamHI restriction sites, respectively. The synthesized sequence was cloned at the above sites into a vector in which the expression of the protein from the cloned sequence is under the control of the T7 promoter. The resulting vector is hereafter referred to as pGNR69-001.

E. coli strain BL21(DE3) was transformed with vector pGNR69-001. The resulting producer strain BL21(DE3)pGNR69-001 was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) under the accession number - VKPM B-13251.

To obtain the target protein romiplostim, E. Coli VKPM B-13251 was cultivated in a Biostat Bplus Tween 5L reactor. The process of cultivating the VKPM B-13251 cell bank was aimed at obtaining a high amount of biomass with targeted localization of the romiplostim protein in inclusion bodies and proceeded in two stages. At the first stage, VKPM B-13251 cells were cultivated in Erlenmeir flasks in a volume sufficient for use at the second stage of production as a seed culture. The seed culture was packaged in test tubes and frozen at -70°C. One tube with a working bank of VKPM B-13251 cells was inoculated with 250 ml of sterile ECM medium (Experimental Cell Medium) and grown at 37°C for 16 hours at 180 rpm.

The second stage of fermentation included the following steps:

(1) 250 ml of the starting culture VKPM V-13251, grown in the first stage, was loaded into a Biostat Bplus Tween 5L reactor with 3 liters of ECM medium;

(2) the fermenter pH was maintained at 7.0 using solutions of 12.5% ammonia and 10% glacial acetic acid;

(3) DO in the reactor was set at 40% saturation, which was adjusted by stirrer speed and dissolved oxygen percentage until induction, and then decreased to 20% in the post-induction phase of cultivation;

(4) fermentation temperature is set to 37°C;

(5) supplementation of nutrient solutions (40% yeast extract and 10% glycerol) was added exponentially after complete consumption of glucose in the medium (usually after 8 hours) at the rate of:

V=V0*e0.18t

where V is the volume of nutrient solution added in ml/hour;

V0 - 1/100 of the starting volume of the medium in the reactor in ml;

t is the fermentation time after the start of the phase of adding nutrient solutions (hour).

The expression of romiplostim was induced by the addition of IPTG to a final concentration of 1.0 mM, when the optical density of the VKPM B-13251 culture was 50-70 optical units. The post-induction fermentation phase continued for another 3 hours with the addition of nutrient solutions and hourly sampling to monitor the optical density of the culture. The final optical density of the culture 3 hours after the post-induction phase of fermentation was 100-150 p.u. The biomass yield ranged from 80-100 g/l of culture. The romisplostim protein has a molecular weight of 29,543 Da, is expressed at a level of 20-30% of the total protein and is found exclusively in the form of inclusion bodies. Bacterial biomass was collected by centrifugation and stored at -70°C.

Frozen E. coli cell biomass was suspended in disintegration buffer 20 mM Tris HCl, 300 mM NaCl, pH 7.5 with the addition of a protease inhibitor to inhibit proteolysis phenyl-methyl-sulfonyl-fluoride (PMSF). For cell disintegration, the standard ultrasonic disintegration method was used, which was replaced by high-pressure disintegration when scaled up. Inclusion bodies were obtained by centrifugation from a disintegrated cell suspension at 25,000xg at 4°C for 40 minutes. The resulting inclusion bodies were sequentially washed with 50 mM Tris HCl, pH 7.5, buffer solutions containing 2% Triton X-100, 2 M urea, 2 M NaCl and 5 mM EDTA. The washed inclusion bodies were dissolved in a buffer solution of 7 M guanidine hydrochloride, 50 mM Tris HCl, 10 mM DTT, pH 8.2 at room temperature for 1.5-2 hours. The content of the monomeric unit of romiplostim was determined by GP HPLC of the reduced inclusion bodies of romiplostim and the reduced solution of LP Enplate, from which a calibration curve was constructed (Fig. 6). Refolding of the target protein romiplostim was carried out by dilution method 21 in 50 mM Tris-HCl buffer; 2.1 M urea; 0.168 M arginine; 3 mM cystine; 4 mM cysteine; pH 9.0, at protein concentration (from 0.7 to 1 mg/ml) for 60-72 hours at 4-8°C with constant stirring. In FIG. Figure 7 shows an electropherogram of samples of the refolded mixture under reducing and non-reducing conditions (sample 1, 2) in comparison with LP Enplate (sample 3, 4); the preparations show almost identical sets of bands on the gel, which indicates the absence of significant differences in their organization and indicates complete completion of renaturation. The quantitative content and quality of refolded romiplostim were controlled by GP HPLC and RP HPLC (Fig. 6, Fig. 8). The yield of refolded target protein was maintained when scaling up the process from 3 L to 30 L of refoldable mixture and ranged from 40% to 50% of the correctly folded dimeric form of romiplostim (Figure 8). Further purification of romiplostim was carried out by separating correctly folded and incorrectly folded conformations of this protein, separating remnants of contaminating impurities of cellular proteins, lipopolysaccharides, DNA from the refolded solution by anion exchange chromatography in breakthrough mode on Q Sepharose FF, followed by affinity chromatography on MabSelect SuRe under conditions of washing closely related impurities with buffer a solution of 30 mM sodium citrate containing 0.2 M arginine, pH-4.4 and soft elution of the target correctly folded protein with a linear gradient over 10 column volumes from 100% washing buffer to 100% elution buffer 30 mM sodium citrate containing 0.5 M arginine, pH-4.0.

Q Sepharose FF and MabSelect SuRe vehicles are equilibrated with the same 20 mM sodium phosphate buffer with 1.5 M urea pH-7.2. Elution of carrier-bound compounds occurs with different buffers. Elution with Q Sepharose FF was carried out with a buffer solution of 20 mM Tris HCl, pH 7.8 with 1 M NaCl and a large peak in optical density was observed in the UV region, which largely corresponds to the absorption of DNA, as well as the admixture of cellular proteins, in addition to Q Sepharose FF bacterial endotoxins are separated. In FIG. Figure 9 shows an electropherogram of fractions obtained from chromatography on Q Sepharose FF and MabSelect SuRe under reducing and non-reducing conditions. Sample 4 corresponds to the eluate sample from Q Sepharose FF, sample 3 corresponds to the eluate from the MabSelect SuRe sorbent, sample 2 corresponds to the wash fraction, sample 1 to the fraction that has not contacted the carrier. The electropherogram clearly demonstrates that the target protein, without binding to the Q Sepharose FF sorbent, is completely retained on MabSelect SuRe; only impurity proteins, DNA and lipopolysaccharides are retained on the Q Sepharose FF carrier. Removing such impurities helps reduce non-specific sorption of MabSelect SuRe, thereby enhancing the efficiency of affinity chromatography and increasing the service life of the expensive media. The eluate from MabSelect SuRe was collected into fractions that were analyzed by RP HPLC.

Analysis of the obtained fractions with MabSelect SuRe by RP HPLC is shown in Fig. 10V. In FIG. 10A shows a chromatography profile. A large peak of optical density in the UV region (peak 1, washout peak) was observed at pH 4.4 and 0.2 M arginine; in addition to the main substance, it contained less hydrophobic forms of the main substance, which were less well retained on the affinity sorbent than the main substance substance. (Fig. 10B, forms 1-2) and more hydrophobic forms (Fig. 10B, forms 3-4). Since all of these impurities eluted from the carrier before the main substance, most likely these are misfolded forms, monomeric units that have not formed a dimer, but also have an Fc domain. When eluting with a gradient of pH and arginine, upon reaching pH 4.3 and 0.25 M arginine, a second peak begins, corresponding to the main correctly folded substance (Fig. 10A., peak 2, gray color), containing the correctly folded Fc domain, remaining more tightly The sorbent-bound forms having an Fc domain were eluted at pH 3.2, in the presence of 0.5 M arginine (Fig. 10A, peak 3). A lower pH, for example pH 3.2, was necessary to elute compounds tightly bound to the sorbent. The fact that these molecules bound to protein A means that they were also Fc fusion proteins. Because these compounds eluted at a lower pH compared to correctly folded romiplostim, they bound more tightly to protein A. These molecules were more hydrophobic, as shown by RP HPLC analysis (Figure 10B), and were misfolded romiplostim molecules, most likely soluble aggregates, misfolds, or oxidized forms. A large absorbance peak in the UV region (Fig. 10A, peak 4, regeneration peak) was observed when the sorbent was regenerated with 0.5 M NaOH. The fact that these compounds were so tightly bound to protein A that they could only be removed by an alkaline solution of fairly high molarity suggests that these compounds are most likely insoluble aggregates and unrefolded protein. Based on the results of RP HPLC analysis, fractions containing at least 60% of the main form, no more than 2% of form 1, and no more than 10% of form 4 were selected for further purification of the correctly folded target protein from remaining aggregates, misfolds, oxidized and deamidated forms.

A summary table of the MabSelect SuRe affinity chromatography results is presented in Table 1.

Further purification was carried out by two cation exchange chromatographies with different volumes of YMC S30 sorbent under conditions of sorption and elution with a long-term (40 column volumes or more) linear gradient of NaCl (Fig. 11A, Fig. 12A, table 2, table 3), the first of which is separating, the second is polishing and concentrating.

To carry out the first chromatography on the YMC S30 sorbent, the fractions selected with MabSelect SuRe were combined, diluted with purified water to an electrical conductivity of 9-10 mSm/sm, and applied to a YMC S30 column equilibrated with a buffer of 20 mM sodium acetate, 0.1 M NaCl, pH 5.1. Eluted with a gradient from buffer 20 mM sodium acetate, 0.1 M NaCl, pH 5.1 to buffer 20 mM sodium acetate, 0.3 M NaCl, pH 5.1 over 40 column volumes. The eluate was collected into fractions, which were analyzed by RP HPLC. In FIG. 11A shows a chromatography profile with YMC S30-1, where 4 pre-peaks corresponding to impurity forms and a large optical density peak corresponding to the main form of the target protein (peak 1, gray color) can be observed when the electrical conductivity reaches 22-23 mSm/sm. In addition, a high narrow peak is observed, corresponding to the elution buffer of 20 mM sodium acetate, 1 M NaCl, pH 5.1, in which more charged forms are eluted (peak 2), and a narrow regeneration peak, most likely corresponding to impurities with the highest charge ( peak 3). A summary table of the results of chromatography on YMC S30-1 is presented in Table 2.

For further purification of the target protein, fractions were selected based on the results of RP HPLC analysis containing at least 87% of the main form, no more than 8% of form 3, and no more than 4% of form 4.

To carry out the second chromatography on the YMC S30 sorbent, which is both polishing and concentrating for the next process - gel filtration, the fractions selected from the YMC S30-1 chromatography were combined, diluted with purified water to an electrical conductivity of 10 mSm/sm and applied to a column with YMC S30 , equilibrated with a buffer of 20 mM sodium acetate, 0.1 M NaCl, pH 5.1. Eluted with a gradient from buffer 20 mM sodium acetate, 0.1 M NaCl, pH 5.1 to buffer 20 mM sodium acetate, 0.3 M -0.35 M NaCl, pH 5.1 over 40-42 column volumes. The eluate was collected into fractions, which were analyzed by RP HPLC and capillary zone electrophoresis for the content of the main isoform. In FIG. 12.A. a chromatography profile is presented, where one peak of optical density can be observed, corresponding to the main form of the target protein when the electrical conductivity reaches 22-23 mSm/sm (peak 1). The peak has a slight stretched right shoulder, corresponding to the admixtures of acidic forms (Fig. 12.B., Fig. 12.C, Table 3.). Small narrow peaks are also observed, corresponding to elution of the buffer 20 mM sodium acetate, 1 M NaCl, pH 5.1, and regeneration (peaks 2, 3).

A summary table of the YMC S30-2 chromatography results is presented in Table 3.

To transfer the target protein into the buffer of the finished form using gel filtration chromatography on Superdex G25, fractions from chromatography on YMC S30-2 were selected according to the results of RP HPLC analysis with a content of the main form of at least 90%, according to the results of capillary zone electrophoresis with a content of the main charged form of not less than 87%. Fractions 6 to 8 were combined and applied to a column with Superdex G25 carrier, eluted with the prepared form buffer: 10.0 mM histidine, 4% mannitol, 2.0% sucrose, 0.0045% polysorbate 20, pH 5.0 at room conditions, results chromatography are presented in Table 4.

The resulting drug was diluted to the desired concentration and sterile filtered. The protein yield is at least 0.9 g per liter of bacterial culture.

Measurements of the parameters of various batches of the finished form of the target protein romiplostim showed that as a result of purification by the claimed method, the target protein is obtained with a main substance content of at least 88%, determined by the RP HPLC method (Fig. 13.A.), with a monomer content of at least 98%, determined by the GP HPLC method (Fig. 13.B) and the content of the main charged form of at least 70%, determined by capillary zone electrophoresis (Fig. 13. The specific biological activity of the drug, determined by the competitive enzyme-linked immunosorbent assay, averaged 95.3 ± 5.3% (results are presented in Fig. 14.A.) and the level of concentration of phosphorylated tyrosine kinase JAK2 released from human platelets averaged 100±6% (results are presented in Fig. 14.B).

A pharmaceutical substance may contain various biological impurities associated with the producer: (endotoxins, residual DNA of the producer strain, host cell proteins). The content of these impurities may affect the safety of the drug and should therefore be assessed in the purified product. The content of bacterial endotoxins, pH and osmolality were determined in accordance with the standards of the State Pharmacopoeia of the Russian Federation (State Pharmacopoeia of the Russian Federation http://femb.ru/femb/pharmacopea.php). The results of the analysis are presented in Table 5. Quantitative determination of the proteins of the producer strain (E. coli) determined by enzyme immunoassay in various batches of the finished form of romiplostim was less than 4 ng/mg with a norm of no more than 10 ng/mg protein ("Draft Guidance on Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products", (FDA, CBER, ICH), 24 July 1998., Jonathan Briggs and Peter R. Panfili, Quantitation of DNA and Protein Impurities in Biopharmaceuticals // Perspective: Analytical Biotechnology, Anal. Chem . - 1991. - 63. - p. 850-859).

Real-time polymerase chain reaction (PCR) was used to determine residual DNA. It is based on amplification of a section of DNA with simultaneous measurement of an increase in the fluorescent signal. By measuring the number of cycles required to reach a threshold level of fluorescence, the initial amount of DNA in the sample can be calculated. Calibration standards of known concentration are used to interpret measurement results.

When studying batches of GF romiplostim, the measured DNA concentration for all samples was below the detection limit (2.1 pg/ml).

These values correspond to international recommendations, according to which the content of residual DNA should not exceed 10 ng per 1 dose of the drug (WHO. Guidelines on the quality, safety and efficacy of biotherapeutic protein products prepared by recombinant DNA technology. Replacement of annex 3 of WHO technical report series - 2013. - N.814. http://www.who.int/biologicals/biotherapeutics/rDNA_DB_final_19_Nov_2013.pd f>.

For a better understanding of the essence of the invention, examples of its implementation follow below.

Example 1. Microbiological protein synthesis

The inventive method is implemented for the producer strain VKPM B-13251 when cultivated in a fermenter (Sartorius Biostat B Plus Twin 5L, Germany) in a medium volume of 3 liters. The entire cultivation process is carried out at a temperature of 37°C. The pH of the medium is maintained at 7.0-7.1 by titration with a 10% acetic acid solution and a 12.5% ammonia solution. The aeration level is 3 l/min and the dissolved oxygen concentration is 40%, which is regulated by a cascade of stirrer speeds.

The grown inoculum in a volume of 250 ml is introduced into a bioreactor with a sterile ECM nutrient medium. As a starting substrate, glucose is added to the medium in an amount of 10 g/l and glycerol 30 g/l.

During the pre-induction phase of growth, cells utilize glucose from the medium. The pH level will drop. After 8-9 hours, the cells reach an optical density of 50-70 optical units. Romiplostim expression is induced by the addition of IPTG to a final concentration of 1.0 mM. Next, the post-induction phase of fermentation continues for another 3 hours with the addition of nutrient solutions (40% glycerol, 1% yeast extract) and hourly sampling to control the optical density of the culture. After 3 hours, the final optical density of the culture reaches 100-150 p.u. The fermentation process stops. Bacterial biomass is collected by centrifugation at 10,000 rpm for 20 minutes and stored at -70°C.

Example 2. Isolation and purification of romiplostim protein from bacterial biomass.

2.1 Destruction of cells of the producer strain, production of inclusion bodies.

Buffer A - 20 mM Tris HCl, 300 mM NaCl, 0.001% PMSF pH 7.5;

The biomass sediment (m = 30.0 ± 1.0 g) is placed from the refrigerator into a 0.5 liter plastic measuring glass, 300 ± 0.1 ml of freshly prepared buffer solution A is added to the cell sediment. A dispersant nozzle is placed in the glass and dispersed within 10-15 minutes. A glass with a homogenized mass of producer cells is placed in an ice bath under the probe of an ultrasonic disintegrator. They are treated with ultrasound on a large probe for 15 minutes at an amplitude of 25% (cycle: 30 sec - ultrasound, 40 sec - cooling). Pour the suspension into centrifuge beakers and centrifuge 25000xg at 4°C for 40 minutes. After centrifugation is complete, the supernatant is discarded.

2.2 Washing inclusion bodies

Buffer B: 50 mM Tris HCl, 2% Triton X-100, 5 mM EDTA, pH 7.5;

Buffer C: 50 mM Tris HCl, 2 M urea; 5 mM EDTA, pH 7.5;

Buffer D: 50 mM Tris HCl, 2 M NaCl; 5 mM EDTA, pH 7.5;

Buffer E: 20 mM Tris HCl, 1 mM EDTA, pH 7.5;

The sediment of inclusion bodies is placed in a container with a volume of 0.25 l, 200 ± 0.1 ml of buffer solution B is added to the sediment. A dispersant nozzle is placed in the glass and dispersed for 3-5 minutes. A centrifuge beaker with a solution of inclusion bodies is centrifuged at 30,000 x g (14,000 rpm) at 5°C for 35 minutes. After centrifugation is complete, the supernatant is discarded.

The above procedure is repeated sequentially with buffers C, D, E.

As a result, no less than 13.6±2 g of washed inclusion bodies are obtained.

The resulting product is stored at minus 80°C.

2.3. Dissolution of inclusion bodies

Buffer L: 50 mM Tris HCl, 10 mM DTT, 7 M guanidine chloride, pH 8.2;

The inclusion body sediment (m=15.0±0.1 g) from the refrigerator is placed in a 0.2 liter plastic measuring glass, 150±0.1 ml of freshly prepared solution L is added to the sediment. Disperse for 5 minutes. The glass is placed on a magnetic stirrer and stirred at room temperature for 90-120 minutes. The suspension is centrifuged at 40,000 x g at 8°C for 120 minutes. After centrifugation is complete, carefully remove the supernatant.

2.4. Refolding.

Buffer R: 2.1 M urea; 0.168 M arginine; 50 mM Tris-HCl; 3 mM cystine; 4 mM cysteine; pH-9.0;

A solution of inclusion bodies with a volume of 150±10 ml is diluted 20-21 times with a 3000 ml volume of buffer R cooled to 8±1°C, for which the container with buffer R is placed on a magnetic stirrer and the solution of inclusion bodies is slowly added dropwise with constant stirring, measured The pH of the solution, if the pH deviates from 9.0, is adjusted with hydrochloric acid or sodium hydroxide solution. The container is placed on a stirrer and the process of protein folding is continued at a temperature of 4-7±1°C with constant stirring for 70-72 hours.

2.5. Affinity chromatography on MabSelect SuRe.

Buffer F - 20 mM sodium phosphate, 1.5 M urea, pH-7.2;

Buffer W1 - 30 mM sodium citrate, 0.2 M arginine, pH-4.4.

Buffer G - 30 mM sodium citrate, 0.5 M arginine, pH-4.0;

Buffer R - 30 mM sodium citrate, 0.5 M arginine, pH-3.2;

Buffers for column regeneration:

100 mM Tris, pH 7.5;

0.5 M NaOH

20% ethyl alcohol

The first stage of purification of the target protein is carried out on sequentially assembled columns: a column filled with 80 ml Q Sepharose FF sorbent and a column filled with 60 ml MabSelect SuRe sorbent, using the Akta Pure 25 chromatography system at room temperature.

The columns are washed with 10 column volumes of buffer solution F at a flow rate of 113 cm/h. 6300 ml of buffer F are added to a 10-liter container and 3150 ml of folded protein solution is added, the pH of the solution is adjusted to 7.2 ± 0.01 with concentrated phosphoric acid and applied to the columns. After application, the sorbents are washed with 3 column volumes of buffer solution F, then the column with the Q Sepharose FF sorbent is disconnected and transferred for regeneration, and the column with the MabSelect SuRe sorbent is successively washed with 2 column volumes of buffer solution F, 10-12 column volumes of buffer solution W1, with a decrease optical density at the column outlet up to 100 mA for elution and fractionation of the target protein and its derivatives bound to the sorbent, a linear gradient from 0% buffer solution W to 100% buffer solution G is used over 10 column volumes. Fractions are collected in 40 ml increments when the optical density of the solution exiting the column increases to 120 mAU relative to the baseline; when the optical density decreases below 120 mAU, the collection of fractions is stopped. Immediately add 1 ml of 1 M Tris solution pH 8.0 to each fraction and adjust the pH to 5.0 ± 0.1 with 5 M sodium hydroxide solution. Upon completion of the gradient, the sorbent is washed with 1.8 column volumes of buffer solution P and 1.8 column volumes of 0.5 M sodium hydroxide solution and transferred to the conservation stage.

Fractions containing the target protein, with a content of the main substance of at least 60% and with a content of impurity peak No. 1 of no more than 2%, determined by RP HPLC, are combined.

To preserve the column with MabSelect SuRe, the sorbent is successively washed with 5 column volumes of buffer solution 01, M Tris HCl, pH 7.5 and 3 column volumes of 20% ethyl alcohol solution.

To regenerate a column with Q Sepharose FF, the sorbent is washed with 3 column volumes of a solution of 0.5 M sodium hydroxide, kept in this solution for at least 60 minutes, washed again with 1.8 column volumes of the same solution, then successively washed with 5 column volumes of buffer solution 0. 1 M Tris HCl, pH 7.5 and 3 column volumes of 20% ethyl alcohol solution.

2.6. Cation exchange chromatography on YMC S30 - 1

Buffer H - 20 mM Na acetate, 0.1 M NaCl, pH 5.0;

Buffer Y - 20 mM Na acetate, 1.0 M NaCl, pH 5.0;

The second stage of purification of the target protein is carried out on a column filled with YMC S30 sorbent with a volume of 70 ml, using the Akta Pure 25 chromatographic system at room temperature.

The column is washed with 3 column volumes of buffer solution Y, then 10 column volumes of buffer solution H at a flow rate of 226 cm/h.

The combined fractions from chromatography on MabSelect SuRe are diluted 4 times with purified water cooled to 4°C, the pH of the solution is adjusted to 5.0±0.01 with glacial acetic acid or 5 M sodium hydroxide solution, the electrical conductivity is adjusted to 8.5±0.5 mS/cm 5M sodium chloride solution and applied to the column. After application, the sorbent is washed with 5 column volumes of buffer solution H; a linear gradient from 0% buffer solution H to 30% buffer solution Y is used for 40 column volumes to elute and fractionate the target protein and its derivatives associated with the sorbent. Fractions are collected when the optical density of the solution exiting the column increases to 136 mAU relative to the baseline; when the optical density decreases below 70 mAU, the collection of fractions is stopped. Collection of fractions for analysis can begin after the release of four prepeaks. Upon completion of the gradient, the sorbent is washed with 1.8 column volumes of 100% buffer solution Y and transferred to the regeneration stage.

Fractions containing the target protein with a content of the main substance of at least 87%, determined by RP HPLC, are combined.

To regenerate the column, the sorbent is successively washed with 3 column volumes of a 0.5 M sodium hydroxide solution, 5 column volumes of a 0.1 M Tris HCl buffer solution, pH 7.5, and 3 column volumes of a 20% ethyl alcohol solution. Store the column with the inlet and outlet holes closed at a temperature of 2...8°C.

2.7. Cation exchange chromatography YMC S30 - 2

The third stage of purification of the target protein is carried out on a column filled with YMC S30 sorbent with a volume of 35 ml, using the Akta Pure 25 chromatographic system at room temperature.

The column is washed with 3 column volumes of buffer solution Y, then 10 column volumes (to ensure complete equilibration) of buffer solution H at a flow rate of 239 cm/h.

The combined fractions from chromatography on YMC S30 - 1 are diluted 2.5 times with purified water cooled to 4°C, the pH of the solution is adjusted to 5.0±0.01 with glacial acetic acid or 5 M sodium hydroxide solution, the electrical conductivity is adjusted to 8.5 ±0.5 mS/cm 5M sodium chloride solution, if necessary, and applied to the column. After application, the sorbent is washed with 5 column volumes of buffer solution H; for elution and fractionation of the target protein and its derivatives associated with the sorbent, a linear gradient is used from 0% buffer solution H to 30% buffer solution Y over 40-42 column volumes. Fractions are collected when the optical density of the solution exiting the column increases to 60 mAU relative to the baseline; when the optical density decreases below 40 mAU, the collection of fractions is stopped. Upon completion of the gradient, the sorbent is washed with 1.8 column volumes of 100% buffer solution Y and transferred to the regeneration stage.

Fractions containing the target protein with a main substance content of at least 88%, determined by RP HPLC, with a monomer content of at least 98%, determined by GP HPLC, and a main form content of at least 70%, determined by capillary zone electrophoresis, are combined.

To regenerate the column, the sorbent is successively washed with 3 column volumes of a 0.5 M sodium hydroxide solution, 5 column volumes of a 0.1 M Tris HCl buffer solution, pH 7.5, and 3 column volumes of a 20% ethyl alcohol solution. Store the column with the inlet and outlet holes closed at a temperature of 2...8°C.

2.8 Gel filtration chromatography on Superdex G 25

Buffer U - 10.0 mM histidine, 4% mannitol, 2.0% sucrose, 0.0045% polysorbate 20, pH 5.0;

The fourth stage of purification of the target protein (buffer replacement) is carried out on a column filled with Superdex G 25 Fine sorbent with a volume of 240 ml, using the Akta Pure 25 chromatographic system at room temperature.

The column is washed with 2 column volumes of buffer solution U at a flow rate of 20-24 cm/h.

From the combined fraction from chromatography on YMC S30 - 2, half of the solution is taken and applied to a column with Superdex G 25 Fine sorbent at a flow rate of 24 cm/h, the target protein is released when 0.3-0.33 column volume of buffer U flows through the sorbent (10±1 min/80±1 ml), the target protein begins to be collected with an increase in optical density above 65 mAU relative to the baseline. When the optical density decreases below 70 mAU, protein collection is stopped. After 1.4 column volumes of buffer solution U have passed through the carrier, purification of the second portion begins. The eluates are combined, stirred for 2-3 minutes, and the volume is measured.

To regenerate the Superdex G 25 column, the sorbent is washed with 3 column volumes of 0.5 M sodium hydroxide, 3 column volumes of purified water and 3 column volumes of a 20% ethyl alcohol solution.

2.9 Preparation of ready-made working solution

Buffer U - 10.0 mM histidine, 4% mannitol, 2.0% sucrose, 0.0045% polysorbate 20, pH 5.0;

Calculate and add the required amount of buffer U to obtain a protein concentration of 0.53 to 0.55 mg/ml. The working protein solution is carefully mixed and left at a temperature of 2...8°C for 60 minutes. Measure the solution volume and protein content and add the required amount of buffer U to obtain a concentration of 0.53 to 0.55 mg/ml.

2.10 Sterilizing filtration

Sterilizing filtration of the working solution of the finished form is carried out in a laminar air flow. For sterilizing filtration, use the FILTERMAX sterilizing system, 250 ml, PES, with a pore size of 0.22 microns, using a membrane vacuum pump.

After filtration, a sample is taken for technological control. The resulting solution is the finished form of romiplostim.

Claims (7)

1. A method for purifying recombinant romiplostim obtained in E. coli, including isolation and washing of inclusion bodies, protein refolding and chromatographic purification, characterized in that the main removal of contaminating impurities of cellular proteins, nucleic acids, lipids and endotoxins is carried out at the stage of isolation of inclusion bodies by their sequential washing, and additional removal of impurities includes carrying out 2 successive stages of chromatography on the same cation-exchange sorbent, where the first stage is separating, and the second is polishing and concentrating. 2. The method according to claim 1, where chromatographic purification is carried out sequentially, first in breakthrough mode on a strong anion-exchange sorbent, followed by sorption of the target protein on an affinity sorbent modified with recombinant protein A, and then two stages of chromatography are carried out on a cation-exchange sorbent. 3. The method according to claim 2, where the cation exchange sorbent is a hydrophilic polymer matrix with a particle size of approximately 30 to 75 μm and a pore size of approximately 100 nm. 4. The method according to claim 1, where the purified protein is obtained by transferring the finished form into a buffer by gel filtration. 5. The method according to claim 1, where the buffer for washing inclusion bodies contains Triton X-100, urea, NaCl and EDTA. 6. The method according to claim 1, where the refolding buffer includes 3 mM cystine and 4 mM cysteine. 7. The method according to claim 1, where refolding is carried out at a protein concentration of 0.7 to 1 mg/ml.

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