RU2473556C1 - Method for commercial production and purification of human recombinant growth hormone of inclusion bodies - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, particularly a method for production and purification of human recombinant growth hormone (rHGH). The presented method for production and purification of rHGH involves dissolution of inclusion bodies in 2 M urea at pH=11.0 followed by renaturation in a buffer solution 20 mM "Трис"-HCl at pH=8.0. Hydrogen peroxide is used as an rHGH sulphhydryl group oxidiser. N-terminal methionine is split from leucinic aminopeptidase. Chromatographic purification of rHGH is enabled with two-stage ion-exchange chromatography on the sorbent Q Sepharose FF and the stage of purification by hydrophobic chromatography on the sorbent Butyl Sepharose 4 FF. Gel filtration on Sephadex G-25 and ion-exchange chromatography on Q Sepharose FF, pH=6.5. It is followed by ion-exchange chromatography on Sephacryl S-100HR.

EFFECT: invention enables producing a preparation of high-purity rHGH protein of high compendial grade applicable for preparing drug preparations.

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Description

The invention relates to the field of biotechnology, in particular to an industrial method for producing recombinant human growth hormone (hGH) human or somatropin.

The objective of the present invention is to develop a technological easily scalable and cost-effective method for producing highly purified hrh, with a purity of not less than 98%.

Somatropin (growth hormone-STH) is a protein hormone produced by the anterior pituitary gland. It is secreted into the blood under the influence of somatostatin and STH-releasing factor - somatoliberin. The time and frequency of excretion are regulated by somatostatin, and the amount of hGH secreted is regulated by somatoliberin. HGH belongs to the group of anabolic hormones [Pavlovsky A.G. et al. // Human Growth Hormone. Collection of scientific papers. Scientific Center for Biological Research, Academy of Sciences of the USSR, Pushchino, 1988., T. 305, No. 4., S.861-864.]. The hGH molecule consists of 191 amino acid residues with a molecular weight of 22125 Da. Keeping the structure scheme common to other mammals (contains two tryptophan residues, three tyrosine residues and four cysteine residues, which form two disulfide bridges in the molecule and form two loops - a large one, including the central portion of the amino acid sequence between Cys ^54- Cys ¹⁶⁵ , and a small (at the C-terminal portion) between Cys ¹⁸² -Cys ¹⁸⁹ ), hGH differs in amino acid sequence from the studied animal somatropins by 34-35%. This explains the inefficiency of somatropins of animal origin as growth stimulants when administered to people [Ovchinnikov Yu.A. // Bioorganic chemistry. M.: Education, 1987, p.815.].

Recombinant human growth hormone is widely used in medical practice for the treatment of various kinds of diseases associated with decreased secretion of this hormone in the human body - small growth, Shereshevsky-Turner syndrome, pituitary nanism [Abdelaziz Elamin, Tuvermo A. // Growth hormone treatment in children and adolescents . Annals of Saudi Medicine, 2002, V.22, P.47-55]. According to scientific research, hGH can be used in the treatment of diseases such as type II diabetes, hypertension, atherosclerosis, cardiovascular failure, obesity and other pathologies that occur as a result of a decrease in hGH in the human body with age [Wilson M.E. , Gordon T.R., Chikazawa K., Gust D., Tanner J.M., Rudman C.G. // Effects of growth hormone on neonatal growth in nursing rhesus monkeys. J. Clin. Endocrinol. Metab., 1991, V.72 (6), P.1302-1307.].

The creation of a strain producing hGH made it possible to obtain a biologically active protein in the amount necessary for conducting various structural and functional studies, as well as for use in medical practice. Abroad, drugs based on rHRG (genotropin, protropin, humatrop, etc.) have been created. The growth hormone obtained in the bacterial cells of E. coli contains an additional N-terminal methionine residue (Met-rGHF), which must be cleaved to obtain the desired drug (rGHF).

The hRHG thus obtained is purified by standard methods mentioned, for example, in US patents: US 4,601,980, US 5,424,199, US 4,658,021 and others. In these methods, protein solubilization is usually used in buffer systems containing chaotropic agents or ionic detergents at sufficiently high concentrations that greatly complicate protein purification in subsequent stages. Also known is a method for carrying out the solubilization of hGH with urea at high pH [Ashok K. Patra et all // Optimization of inclusion body solubilization and renaturation of recombinant human growth hormone from Escherichia coli, Protein Expression and Purification, 2000, V 18, p. 182-192]. Protein renaturation is carried out by lowering the concentration of a chaotropic agent or by adding a redox pair. Further purification of hGH is carried out by the following methods: fractionation with polyethyleneimine, gel filtration on Sephacryl S-200, fractionation with ammonium sulfate, ion exchange chromatography, affinity chromatography using antibodies against growth hormone. Such methods are laborious and expensive.

Also known are methods of producing hGH, [US Pat. EP No. 0114506, RF 1596731, RF 2099348], in which it is proposed to purify the protein by precipitation with various metal salts, thus achieving the disposal of high molecular weight protein impurities. But this approach leads to lengthy centrifugation or filtration processes. Such methods complicate the process of protein purification and lead to a rise in the cost of technology.

Closest to the claimed method is a method [Vorobev I.I. et al. // Highly efficient process for producing human growth hormone, Biotechnology, 2005, No. 5, P.44-48], which allows one to obtain an intact native hRHF, which includes several stages: solubilization of inclusion bodies with 6 M guanidine hydrochloride, refolding with by adding 1 mM cysteamine and 0.1 mM cystamine to the reaction mixture, protein purification on a Source 15Q ion-exchange sorbent, cleavage of the N-terminal methionine with aminopeptidase. Final purification was performed using reverse phase chromatography on a sorbent C4, Nucleosil. Exclusive gel filtration on Superdex 75 gel was used to transfer the protein to the pharmacological buffer solution. This method allows one to obtain hGH with a yield of 0.1 g from 1 g of the target protein solubilized from inclusion bodies.

The disadvantage of the prototype method is that the use of 6 M guanidine hydrochloride for solubilization reduces the yield of Met-hRHC after the renaturation process and leads to large protein losses up to 50% of the initial amount. Another disadvantage of this method is the need to concentrate the protein after renaturation by precipitation with a 50% solution of ammonium sulfate, which, in turn, when scaling the process leads to large protein losses and laboriousness at the centrifugation stage.

Thus, there is a need for new improved and cheaper methods for purification of hGHR, allowing to obtain highly purified protein with high yields, as well as reduce the time of cleaning.

The technical result of the claimed invention is to simplify the technology of purification of recombinant human growth hormone.

The specified technical result is achieved by the implementation of the claimed method of purification of recombinant human growth hormone. The proposed method for the preparation and purification of hGH, synthesized in E. coli cells in the form of insoluble inclusion bodies, consists of several successive stages.

The inclusion bodies of Met-hRHF are solubilized in a buffer solution containing 20 mM Tris-HCl, 2 M urea, pH 11.0 and allowed to mix at room temperature.

The protein solution obtained after solubilization is diluted with a buffer solution containing 20 mm Tris-HCl. Then the pH of the solution was adjusted to a value of 8.0, 37% H ₂ O _{2 was} added and stirring was continued at room temperature. The amount of correctly laid protein is monitored by RP-HPLC on a YMC C4, 250 x 4.6, 5 μm, 300 A column (YMC Co., LTD, Japan).

The first stage of chromatographic purification of Met-hRHC is carried out at room temperature on an axial column packed with Q Sepharose FF ion-exchange sorbent (GE Healthcare, USA). The protein solution after renaturation is applied to the column, balanced with the starting buffer solution, which contains 20 mm Tris-HCL, pH 8.0. Elution of protein from the column is a linear gradient of the concentration of sodium chloride from 0-40%.

After the first purification step, Q Sepharose FF is used to cleave the N-terminal methionine to obtain the native protein (hGH). For this, a leucine aminopeptidase is added to the Met-rGH solution. The cleavage reaction is carried out at room temperature and is monitored by isocratic RP HPLC on a Symmetry 300, C ₁₈ , 300 Å (4.6 × 250 mm) column (Waters, USA).

In the second stage of chromatographic purification of hGH, the protein with cleaved methionine is applied to an axial column packed with Butyl Sepharose 4 FF (GE Healthcare, USA) and balanced with a starting buffer solution containing 20 mM Tris-HCL, 2M NaCl, pH 7.0. Elution of the protein from the column is carried out by a decreasing linear gradient of the concentration of sodium chloride from 100% to 0.

After hydrophobic chromatography, the third stage of chromatographic purification of hGH is carried out on an axial column packed with Q Sepharose FF ion-exchange sorbent (GE Healthcare, USA). The protein solution was transferred by gel filtration on a Sephadex G-25 Fine gel (GE Healthcare, USA) into a buffer solution containing 50 mM Histidine, pH 6.5. Then, the resulting protein solution is applied to a column with an ion-exchange sorbent balanced with a starting buffer solution that contains 50 mM Histidine, pH 6.5. Elution of protein from the column is a linear gradient of the concentration of sodium chloride from 0-30%.

The final stage of chromatographic purification of hGH is carried out on a column filled with Sephacryl S-100 HR gel (GE Healthcare, USA). Pre-balance the column with a buffer solution that contains 20 mm Na ₂ HPO ₄ , 0.2% glycine, 4% mannitol, pH 6.8.

The result is 0.45 g rhrh with a purity by HPLC 98-99% of 1 g of the target protein obtained from inclusion bodies.

The application of the proposed invention has the following advantages.

- Solubilization of Met-rGHC is carried out in 2 M urea at pH 11.0, which allows you to save the formed secondary structure of the protein at the stage of formation of inclusion bodies in the E. coli cell. The use of higher pH values leads to the conversion of the Cys ^182- Cys ¹⁸⁹ disulfide bond to a thioether bond, which in turn entails the formation of related impurities that are difficult to separate under industrial production conditions and a decrease in the activity of the target product.

- The process of protein renaturation is carried out at a concentration of 1.2 mg / ml, which allows this stage to be carried out in small volumes. For the first time, hydrogen peroxide is used as an oxidizing agent of sulfohydryl groups of a protein. Under such conditions, the time of the renaturation process was reduced to 2 hours at an industrial scale of production.

- Thanks to the selected conditions, the yield of the target protein after the renaturation process was able to increase to 75-80%.

- The stages of protein concentration before chromatographic purification by precipitation of the protein with 50% ammonium sulfate solution are completely excluded.

- The stage of protein purification on a reverse-phase sorbent has been replaced by a stage of purification using hydrophobic chromatography, which has led to a significant simplification and cheapening of the process.

The invention is illustrated by the following figures.

Figure 1 shows the amino acid sequence of hGH.

Figure 2 presents the control of the cleavage of the N-terminal methionine rGH using RP HPLC

Figure 3 presents the analysis of hGH by RP HPLC after solubilization, renaturation.

The invention is illustrated by the following examples.

Example 1. Obtaining highly purified hRHC.

4 L of a buffer solution containing 20 mM Tris-HCl, 2 M urea, pH 11.0 was added to 200 g of wet bodies of rhGH inclusion, and left to mix for 30 min at room temperature. The protein solution obtained after solubilization is diluted with a buffer solution containing 20 mm Tris-HCl. The pH of the solution was adjusted to 8.0, 37% H ₂ O _{2 was} added. Then the reaction mixture was allowed to stir at room temperature for 2 hours.

The amount of correctly laid protein is monitored by RP-HPLC on a YMC C4 column. The first stage of chromatographic purification of hGH is carried out at room temperature on an axial column of the BPG series 140 × 500 mm filled with ion exchange sorbent Q Sepharose FF. The protein solution after renaturation is applied to the column, balanced with the starting buffer solution, which contains 20 mm Tris-HCL, pH 8.0. The load on the sorbent can reach 80 mg of total protein per 1 ml of carrier. After applying the entire volume of protein, the sorbent is washed with three volumes of the starting buffer solution. The elution of the protein from the column is carried out by a linear gradient of the concentration of sodium chloride from 0 to 40%. Protein detection is carried out at a wavelength of 280 nm. The fractions containing the target protein, according to the results of analysis on a reversed-phase C ₄ column, are combined.

In the next step, to the solution of Met-rGHC obtained after purification with Q Sepharose FF, 7.5 leucine aminopeptidase was added. per 1 g of protein. The cleavage reaction is carried out for 16 hours at room temperature. The reaction is monitored by isocratic RP HPLC on a Symmetry 300, C ₁₈ , 4.6 × 250 mm, 5 μm, 300 A column (Waters, USA), FIG. 2.

After cleavage of the N-terminal methionine, rGHC chromatographic purification was performed using hydrophobic chromatography at room temperature on an axial column of the BPG series 140 × 500 mm filled with Butyl Sepharose 4 FF. After ion exchange chromatography, sodium chloride is added to the protein solution to a final content of 2 M, the pH is adjusted to 7.0, and applied to the column balanced with the starting buffer solution. After applying the entire volume of protein, the sorbent is washed with three volumes of the starting buffer solution, which contains 20 mm Tris-HCL, 2M NaCl pH 7.0. Elution of the protein from the column is carried out by a decreasing linear gradient of the concentration of sodium chloride 100 to 0%. Protein detection is carried out at a wavelength of 280 nm. The fractions containing the target protein, according to the results of analysis on a reversed-phase column With ₄ , are combined.

The combined protein fraction was transferred by gel filtration on Sephadex G-25 Fine gel to a buffer solution containing 50 mM Histidine, pH 6.5. Then, the resulting protein solution is applied to the column with the ion-exchange sorbent Q-Sepharose FF, balanced with the starting buffer solution, which contains 50 mm Histidine, pH 6.5. Elution of protein from the column is a linear gradient of the concentration of sodium chloride from 0-30%. Protein detection is carried out at a wavelength of 280 N. m. Fractions containing the target protein according to the results of analysis on a reversed-phase column With ₄ are combined.

The final stage of chromatographic purification of hGH is carried out on a column filled with Sephacryl S-100 HR gel. Preliminarily, the column is equilibrated with a buffer solution that contains 20 mM Na ₂ НРО ₄ , 0.2% glycine, 4% mannitol, pH 6.8. Next, gel filtration of the obtained protein solution is carried out after the third stage of chromatographic purification. Protein detection is carried out at a wavelength of 280 nm. In the obtained protein fraction, the concentration is determined by reverse phase chromatography on a YMC, C4 column.

The yield is 0.45 g of highly purified hGH with 1 g of the target protein obtained by solubilization from inclusion bodies. Protein purity - not less than 98%. The material balance of the industrial process of cleaning hrh is presented in table 1.

Table 1 The material balance of the main stages of the industrial process of cleaning hrh STAGES OF CLEARING OF HRCG EXIT RGRG, g RGRG OUTPUT,% Chromatographic purity rhr,% CONTENT Protein E. coli, ng / mg _protein Endotoxins, U / mg _protein Protein extraction from 20 g inclusion Taurus 40 one hundred - > 250 > 250 Renaturation 30-32 75-80 75-80 Ion exchange chromatography on 26-31 66-77 85-90 50-100 100-250 Q Sepharose FF, pH8.0 Hydrophobic chromatography on Butyl Sepharose 4 23-25 58-63 89-95 FF Gel Filtration on the Sephadex G-25 22-23 55-59 Ion exchange 1-3 ≤1 chromatography on 18-20 46-50 95-99 Q Sepharose FF, pH 6.5 Gel filtration on Sephacryl S-100HR 17-18 44-47 97-99

Example 2. Control of the cleavage of the N-terminal methionine of hGH using RP-HPLC on a Symmetry 300 column, C ₁₈ .

Chromatography was performed isocratically on a reversed-phase column Symmetry 300, C _18, 5 micron, 300 A (4,6 × 250 mm) (Waters, USA). The mobile phase contains 0.5 M KN ₂ PO ₄ , pH 6.5, 24% 1-propanol. The flow rate is 0.5 ml / min, the temperature is 45 ° C, and the wavelength is 220 nm. As an external standard, a European Standard (CRS) with a known concentration is used. The product elutes with one peak (figure 2), the purity of the product is at least 98%.

Example 3. The analysis of hGH using RP HPLC on a YMC column, C4.

The analysis of the obtained hGH is carried out using reverse phase chromatography in a concentration gradient of 1-propanol: acetonitrile (20:80) from 30 to 50% in the presence of 0.1% TFA on a YMC C ₄ column, 250x4.6 mm, 5 μm, 300A (YMC Co., LTD, Japan) on an Agilent 1200 series chromatograph (Agilent Corporation, USA). The product elutes with one peak (figure 3), the purity of the product is at least 98%.

Example 4. Scaling of the purification process of somatropin rGRC.

The data shown in table 2 were obtained on 10 pilot industrial series of the drug.

table 2 STAGES OF CLEARING OF HRCG EXIT RGRG, g RGRG OUTPUT,% Chromatographic purity hrh,% CONTENT Protein E. coli, ng / mg _protein Endotoxins, U / mg _protein Protein extraction from 2000 g inclusion Taurus 400 one hundred - > 250 > 250 Renaturation 300-320 75-80 75-80 Ion exchange chromatography on Q Sepharose FF 265-310 66-77 85-90 50-100 100-250 Hydrophobic chromatography on Butyl Sepharose 4 235-250 58-63 Ff 89-95 Gel filtration on the Sephadex G-25 220-237 55-59 Ion exchange 1-3 ≤1 chromatography on Q Sepharose FF 187-200 46-50 95-99 Gel filtration on Sephacryl S-100 HR 172-185 44-47 97-99

Claims (1)

1. The method of obtaining and purification of recombinant human growth hormone, comprising the following stages:
1) dissolution of inclusion bodies in 2M urea at pH 11;
2) protein renaturation in a buffer solution of 20 mM Tris-Hcl at pH 8.0 using hydrogen peroxide protein as an oxidizing agent;
3) ion exchange chromatography on Q Sepharose FF, pH 8.0;
4) cleavage of the N-terminal methionine with leucine aminopeptidase;
5) hydrophobic chromatography on Butyl Sepharose 4 FF;
6) gel filtration on Sephadex G-25;
7) ion exchange chromatography on Q Sepharose FF, pH 6.5;
8) gel filtration on Sephacryl S-100HR.

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