EA044422B1 - METHOD FOR CLEANING IFR-1 - Google Patents

Description

Field of technology to which the invention relates

The invention relates to the production of IGF-1 protein for the needs of the pharmaceutical and veterinary industries and other areas where high-quality pure IGF-1 protein is required. In particular, a method for purifying the IGF-1 protein and a protein preparation obtained by this method are disclosed.

Information about the prior art

IGF-1 (insulin-like growth factor 1, somatomedin C, IGF1, insulin-like growth factor 1) is a hormone from the family of insulin-like growth factors, similar in structure and function to insulin. It is involved in the endocrine, autocrine and paracrine regulation of the processes of growth, development and differentiation of cells and tissues of the body, primarily bone and muscle tissue, and also has various other functions. The protein is used as a marker to help diagnose childhood growth disorders and acromegaly and monitor growth hormone replacement treatment. In addition, IGF-1 is used as a drug for long-term therapy for growth disorders in children and adolescents (Increlex, Ipsen Biopharmaceuticals, Inc), and the possibility of its use as a neuroprotective agent for stroke has also been shown (RU2711111, WO 93/08828) and for the treatment of difficult-to-manage wounds (Garoufalia Z, et al. Insulin-like growth factor-I and wound healing, a potential answer to non-healing wounds: A systematic review of the literature and future perspectives. Biomed Rep. 2021 Aug; 15(2):66).

IGF-1 preparations are used by athletes to gain muscle mass and increase strength indicators and are used in veterinary medicine, for example, to increase the weight gain of cattle, small ruminants, and pigs. IGF-1 is included in cosmeceutical products designed to combat signs of skin aging (DermaHeal Cosmeceutical Mask Pack, YBK Investment, Inc.).

In modern industry there is an increasing need to obtain IGF-1 protein in large quantities with acceptable quality.

In industry, IGF-1 is produced by biotechnology using various expression systems and using recombinant nucleic acid molecules. Typically, bacteria, such as Escherichia coli, and yeast, such as Pichia pastoris, transformed with a plasmid encoding IGF-1 are used. The goal of maximizing protein yield is to overexpress IGF-1 in bacteria, resulting in protein aggregation into inclusion bodies that contain tightly packed target protein (and small amounts of other proteins) and are easily separated from cells. The problem is that the protein in the inclusion bodies does not have the correct tertiary structure, and the inclusion bodies themselves are insoluble, and therefore they must be solubilized under conditions that lead to denaturation of the protein, and then subjected to folding (refolding, folding), i.e. i.e. folding into the correct tertiary structure.

Unlike bacteria, yeast tend to secrete the protein they produce into the culture medium, which reduces problems with misfolded protein. However, this is a more difficult producer to optimize, giving on average a lower yield of the target protein.

Denaturation (solubilization) and folding technologies differ in overall yield, in the yield of protein of the required conformation, in the ability to scale the process, and in cost-effectiveness. There is no one-size-fits-all approach for all target proteins; during the folding process, in addition to correct folding, incorrect folding and aggregation of the protein may be observed, and, therefore, fine tuning of this process is required to increase efficiency and yield (Eiberle M., Jungbauer A. Technical refolding of proteins: Do we have freedom to operate? Biotechnology Journal , 2010, 5 (6), pp. 547).

Solubilization is achieved by using, for example, chaotropic agents such as urea or guanidine salts in high concentrations. This is followed by folding, in which conditions are created that promote proper folding and the formation of disulfide bonds between the target amino acid residues. For this purpose, various agents are used, including urea and guanidine salts (but in lower concentrations), cyclodextrins, ethyl ammonium nitrate, etc. The folding buffer must contain a redox component (redox couple) to ensure the formation of disulfide bonds , for example oxidized-reduced glutathione, cysteine-cystine, etc.

There is a known method for producing recombinant IGF-1 in Escherichia coli, in which the bacterium is transformed with the IGF-1 encoding plasmid pET15, the bacterium is cultivated, inclusion bodies are isolated, solubilized in a buffer with urea for 12 hours, and folded with a buffer containing a redox couple. glutathione-glutathione disulfide, then centrifuged and purified by gel filtration (Jafari S. et ai., Recombinant production of mecasermin in E. coli expression system. Res Pharm Sci. 2014 Nov-Dec;9(6):453-61) . This document does not disclose details regarding the yield and activity of the resulting protein.

US5650496 discloses the production of properly folded IGF-1 in the yeast Pichia pastoris using a method including cation exchange resin protein purification, unfolding/folding, hydrophobic interaction chromatography, cation exchange resin protein purification, reverse phase chromatography, gel permeation chromatography, diafiltration. The preferred unfolding/folding buffer composition includes 2 M urea, 1.5 mM sodium chloride, 15% ethanol, 5 mM sodium borate and 0.2 mM dithiothreitol at pH 9.0-9.5. This method is labor-intensive and costly; In addition, the effectiveness of this method when working with inclusion bodies is unclear.

There remains a need for methods for purifying recombinant IGF-1 with higher efficiency, high overall protein yield, high yield of properly folded functional protein, cost-effectiveness and scalability, and versatility with respect to raw materials. In addition, a method is needed that is suitable for purifying the protein produced in the bacterial cell.

The essence of the invention

The above problems are solved by the present invention, namely, a method for purifying the IGF-1 protein, including:

a) protein denaturation using a solubilizing buffer containing 7-8 M urea and 210 mM dithiothreitol, with a pH of at least 12.5;

b) protein folding;

c) chromatographic purification;

d) changing the buffer solution;

wherein the folding step (b) includes:

b1) mixing the mixture obtained in step (a) with a folding buffer in a ratio of 1:2-1:3 v/v, wherein the folding buffer contains water, a stabilizer, which is sugar or a polyol, and propanol; and with a redox component of cysteine and cystamine, which is contained in said folding buffer or added separately; and incubating the resulting mixture for at least 5 days;

b2) diluting the mixture obtained at stage (b1) with acetate buffer in a ratio of 1:3-1:3.5 v:v. so that the pH is 5.4-5.7;

b3) adding hydrogen peroxide to the mixture obtained in step (b2) at a concentration of 0.03-1.96 mmol/l, incubating for at least 30 minutes;

b4) centrifuging the resulting mixture and filtering the supernatant, which is then sent to step (c).

The proposed method provides the following advantages:

greater product yield;

the product is characterized by a smaller number of incorrectly folded forms of IGF-1, i.e., a larger proportion of correctly folded IGF-1;

it is possible to obtain protein in high concentrations;

reduced content of producer impurities (proteins, DNA and/or LPS).

In addition to the above, the purification method is cost-effective, since it allows, given the existing protein yield, to spend less reagents, use less valuable reagents (for example, avoid the use of arginine), and carry out the process at room temperature.

In particular, high yield is achieved by reducing losses, primarily precipitation, at the stages of denaturation, folding and chromatographic purification, and high yield of the correctly folded form of IGF-1 is achieved due to the essential folding conditions indicated above.

In preferred embodiments, the method is characterized by one or more of the following features:

propanol is isopropanol;

sugar is sucrose;

step (c) includes chromatographic purification on sulfopropyl sepharose and butyl sepharose;

step (d) of changing the buffer solution is performed by ultrafiltration, size exclusion chromatography or dialysis;

the concentration of each of cysteine and cystamine in the mixture formed in step (b1) is 1-4 mM;

The pH in step (b1) is 10.5.

The invention also relates to an IGF-1 product, which is obtained by the method according to the invention and is, without limitation, a pharmaceutical, veterinary and/or cosmeceutical substance or laboratory reagent; and to the use of said product for medical, veterinary, and/or cosmeceutical purposes, or for scientific research.

In addition, the present invention relates to a method for producing an IGF-1 product, comprising producing IGF-1 in a producer cell, such as a bacterial, yeast, plant or animal cell, and purifying using the purification method described above. The preferred producer cell is a bacterial cell, most preferably Escherichia coli.

List of graphic materials

Fig. 1: Diagram showing protein yield (vertical axis, g) as a function of folding time (horizontal axis, day) for the method according to the invention in comparison with a method in which

- 2 044422 do not use the cysteine-cystamine pair, and in a manner that does not use hydrogen peroxide. The initial mass of protein supplied for purification was 10 g.

Fig. 2: HPLC chromatogram for the product obtained by the method according to the invention (horizontal axis - retention time, min; vertical axis - millioptic units).

Fig. 3: HPLC chromatogram for the product obtained by the comparative method (horizontal axis - retention time, min; vertical axis - millioptic units).

Fig. 4: Study of the activity of the protein purified by the method according to the invention. A and B: experiment with the conversion of resazurin to resofurin, B - visually, A - assessment using a fluorimeter (vertical axis - OE, optical units); B - assessment of the acceleration of cell growth (the order of the samples in Fig. 4A-4B is the same).

Detailed Description of the Invention

Synthesis of IGF-1.

Crude IGF-1 obtained by any method is suitable for carrying out the invention. Typically, IGF-1 is produced in expression systems, such as bacteria (Escherichia coli, Lactococcus lactis, Bacillus subtilis, etc.), yeast (Pichia pastoris, Saccharomices cerevisiae, etc.), animal cells (CHO cells Chinese hamster ovary), etc.) and even plants (Nicotiana tabacum plant, Oryza sativa rice callus suspension, etc.). Expression systems differ in protein yield, its activity (determined primarily by the correctness of the tertiary structure), and the labor intensity of the process. The purification method proposed in the application is fundamentally suitable for the purification of IGF-1 obtained in any of the expression systems, since efficient folding is carried out to obtain the correct tertiary structure, regardless of the initial state of the expressed protein.

Various methods for producing IGF-1 are disclosed, without limitation, in US5324639, US5650496, RU2372941, RU2711111. Additional tools and methods for producing recombinant proteins are known to one skilled in the art, for example, from Sambrook J. & Russell D. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, John & Sons, Inc. (2002); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1998); and Coligan et al., Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003).

The method according to the invention can purify various variants of IGF-1 that have biological activity of IGF-1 and are therefore suitable for use in human medicine, veterinary medicine, cosmeceuticals and scientific research. Preferably, the IGF-1 is human IGF-1 (hIGF-1), having 70 amino acid residues (7.6 kDa), also known as mecasermin (Increlex): GPETLCGAELVDALQFVCGDRGFYFNKPTGIGSSSRRAPQTGIVDECCFRSCDLRRLEMY CAPLKPAKSA (DB01277; variants of mecasermin with I at position 4 are also known or with Y at position 43 relative to the specified sequence, which can be used for purification). For example, a variant of 71 amino acid residues with the first amino acid M added (GenBank: CAA01954.1), or des(1-3)IGF-1, which is a shortened version (67 amino acid residues) in which the first three are missing amino acids (GPE). In non-limiting embodiments, IGF-1 can be used with selenocysteine at positions 4, 29, 41, 43, as known from WO2018204764; or with alanine, glycine or serine at one or more positions 3, 4, 5, 10, 14, 17, 23, 24, 25, 43, 49 or 63 and any amino acid at position 7, as known from WO2000040612; or with methionine at position 43 and, optionally, with the first amino acid M added, as known from RU2711111; with isoleucine at position 4 and any other options that can be used as biologically active IGF-1.

When deciding which organism to use to produce IGF-1, one tends to lean toward bacteria, particularly Escherichia coli, since its ease of handling and high protein yield seem to outweigh other disadvantages, such as the need to getting rid of bacterial toxins and the lack of correct tertiary structure of the protein expressed in the bacteria. It is from Escherichia coli that mecasermin is obtained.

As noted earlier, overexpression in bacteria leads to the accumulation of protein in inclusion bodies, which can be quite simply isolated by cell lysis, centrifugation and washing, as is known to specialists (Palmer I, Wingfield PT. Preparation and extraction of insoluble (inclusion-body) proteins from Escherichia coli. Curr Protoc Protein Sci. 2004 Nov; Chapter 6:6.3.1-6.3.18). Lysis can be carried out, for example, by ultrasound, pressure, enzymatically, chemically and other methods, as well as combinations thereof. It is preferable to use an ultrasonic or high pressure homogenizer, even more preferably in combination with chemical or enzymatic lysis. The lysis buffer may contain, for example, benzamidine hydrochloride, DTT, Tris and EDTA; Optionally, one or more protease inhibitors may be included in the buffer.

After lysis and centrifugation, the precipitate is washed with solutions of detergents and chaotropes in low concentrations (urea or guanidine hydrochloride) with further centrifugation to obtain a precipitate containing inclusion bodies, which can then be fed into the purification method according to the invention.

In the case where the crude IGF-1 preparation is a solution, for example a culture medium in the case of secretory expression, the protein is pre-concentrated, for example by precipitation, in particular by the addition of ammonium sulfate, or by any other methods known to those skilled in the art. The precipitate can be washed by known methods before being introduced into the purification method of the invention. It is most preferable for the implementation of the invention to use inclusion bodies as the initial IGF-1 preparation to be purified. In the following presentation, inclusion bodies are mentioned only for convenience of presentation, and it is understood that they can be replaced with another IGF-1 drug. Solubilization is carried out by adding a solubilizing buffer, which is a concentrated urea solution (7-9 M, preferably at least 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7 .8, 7.9 M and/or no more than 8.9, 8.8, 8.7, 8.6, 8.5, 8.4, 8.3, 8.2, 8.1 M, preferably 7-8 M, most preferably about 8 M) in a buffer with a pH of at least 12.5 (in various embodiments may be about 12.6, 12.7, 12.8, 12.9, 13 or higher), and DTT (dithiothreitol) in an effective amount, preferably 2-10 mM. In particular, the lower limit of the DTT content range can be at least 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3 ,1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4 ,4, 4.5, 4.6, 4.7, 4.8,4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5 .7, 5.8, 5.9 or 6 mM; upper limit - no more than 9.9, 9.8, 9.7, 9.6, 9.5, 9.4, 9.3, 9.2, 9.1, 9, 8.9, 8.8 , 8.7, 8.6, 8.5, 8.4, 8.3, 8.2, 8.1, 8, 7.9, 7.8, 7.7, 7.6, 7.5 , 7.4, 7.3, 7.2, 7.1, 7, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2 , 6.1, 6, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1 or 5. In a preferred embodiment, DTT content in buffer is 2, 3, 4, 5, 6, 7, 8, 9 or 10 mM. DTT can be added to the solubilization buffer before the IGF1 preparation to be purified is mixed with the buffer; in an alternative embodiment, the drug is first mixed with a buffer containing urea and then, after careful resuspension, DTT is added. The buffer can be based on an aqueous solution of Tris HCl or another substance known to specialists for such purposes. The pH of the buffer is adjusted to the above value using a suitable alkali, such as, but not limited to, sodium or potassium hydroxide.

The ratio of the protein drug and the solubilizing buffer is not fundamentally limited and is selected in such a way as to achieve effective solubilization, for example, at least 10,15,20, 25, 30, 35,40 ml per 1 g of the drug.

The mixture of the IGF-1 drug with the solubilizing buffer is incubated with stirring for at least half an hour, for example, at least 45 minutes, 1, 1.5, 2, 2.5 or 3 hours. The purpose of this stage (denaturation stage) is to dissolve as completely as possible protein preparation with destruction of its spatial structure.

At the next stage (fodding stage), the solution obtained at the previous stage is mixed with the folding buffer in a ratio of 1:2-1:3 (by volume). The folding buffer contains water, a stabilizer, which is a sugar or polyol, and propanol.

The sugar may be selected from monosaccharides or disaccharides, for example sucrose, trehalose, galactose, lactose, glucose, or mixtures thereof. The polyol may be a diol, triol, tetraol, pentaol, hexaol, or mixtures thereof; in non-limiting embodiments, the polyol is selected from, for example, glycerol, propylene glycol, erythritol butanediol, pentaerythritol, xylitol, sorbitol, mannitol, ribitol, or mixtures thereof. Preferably, the stabilizer is selected from glycerol, sucrose, glucose, trehalose or a mixture thereof, most preferably sucrose. The stabilizer is used in an effective amount known to the skilled person (Butler SL, Falke JJ. Effects of protein stabilizing agents on thermal backbone motions: a disulfide trapping study. Biochemistry. 1996 Aug 20; 35(33): 10595-600). The amount of stabilizer in the folding buffer is preferably from 1 mM to 5 M, for example at least 2, 3.4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, 60, 70, 80, 90 , 100, 200, 300,400, 500, 600, 700, 800 or 900 mM, 1.2 or 3 M; for example not more than 4, 3, 2 or 1 M; and most preferably is 1 M. The propanol may be isopropanol, n-propanol, or a mixture thereof. The amount of propanol can be 30-50% (v/v), for example at least 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 , 47, 48, 49, 50%; for example no more than 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31%.

The redox component is selected from the cysteine-cystamine pair. The concentration of each substance in the mixture of the folding buffer with the solution obtained at the solubilization stage is usually 0.1-10 mM, for example from 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 mM or higher ; for example 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1 .5, 1.4, 1.3, 1.2, 1.1 or 1 mM or lower.

Preferably, 1-4 mM of each is used, most preferably 1 mM of each. The ratio between cysteine and cystamine is not fundamental and can range from 1:4 to 4:1, for example 1:4,1:3, 1:2,1:1,2:1,3:1 or 4:1. The cysteine-cystamine pair can be pre-added to the folding buffer, which is then mixed with the solution obtained from the solubilization step. Typically, in such an embodiment, the buffer is used immediately after manufacture. In an alternative embodiment, the cysteine-cystamine pair is added during or after mixing the solution obtained from the solubilization step with the folding buffer. The pH of the folding buffer is adjusted to be in the alkaline range, preferably greater than 10, for example 10-11, in particular

- 4 044422

10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8 or 10.9. If cysteine-cystamine is added separately to the folding buffer, in one embodiment the pH is adjusted, if necessary, before adding cysteine-cystamine.

The mixture obtained at the folding stage is incubated for at least 5 days. In particular, the duration of incubation can be 5, 6, 7, 8, 9 or 10 days. Incubation is preferably carried out at room temperature and constant stirring.

After incubation, the resulting mixture is diluted with acetate buffer in a mixture:buffer ratio of 1:3-1:3.5 (by volume). In this case, the pH is controlled so that it is no more than 5.7, preferably in the range of 5.4-5.7, for example 5.4, 5.5, 5.6 or 5.7, adjusting it by adding, for example, acetic acid after mixing the mixture and buffer. The acetate buffer can be made, for example, based on an aqueous solution of sodium acetate. Preferably, a surfactant (surfactant), preferably a nonionic surfactant, is additionally added to the buffer, in particular fatty alcohol ethoxylates, fatty acid ethoxylates, alkylphenol ethoxylates, ethoxylated fatty acid amines or amides, fatty acid esters of sorbitol, fatty acid esters of glycerol, fatty acid esters acids and polyhydroxy compounds, poloxamers, etc. Surfactants. Non-limiting examples of surfactants include the Tween family, such as Polysorbate-20, or Triton X, such as Triton X-100. Polysorbate-20 is most preferred. The amount of surfactant in the acetate buffer may be, but is not limited to, 0.05-5%, for example 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0. 8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0% or within any subrange formed by any of the specified values.

After the dilution stage, hydrogen peroxide is added to the mixture in an amount that ensures a hydrogen peroxide concentration of 0.03-1.96 mmol/l in the mixture. In particular, this concentration can be from 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0, 55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.05, 1.1, 1.15, 1, 2, 1.25, 1.3, 1.35, 1.4, 1.45, 1.5, 1.55, 1.6, 1.65, 1.7, 1.75, 1.8, 1.85 or 1.9; and/or up to 1.9, 1.85, 1.8, 1.75, 1.7, 1.65, 1.6, 1.55, 1.5, 1.45, 1.4, 1, 35, 1.3, 1.25, 1.2, 1.15, 1.1, 1.05, 1, 0.95, 0.9, 0.85, 0.8, 0.75, 0, 7, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15 or 0.1. When using a 30% aqueous solution of hydrogen peroxide, you can be guided by a hydrogen peroxide solution:mixture ratio of 1:250000 to 1:5000 (v/v), for example 1:250000, 1:240000, 1:230000, 1 :220000, 1:210000, 1:200000, 1:190000, 1:180000,1:170000, 1:160000, 1:150000, 1:140000, 1:130000, 1:120000, 1:110000, 1:1 00000 , 1:90000, 1:80000, 1:70000, 1:60000, 1:50000, 1:40000, 1:30000, 1:20000, 1:10000 or 1:5000 or in any subrange formed by any of the above values. The use of a solution of a different concentration of hydrogen peroxide is within the scope of the present invention, and the above ratio will be proportionally changed, as known to those skilled in the art. After adding hydrogen peroxide, incubate the resulting mixture for at least 30 minutes; in some embodiments, the duration may be at least 45 minutes, 1, 1.5, 2, 2.5, or 3 hours. It appears that the addition of hydrogen peroxide avoids sticking of the protein on the column, thereby further increasing yield.

After incubation, the resulting mixture is centrifuged, and the supernatant is separated and filtered.

The resulting solution is sent to the stage of chromatographic purification (HPLC). The choice of chromatographic purification methods is not essential for implementing the method according to the invention and remains at the discretion of the specialist. The types of chromatographic methods for the purification of recombinant proteins, the reagents and equipment used are well known to specialists in this field; the practical aspects of these methods are covered, for example, in the Protein Purification Handbook. GE Healthcare Bio-Sciences AB 18-1132-29 AD 02/2007. The general approach in protein purification is to minimize the number of steps (since loss of the target protein is inevitable at each step) and to use different techniques if more than one step is used. It should be noted that the selection of purification methods may depend on: (a) the nature of the impurities, which in turn depends on the initial protein preparation supplied to the method for purification; and (b) applications of the purified protein. For example, when producing protein in bacterial systems, the resulting drug may contain pyrogens - bacterial exo- and endotoxins, the content of which must be controlled if the protein is to be used in medicine or veterinary medicine, especially in the form of forms for parenteral administration. As a rule, the level of lipopolysaccharides (LPS) is monitored using, for example, the LAL test (OPS. 1.2.4.0006.15 Bacterial endotoxins).

In various embodiments of the method according to the invention, the following chromatographic techniques can be used: ion exchange (IEX), gel filtration (or (size) exclusion; GF or SEC), hydrophobic interaction (GC, HIC), reverse phase (RPC, RPC), affine (AC, AX) and other types. The invention includes 1 or more than one (2, 3 or 4) chromatographic purification steps, and/or 1 or more than one (2, 3 or 4) types of chromatographic purification. Preferably, the number of chromatographic purification steps and/or types is minimized, for example 1 or 2 steps/types. Most preferably, 2 purification steps are used, representing 2 types of chromatographic purification, for example, ion exchange chromatography and hydrophobic interaction chromatography or any combination of any two of the above techniques. Even more preferably, IOC is carried out first, then GC. The above-mentioned one type of chromatographic purification also means the use of different types of mobile and/or stationary phases, for example, in the case of IOC, the use of different ion exchangers (for example, strong/weak).

In the case of IOC, the stationary phase may be selected from, without limitation, diethylaminoethylcellulose (DEAE cellulose), quaternary aminoethyl Sephadex (QAE Sephadex), quaternary ammonium Sepharose (Q Sepharose), carboxymethylcellulose (CM cellulose), sulfopropyl Sepharose (SP Sepharose ), polystyrene/divinylbenzene polymer with methyl sulfate groups (SOURCE C), etc. Preferably, SP Sepharose is used.

In the case of GC, the stationary phase has, without limitation, phenyl, octyl, butyl, etc. groups, the preferred stationary phase being butyl sepharose.

Various examples of chromatographic purification of IGF-1 are known to specialists in this field (see, for example, Ishchuk S. A. et al. Preparation of recombinant insulin-like growth factor-1 and its effect on neuroblastoma cells in vitro. Medical academic journal. 2018. Volume 18 No. 4).

After the chromatographic purification stage, the buffer solution in which IGF-1 is located is changed by any method known to specialists to obtain an IGF-1 solution suitable for use for any purpose. In non-limiting embodiments, any of the following methods may be used: ultrafiltration, dialysis, size exclusion chromatography or other method and combinations thereof (Flickinger, M.C., Reis, R.V. and Zydney, A.L. (2010). Protein Ultrafiltration. In Encyclopedia of Industrial Biotechnology, M.C. Flickinger ( Ed.); Size Exclusion Chromatography. Principles and methods. Cytiva. CY12707-03Dec20-HB; Andrew, S. M., Titus, J. A. and Zumstein, L. (2001), Dialysis and Concentration of Protein Solutions. Current Protocols in Toxicology, 10: A.3H.1-A.3H.5). The final composition of the IGF-1 solution depends on the purpose of the protein being obtained, and may consist of (in addition to protein) water or a suitable buffer (phosphate, acetate, etc.). In various embodiments, the IGF-1 solution may also optionally contain a surfactant (eg, Tween-20) and/or a preservative (eg, benzyl alcohol). IGF-1 (i.e. preparation, solution, product, composition of IGF-1) obtained by the purification method according to the invention is suitable for any purpose, for example, storage, processing, production of pharmaceutical, veterinary, cosmeceutical, laboratory and other drugs, including by lyophilization; or direct use as a biologically active substance. The present invention also relates to the IGF-1 preparation obtained by the purification method according to the invention, and to the use of this preparation in medicine, the sports industry, veterinary medicine, cosmeceuticals, and laboratory research.

Unless otherwise defined in this application, scientific and technical terms used in connection with the present invention have meanings customary in the art and understood by those skilled in the art. In general, the nomenclature and methods used in cell and tissue culture, molecular biology, microbiology and protein and nucleic acid chemistry, hybridization, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described in this application represent nomenclature and methods well known and commonly used in the art.

The terminology used herein is intended for the purpose of describing specific embodiments of the invention and is not limiting. Whenever the singular number appears in the present specification and claims, it is also to be understood to alternatively imply the plural unless the context clearly indicates otherwise.

Any numerical values disclosed herein are subject to variation within the measurement uncertainty associated with the methods and equipment typically used for each specific parameter. Thus, numerical values can also be considered to be characterized by the term approximately, about, or roughly.

The terms including, containing, having, etc. mean that in addition to the specified elements (for example, ingredients), other unspecified (non-essential) elements may be contained. In each case, these terms also include the meaning consisting of, i.e., in one embodiment, they imply that the object described is characterized by the presence of only the specified elements.

Information confirming the possibility of implementing the invention.

The examples presented below do not limit the scope of the claims, which are determined by the claims. One skilled in the art will appreciate that, subject to the limitations of the claims, the invention is capable of various embodiments, all of which are included within the scope of protection provided by the patent.

Example 1. Preparation and purification of recombinant IGF-1.

Obtaining biomass containing IGF-1.

IGF-1 was obtained (GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGMVDE CCFRSC DLRRLEMYCAPLKPAKSA) by the method described in RU2711111: E. coli BL21 Star (DE3) cells (Invitrogen, USA) were transformed with a vector based on the pET28a(+) plasmid containing an insert encoding IGF-1 1; biomass was cultivated in bioreactors with a volume of 40 and 300 l. During cultivation, indicators of optical density, pH, pO ₂ were monitored. Cells are pelleted by flow centrifugation. The amount of target protein in the biomass was checked by electrophoresis and only

- 6 044422 biomass with satisfactory analysis results.

Isolation and washing of inclusion bodies.

For all work, highly purified water with a conductivity of less than OD µS/cm was used. All weighing procedures were carried out using laboratory scales with a measurement accuracy of 0.01 g.

When preparing solutions, the volumes of added reagents were measured using graduated cylinders.

A sample of biomass was placed in a container containing the first washing buffer (in a biomass:buffer ratio of 1:10 (g:ml)) and resuspended until homogeneous using a submersible mixer, avoiding foaming. Cell disintegration was carried out using a sonicator. The suspension was centrifuged at 12,000 rpm, and at the end of centrifugation, the body pellet was placed in a container containing a second wash buffer (body:buffer ratio of 1:10 (g:ml)) and homogenized using a submersible mixer until smooth.

Washing buffers contain 2 M urea, 100 mM TpucHCI, pH 7.0, 5 mM EDTA, 5 mM DTT, 2% Triton X-100.

Solubilization and folding.

The washed bodies were weighed and then carefully resuspended in a solubilizing solution containing 8 M urea and 25 mM TpucHCI at pH 12.5, in a body:buffer ratio of 1:20 (g:ml). Then 5 mM DTT was added to the mixture and incubated with stirring for at least half an hour until complete dissolution. A folding buffer containing 1 M sucrose, 40% isopropanol in a ratio of 1:3 (mgml) was added to the resulting mixture, the pH was adjusted to 10.5 with sodium hydroxide, then 1 mM cysteine and 1 mM cystamine powder were added (concentration relative to the final volume of the mixture ). The resulting folding mixture was incubated at room temperature and constant stirring for at least 5 days (the total duration of the experiment was 8 days), and a sample was taken for HPLC once a day to monitor the folding process.

After folding, the mixture was diluted with stirring with a dilution buffer containing 15 mM sodium acetate, 1% polysorbate-20, in a mixture:buffer ratio of 1:3 (ml:ml). The pH of the resulting mixture was adjusted to a value in the range of 5.4-5.7 using acetic acid. Then 30% hydrogen peroxide was added in a ratio of 1:5000 (ml:ml) and incubated at room temperature with stirring for 30 minutes.

In parallel, control experiments were carried out: in one of them, folding was carried out without the use of a redox couple, and in the other, the method according to the invention was carried out, but without the addition of hydrogen peroxide. The daily output in each case is shown in Fig. 1. As can be seen, the addition of hydrogen peroxide significantly affects the protein yield, apparently ensuring reversibility of protein binding to the column stationary phase and its more efficient elution. Folding time (more than 5 days) significantly reduces protein loss at subsequent stages, providing high yield. The use of cysteine-cystine allows the protein yield to be increased to a greater extent.

The resulting suspension was centrifuged at 3700 rpm, the supernatant was separated and filtered through fabric and capsule filters.

Chromatography.

IGF-1 was purified on a chromatography column containing SP Sepharose Fast Flow as follows. The column (SP Sepharose Fast Flow sorbent volume is 270 ml, 1 ml of sorbent binds 50 mg of protein) at a flow rate of 40 ml/min was washed with 300 ml of purified water to remove the ethanol solution, then with 1 M NaOH, then again with water. The column was washed with 200 ml of elution buffer (buffer B (15 mM sodium acetate, 1% Tween-20, 1 M NaCl, pH 5.4) at a flow rate of 40 ml/min. The column was equilibrated with 300 ml of starting buffer (buffer A (15 mM sodium acetate, 1% Tween-20, pH 5.4), at a flow rate of 40 ml/min. Apply the protein solution (filtered supernatant from the previous step) to the column at a flow rate of 40 ml/min using a sampling pump. Wash the column 300 ml of starting buffer at a flow rate of 40 ml/min. Then, elution was carried out in a gradient from 0 to 50% of elution buffer B over 35 minutes at a flow rate of 40 ml/min. The peak corresponding to the target protein was collected in a 500 ml container and the result was measured volume. A sample was taken for analysis by spectrophotometry and the amount of protein obtained was assessed. The column was regenerated using the CIP program for SP Sepharose (2 M NaCl, 1 M NaOH) and preserved. Optical density was measured at a wavelength of 280 nm.

Next, the protein was depyrogenated on a chromatographic column containing butyl Sepharose as follows. The column (sorbent volume is 314 ml) was washed at a flow rate of 20 ml/min with 400 ml of purified water, then with 30% isopropanol and water, then with 1 M NaOH, then again with water, and the column was equilibrated with 500 ml of buffer A (1 M ammonium sulfate , 50 mM TpucHCl, pH 8) at a flow rate of 20 ml/min. Before application to the column, the protein solution obtained in the previous step was diluted to a concentration of 5 mg/ml with 3 M ammonium sulfate and 1 M TpucHCl (pH 8) so that the final solution contained 1 M ammonium sulfate and 50 mM TpucHCI. The resulting protein solution was applied to the column at a flow rate of 20 ml/min using a sampling pump. The column was washed with 400 ml of starting buffer (A) at a flow rate of 20 ml/min. The protein was washed away with 100% elution buffer B (15 mM sodium acetate, 1% Tween-20, pH 5.4) for 35 minutes at a flow rate of 20 ml/min. Collected the peak

- 7 044422 corresponding to the target protein, into a 500 ml container and the volume of the resulting protein was measured. A sample was taken for analysis using spectrophotometry and the amount of protein obtained was assessed. The column was regenerated using the CIP program for butyl Sepharose (1 M NaOH) and preserved. Optical density measurements were carried out at a wavelength of 280 nm.

Buffer change.

The protein solution obtained at the previous stage was placed in a dialysis bag with a pore diameter of at least 2 times smaller than the protein size. The protein bag was placed in a large container with a buffer containing 15 mM sodium acetate, 1% Tween. Leave for 6 hours (3 hours of which with stirring). These manipulations were repeated 3-4 times and a protein solution essentially free of ammonium sulfate was obtained. Samples were diluted in a buffer containing 15 mM sodium acetate, 1% Tween, 0.5% benzyl alcohol to a concentration of 5 mg/ml, then filtered through an antibacterial filter into clean dark-colored jars. The resulting preparation was stored at +4°C. A sample was taken for quality control according to the FSP, which included determining the protein concentration by HPLC, determining the amount of proteins of the producer strain, determining the amount of DNA of the producer strain, and determining the residual amount of LPS using the LAL test. The product obtained by the method according to the invention meets the following indicators:

the DNA content of the producer strain is not more than 3 ng/ml, the protein content of the producer strain is not more than 8 ng/ml, the LPS level is not more than 12.5 EU/ml;

and is therefore suitable for its intended use.

HPLC chromatograms for the product obtained according to the invention and the product obtained by the conventional method are shown in FIG. 2 and 3, respectively. In FIG. 3 you can see many protein isoforms presented in approximately equal proportions, which indicates non-optimal folding; on the contrary, in Fig. 2, corresponding to the product according to the invention, one can see the predominance of one peak of the native form of the protein, to confirm which its activity was further tested.

Example 2. Study of the activity of purified protein.

First of all, the acceleration of cell growth was assessed. The effect of low concentrations of the hormone described in the literature (samples diluted to 4 ng/ml) on the growth and differentiation of the mouse neuroblastoma cell line Neuro2a was assessed. As can be seen in the micrographs (Fig. 4B), a low concentration of the hormone affects cell growth: an increase in cell volume and a change in structure are visible (processes characteristic of neurons appear), but the addition of 10% fetal serum, containing all the hormones and substances necessary for growth and differentiation, enhances the effect. Therefore, in further experiments, the hormone concentrations were increased to 100 ng/ml. Next, an experiment was carried out with the conversion of resazurin to resorufin. Resazurin is a phenoxazine redox indicator. Having a blue or violet color, when reduced it becomes a pink fluorescent resorufin. Cells, including the Neuro2a lineage, reduce resazurin during metabolism, and the metabolic activity is proportional to the level of reduction of resazurin into fluorescent resorufin. Thus, the activity of the added hormone can be determined by the intensity of cell glow in ultraviolet light. An equal amount of the hormone sample, diluted 10,000 times, was added to a plate with cells in a serum-free medium. After incubation of the cells, resazurin was added and incubated for 4 h for conversion. The glow was assessed visually.

As can be seen from the photograph (Fig. 4B), the hormone sample activates cell metabolism in relation to the sample without the addition of serum, which is visible even with the naked eye.

To more accurately assess the effect of the product on the cells, the culture medium without cells was transferred to another plate and the luminosity was measured on a BMG Clariostar fluorometer using the standard Resarufin protocol. As can be seen from the data obtained (Figure 4A), the product is highly active (higher than the serum-spiked sample) and has activity comparable to the Sigma standard.

Claims (6)

CLAIM 1. A method for purifying IGF-1 protein, including: a) protein denaturation using a solubilizing buffer containing 7-8 M urea and 210 mM dithiothreitol, with a pH of at least 12.5; b) protein folding; c) chromatographic purification; d) changing the buffer solution; wherein the folding step (b) includes: b1) mixing the mixture obtained in step (a) with a folding buffer in a ratio of 1:2-1:3 v/v, wherein the folding buffer contains water, a stabilizer, which is sugar or a polyol, and propanol; and with a redox component of cysteine and pistamine, which is contained in said folding buffer or added separately; and incubating the resulting mixture for at least 5 days; b2) diluting the mixture obtained at stage (b1) with acetate buffer in a ratio of 1:3-1:3.5 v:v. so that the pH is 5.4-5.7; b3) adding hydrogen peroxide to the mixture obtained in step (b2) at a concentration of 0.03-1.96 mmol/l, incubating for at least 30 minutes; b4) centrifuging the resulting mixture and filtering the supernatant, which is then sent to step (c). 2. The method according to claim 1, wherein the propanol is isopropanol and the sugar is sucrose. 3. The method according to claim 1 or 2, where step (c) includes chromatographic purification on sulfopropyl sepharose and butyl sepharose. 4. The method according to any one of claims 1 to 3, where step (d) of changing the buffer solution is performed by ultrafiltration, size exclusion chromatography or dialysis. 5. Method according to any one of claims 1 to 4, where the concentration of each of cysteine and cystamine in the mixture formed in step (b1) is 1-4 mM. 6. Method according to any one of claims 1 to 5, wherein the pH in step (b1) is 10.5.