RU2765951C1 - Method of purifying hyaluronate from endotoxins - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: present invention relates to chemical and pharmaceutical industry, namely to a method of purifying hyaluronate from endotoxins, which consists in the fact that a solution containing sodium hyaluronate and sodium chloride is prepared, and the hyaluronate is precipitated by centrifugation, characterized by that the solution is prepared by dissolving sodium hyaluronate in 0.45–0.5 % sodium chloride solution to obtain a solution with a hyaluronate concentration of 0.25–0.5 %, 0.5–0.75 % cetylpyridinium chloride solution is added to the solution with an amount of cetylpyridinium chloride equimolar to the amount of hyaluronate by carboxyl groups, leaving the obtained solution until maturation and coagulation of the precipitate, after which the hyaluronate is precipitated by centrifuging the mixture and the crude precipitate of cetylpyridinium hyaluronate is collected, after that, preparing 0.3–0.5 % solution of cetylpyridinium hyaluronate in dimethylsulfoxide, pre-cooled to temperature of 5–7 °C, with stirring, after complete dissolution of the precipitate, the solution is exposed to 1.25–1.5 % sodium chloride solution with an amount of sodium chloride equimolar to the amount of hyaluronate, the formed hyaluronate precipitate is collected and dissolved in 0.9 % sodium chloride solution to obtain a hyaluronate solution with concentration of 0.1–0.15 %, sodium hyaluronate solution is fed into a tangential reverse osmosis system with a membrane corresponding to the molecular weight of the initial hyaluronate, wherein 10-fold volume of 0.9 % sodium chloride solution relative to the volume of the hyaluronate solution is gradually poured into the system, the sodium hyaluronate solution passed through system in 0.9 % sodium chloride solution is concentrated to 1–2 %, filtered through a sterilization membrane and purified hyaluronate is obtained.

EFFECT: present invention ensures preservation of the initial molecular weight of hyaluronic acid (its salts), rheological properties during the purification process, ensures the purity of the end product due to the exclusion of the use of organic solvents, and also provides higher output of the end product.

3 cl, 5 tbl, 5 ex

Description

The invention relates to the pharmaceutical industry and can be used in the purification of sodium hyaluronate salt from endotoxins for medical purposes.

Endotoxins are bacterial toxic substances that are structural components of certain bacteria and are released only during lysis (decay) of the bacterial cell. This distinguishes endotoxins from exotoxins, soluble compounds secreted by a living bacterial cell.

Bacterial endotoxins consist of polysaccharide and lipid fragments. The polysaccharide fragment contains an O-specific chain (O-antigen), which includes a repeating sequence of oligosaccharide units based on glycosyl residues (up to 50), as well as a core. Lipid A and the inner core of the polysaccharide component of endotoxins are partially phosphorylated. This leads to the fact that in solutions with a neutral or basic pH, endotoxins will have a pronounced negative charge (pKa 1.3).

The molecular weight of the monomers of various lipopolysaccharides can vary within fairly wide ranges, which is explained by the variability of the O-specific chain. Known endotoxins with molecular weights from 2.5 kDa (with a shortened O-specific chain) to 70 kDa (with a very long O-specific chain). Most of the lipopolysaccharides have a molecular weight of 10 to 20 kDa.

However, it should be noted that monomeric lipopolysaccharides can form supramolecular structures due to nonpolar interactions between lipid "tails", as well as due to the formation of "crosslinks" of phosphate groups by divalent cations. Thus, in aqueous solutions, endotoxins can aggregate into lamellar, cubic, or inverted hexagonal structures such as micelles or vesicles. The diameter of such structures reaches 0.1 µm, and the molecular weight is 1000 kDa. Bivalent cations, such as Ca ²⁺ and Mg ²⁺ , contribute to the formation of supramolecular structures, while detergents, EDTA and proteins, on the contrary, shift the equilibrium towards the formation of monomeric forms.

The O-specific chain is unique to each prokaryotic strain; it contributes significantly to serological specificity by inducing an immune response in humans and animals.

The main constituents of the core are heptose residues (hexapyranose in the outer part of the core and L-glycero-D-mannoheptose in the inner part), as well as the 2-keto-3-deoxyoctonic acid group.

The lipid fragment of endotoxins or lipid A is the least variable and most conserved part. Lipid A is responsible for endotoxic activity. It manifests itself in stimulating the production and release by granulocytes and macrophages of endogenous mediators, such as bioactive lipids, NO, cytokines (for example, interleukin-1). Large concentrations of such mediators in the body can lead to many different pathophysiological reactions, such as fever, leukopenia, tachycardia, hypotension, diffuse intravascular coagulation, etc.

All of the above confirms the urgent need for the release of hyaluronate preparations from endotoxins.

Taken together, there are a number of ways to reduce the content of endotoxins in biological solutions. But in the case of proteins in particular, there are currently no standard methods for widespread use. The respective methods used are adapted to the particular properties of the respective protein and to the respective methods of obtaining that protein. There are various options for reducing endotoxins, each with specific advantages and disadvantages.

Ultrafiltration (Petsch, D. & Anspach, F.B., 2000, J. Biotechnol. 76, 97-119 and references) is used to reduce endotoxins in water and solutions with small molecular weight components such as salts, sugars and antibiotics, but not suitable for high molecular weight proteins or DNA.

Biphasic extraction (eg WO 0166718, Merck) is designed to separate water soluble proteins and DNA from endotoxin, but results in detergent residues in the purified product. In addition, this method is time consuming due to repeated repetition of the cleaning procedure.

Anion exchange (diethylaminoethyl (DEAE)-cellulose) is also used to reduce endotoxin levels in DNA and basic proteins (e.g. US 5990301, Qiagen; WO 9414837, Enzon), but it requires low ionic strength (less than 50 mM NaCl) and results in in the case of acidic proteins to co-adsorption of proteins.

Another method for reducing the content of endotoxins in DNA and proteins (for example, BSA (bovine serum albumin), myoglobin, gamma globulin, cytochrome C) is affinity adsorption (for example, polymyxin B, histamine, histidine, polylysine) according to, for example, GB 2192633 (HammersmithHospital), which, however, is toxic in the case of polymyxin B and can lead to co-adsorption of proteins in the case of low ionic strengths.

In addition, immunoaffinity chromatography is used, in which specificity for specific endotoxins can only be achieved with expensive antibodies (US 5179018, Centocor; WO 0008463, Bioserv) against measles oligosaccharide.

In addition, factor C S3 delta peptide (WO 0127289) (component of the LAL test) (WO 9915676 both: National University of Singapore) is used with proteins (eg BSA, chymotrypsinogen) and this method has low efficiency in case of high ionic strengths and also requires high production costs.

In the pharmaceutical industry, essentially three methods have found application for biopolymer solutions, adapted to the properties of the target proteins:

- anion exchange chromatography;

- reversed-phase chromatography; its disadvantage is that it is not equally suitable for all proteins, in particular problems arise in the case of hydrophobic proteins; this method, in addition, requires a very large investment of time;

- RemTox (Millipore): The disadvantage of this method is that in addition to the very long incubation time, the non-specific binding component is high and protein recovery is often inadequate.

The methods listed above for the purification of biological preparations from endotoxins involve the use of expensive equipment and chemical reagents, and also have a low yield of the target product.

Closest to the proposed is a method of purification of salts of hyaluronic acid from impurities, including endotoxins, including the following steps: obtaining a partially purified salt of hyaluronic acid from a culture of a microorganism capable of producing hyaluronic acid; contacting the partially purified salt of hyaluronic acid with a solution containing a salt, in particular sodium chloride, and a hydrophilic organic solvent to transfer the impurities contained in the partially purified hyaluronic acid into a liquid phase; and isolating the purified hyaluronic acid salt from the mixture of the solution and the partially purified hyaluronic acid salt product by centrifugation; while the microorganism capable of producing hyaluronic acid belongs to the genus Streptococcus, and the hydrophilic organic solvent is selected from methanol, ethanol, propanol, isopropanol and acetone (US 2009/0162905 A1, Hideki, Kazuo [JP], publ. 25.06.2009).

The disadvantage of this method is the impossibility of maintaining the molecular weight of hyaluronic acid during the purification process, the molecular weight of purified hyaluronic acid is halved, which greatly degrades the characteristics of the final product.

Also, the disadvantage of this method is the fact that organic solvents are used, such as methanol, propanol, isopropanol and acetone, which makes it difficult to use the final product for medical purposes without an additional purification step from the "traces" of these highly toxic compounds.

The proposed invention solves the technical problem of obtaining hyaluronate with high performance.

The technical result that allows solving this problem is to ensure the preservation of the initial molecular weight of hyaluronic acid (its salts), rheological properties during the purification process, ensuring the purity of the final product due to the elimination of the use of organic solvents, as well as increasing the yield of the final product.

The technical problem is solved by a method for purifying hyaluronate from endotoxins, which consists in preparing a solution containing sodium hyaluronate and sodium chloride, and hyaluronate is precipitated by centrifugation, while the method differs in that the preparation of the solution is carried out by dissolving sodium hyaluronate in 0.45-0, 5% sodium chloride solution to obtain a solution with a hyaluronate concentration of 0.25-0.5%, a 0.5-0.75% solution of cetylpyridinium chloride is added to the solution with stirring at an amount of cetylpyridinium chloride equimolar to the amount of hyaluronate by carboxyl groups , leave the resulting solution until the sediment matures and coagulates, after which the hyaluronate is precipitated by centrifuging the mixture and the raw precipitate of cetylpyridinium hyaluronate is collected, after which a 0.3-0.5% solution of cetylpyridinium hyaluronate in dimethyl sulfoxide, pre-cooled to a temperature of 5- 7°C, with stirring, after complete dissolution of the precipitate on the solution 1.25-1.5% sodium chloride solution with an amount of sodium chloride equimolar to the amount of hyaluronate, the resulting precipitate of hyaluronate is collected and dissolved in a 0.9% sodium chloride solution to obtain a hyaluronate solution with a concentration of 0.1-0, 15%, a sodium hyaluronate solution is fed into a tangential reverse osmosis system with a membrane corresponding to the molecular weight of the initial hyaluronate, while a 10-fold volume of a 0.9% sodium chloride solution is gradually poured into the system in relation to the volume of the hyaluronate solution that has passed through the system a solution of sodium hyaluronate in a 0.9% sodium chloride solution is concentrated to 1-2%, filtered through a sterilization membrane and purified hyaluronate is obtained.

In one embodiment, the purified hyaluronate is obtained as a solution passed through a sterilization membrane.

In another embodiment, purified hyaluronate is obtained by precipitating it with 96% ethanol from a solution that has passed through a sterilization membrane and drying the precipitate.

A method is proposed for the purification of hyaluronate preparations from endotoxins using several processes in the technological chain: precipitation, micellization, dissolution in aprotic solvents and ultrafiltration on membranes corresponding to the molecular weight of hyaluronate, followed by washing with sodium chloride solutions.

In the proposed method, inexpensive reagents are used, it is characterized by low time costs, a high yield of the target product and high purification efficiency, and in the purification process, you can use the source material with varying degrees of contamination with endotoxins and proteins and different molecular weights. During the cleaning process, the viscosity and molecular weight remain unchanged.

The method of purification of hyaluronate from endotoxins is carried out as follows.

A portion of a dry preparation of sodium hyaluronate of a certain molecular weight, containing up to 1⋅10 ³ EU/mg of endotoxins, is dissolved in a solution of 0.45 - 0.5% sodium chloride and a solution of sodium hyaluronate with a concentration of 0.5% - 0.25% is obtained. A feature of this method is that in the purification process it is possible to use the starting material with varying degrees of contamination with endotoxins and proteins and different molecular weights, as well as the material stabilized by crosslinking agents. Then, with vigorous stirring, a 0.5-0.75% solution of a quaternary ammonium salt, preferably cetylpyridinium chloride, is used. The hyaluronate is precipitated with an equimolar amount of a quaternary ammonium salt at the carboxyl groups (1 mol of hyaluronate at the carboxyl groups and 1 mol of a quaternary ammonium salt).

The number of carboxyl groups of sodium hyaluronate can be calculated according to the known formula based on the weight of the sample of sodium hyaluronate. The number n of disaccharide units is calculated by the formula

n=m/M,

where m is the weight of the sample of sodium hyaluronate, minus impurities;

M is the molar mass of the disaccharide unit of sodium hyaluronate (equal to 401.22 g/mol).

The number of disaccharide units of sodium hyaluronate in a sample and will be equal to the number of carboxyl groups, which makes it possible to calculate the required amount and mass of cetylpyridinium chloride. For example, in 1.00 grams of sodium hyaluronate containing 97.5% of the target substance, there will be the following amount of carboxyl groups:

the mass of hyaluronate itself will be 0.9750 g;

n=0.975/401.22=0.00243 (mol).

A portion of cetylpyridinium chloride is calculated by the formula:

m=M ⋅ n,

where m is the mass of a sample of cetylpyridinium chloride;

M is the molar mass of cetylpyridinium chloride, which is 358.01 g/mol.

Thus, from the example, if the number of carboxyl groups in 1 g of a sample of hyaluronate is 0.00243 mol, then it is necessary to take the following mass of quaternary ammonium salt for complete precipitation of cetylpyridinium hyaluronate:

m=0.00243 ⋅ 358.01=0.8699 (d).

The solution is left for 8-16 hours for the maturation and coagulation of the precipitate. After a predetermined time, the mixture is centrifuged at 15 thousand revolutions per minute for 30 minutes. The crude sediment is collected and weighed.

Prepare a 0.3% - 0.5% solution of cetylpyridinium hyaluronate in 100% dimethyl sulfoxide. Since the process of dissolving the hyaluronate salt with quaternary ammonium is exothermic, the dimethyl sulfoxide is cooled to 5C° - 7°C. The dissolution proceeds slowly with stirring for 4-7 hours. After complete dissolution of the precipitate, 1.25% - 1.5% sodium chloride solution acts on the solution.

To precipitate sodium hyaluronate from a solution of dimethyl sulfoxide, an equimolar amount of sodium chloride in solution is taken. The precipitate is collected and dissolved in a 0.9% sodium chloride solution at a concentration of 0.1-0.15%. A solution of sodium hyaluronate is poured into a tangential reverse osmosis system with a membrane corresponding to the molecular weight of the hyaluronate initially supplied for purification. To remove residues of cetylpyridinium chloride and dimethyl sulfoxide, a 10-fold volume of 0.9% sodium chloride solution is gradually poured into the system from a volume of 0.1% - 0.15% solution. After washing, the solution of sodium hyaluronate in 0.9% sodium chloride solution is concentrated to 1% - 2%, filtered through a sterilization membrane and the solution is used directly, either precipitated with 96% ethanol, or freeze-dried. The output of sodium hyaluronate is 93.7% - 95.5%. Endotoxins and protein in the preparation after cleaning are not detected. Also, if the material has been stabilized with BDDE, no BDDE and its hydrolysis products are detected after the purification process.

Example 1

We took 10.0 g of dry powder of sodium hyaluronate, bacterial origin, made in China, stabilized with a crosslinking agent BDDE. A 0.3% solution of sodium hyaluronate in a 0.45% sodium chloride solution was prepared, for which 14.999 g of sodium chloride and then sodium hyaluronate powder were dissolved in 3333 ml of distilled water and mixed in a bag on a spatula homogenizer for 2 hours.

Calculate the mass of cetylpyridinium chloride for equimolar precipitation of sodium hyaluronate by carboxyl groups:

n (carboxyl groups)=10.0/401.22=0.0249 (mol),

n (cetylpyridinium chloride)=n (carboxyl groups)=0.0249 mol,

m (cetylpyridinium chloride)=M ⋅ n,

m (cetylpyridinium chloride)=358.01 ⋅ 0.0249=8.9144 (g).

A 0.55% solution of cetylpyridinium chloride was prepared, for which 8.9144 g of the salt was dissolved in 1621 ml of distilled water.

A solution of sodium hyaluronate with a volume of 3333 ml was poured into a stirred reactor. With vigorous stirring, 1621 ml of a solution of cetylpyridinium chloride was poured. The stirrer was stopped and the solution was left for 16 hours to mature and coagulate the precipitate.

After the specified time, the mixture was centrifuged at 15 thousand rpm for 30 minutes. The crude amorphous precipitate was collected.

A 0.4% solution of cetylpyridinium hyaluronate in dimethyl sulfoxide was prepared, for which 2500 ml of dimethyl sulfoxide cooled to 5°C were taken. Dissolved for 5 hours with stirring until complete dissolution of the precipitate.

We calculated the equimolar amount of sodium chloride for the precipitation of sodium hyaluronate from a solution of dimethyl sulfoxide:

n (carboxyl groups)=10.0/401.22=0.0249 (mol),

n (sodium chloride)=n (carboxyl groups)=0.0249 mol,

m (sodium chloride)=M ⋅ n,

m (sodium chloride)=58.44 ⋅ 0.0249=1.4552 (g).

A 1.3% sodium chloride solution was prepared, for which the salt was dissolved in 111.94 ml of distilled water.

To 2500 ml of cetylpyridinium hyaluronate solution, 111.94 ml of sodium chloride solution were added with slow stirring. The resulting fibrous precipitate was collected.

A 0.1% solution was prepared from the precipitate in a 0.9% sodium chloride solution, for which 10 liters of distilled water were taken and 90 g of sodium chloride was dissolved.

A solution of 0.1% sodium hyaluronate was poured into a tangential reverse osmosis system with a membrane having a pore size of 5 × 10 ⁶ nm. To remove residual quaternary ammonium salt and dimethyl sulfoxide, 25 liters of 0.9% sodium chloride solution were gradually poured into the system. After washing, a solution of sodium hyaluronate in 0.9% sodium chloride solution was concentrated to 1.5%, which was 632 ml by volume.

The solution was drained from the tangential installation and filtered through a 0.2 μm sterilization membrane.

The volume of the solution after filtration was 625 ml.

The sodium hyaluronate solution was precipitated with 5 volumes of 96% ethanol cooled to 4°C. The fibrous precipitate was collected, squeezed, crushed and dried to constant weight at 60°C.

The sediment mass was 9.375 g.

Table 1 shows the content of endotoxins, molecular weight, viscosity in samples of sodium hyaluronate before and after cleaning.

Table 1

Indicator Hyaluronate sample before purification (bacterial preparation stabilized with BDDE) Hyaluronate sample after cleaning Sodium hyaluronate content (%) 99.40 99.75 Molecular mass 1.45 ⋅ 10 ⁶ Yes 1.45 ⋅ 10 ⁶ Yes Viscosity of 1% solution (mPa⋅s) 930 930 Content of endotoxins (EU/mg) 15 Not detected

Example 2

We took 10.0 g of dry powder of native (without modifications and stabilization) sodium hyaluronate of bacterial origin, made in China.

The process was carried out as described in example 1.

In a tangential reverse osmosis system, a membrane with a pore size ^of 25 × 106 nm was used.

The solution was concentrated to 1%, which was 952 ml by volume.

The solution from the tangential filtration system was aseptically filtered through a 0.2 μm sterilization membrane. The volume of the solution was 945 ml. The yield of the target product was 94.5%.

Table No. 2 shows the endotoxin content, molecular weight, viscosity in sodium hyaluronate samples before and after cleaning.

table 2

Indicator Hyaluronate sample before cleaning
(bacterial preparation without stabilization and modifications) Hyaluronate sample after cleaning Content of sodium hyaluronate 99.37 99.80 Molecular mass 5.25 ⋅ 10 ⁶ Yes 5.25 ⋅ 10 ⁶ Yes Viscosity of 1% solution (mPa⋅s) 2870 2870 Content of endotoxins (EU/mg) 23 Not detected

Example 3

We took 10.0 g of dry powder of native (without modification and stabilization) sodium hyaluronate of animal origin.

The process was carried out as described in example 1.

In a tangential reverse osmosis system, a membrane with a pore size ^of 50 × 106 nm was used.

The solution was concentrated to 0.5%, which was 1880 ml by volume.

The solution from the tangential filtration system was aseptically filtered through a 0.2 μm sterilization membrane. The volume of the solution was 1874 ml. The yield of the target product was 93.7%.

Table 3 shows the content of endotoxins, molecular weight, viscosity in samples of sodium hyaluronate before and after cleaning.

Table 3

Indicator Hyaluronate sample before cleaning
(drug of animal origin, without stabilization and modifications) Hyaluronate sample after cleaning Content of sodium hyaluronate 99.40 99.80 Molecular mass 10.84 ⋅10 ⁶ Yes 10.84 ⋅ 10 ⁶ Yes Viscosity of 1% solution (mPa⋅s) 4320 4320 Content of endotoxins (EU/mg) eighteen Not detected

Example 4

We took 10.0 g of dry powder of native (without modifications and stabilization) sodium hyaluronate of bacterial origin, made in China.

The process was carried out as described in example 1.

In a tangential reverse osmosis system, a membrane with a pore size ^of 2.5 × 105 nm was used.

The solution was concentrated to 1.5%, which was 645 ml by volume.

The solution from the tangential filtration system was aseptically filtered through a 0.2 μm sterilization membrane. The volume of the solution was 637 ml. The yield of the target product was 95.5%.

Table 4 shows the content of endotoxins, molecular weight, viscosity in samples of sodium hyaluronate before and after cleaning.

Table 4

Indicator Hyaluronate sample before cleaning
(bacterial preparation stabilized with BDDE) Hyaluronate sample after cleaning Content of sodium hyaluronate 98.20 99.77 Molecular mass 2.5 ⋅ 10 ⁵ Yes 2.5 ⋅ 10 ⁵ Yes Viscosity of 1% solution (mPa⋅s) 320 325 Content of endotoxins (EU/mg) 950 Not detected

Example 5

10.0 g dry powder of BDDE stabilized sodium hyaluronate of animal origin was taken.

The process was carried out as described in example 1.

In a tangential reverse osmosis system, a membrane with a pore size ^of 25 × 106 nm was used.

The solution was concentrated to 0.5%, which was 1880 ml by volume.

The solution from the tangential filtration system was aseptically filtered through a 0.2 μm sterilization membrane. The volume of the solution was 1874 ml. The yield of the target product was 93.7%.

Table 5 shows the content of endotoxins, molecular weight, viscosity in samples of sodium hyaluronate before and after cleaning.

Table 5

Indicator Hyaluronate sample before cleaning
(animal product stabilized with BDDE) Hyaluronate sample after cleaning Content of sodium hyaluronate 99.40 99.80 Molecular mass 12.63 ⋅ 10 ⁶ Yes 12.63 ⋅ 10 ⁶ Yes Viscosity of 1% solution (mPa⋅s) 5320 5320 Content of endotoxins (EU/mg) 17 Not detected

Claims (3)

1. A method for cleaning hyaluronate from endotoxins, which consists in preparing a solution containing sodium hyaluronate and sodium chloride, and hyaluronate is precipitated by centrifugation, characterized in that the solution is prepared by dissolving sodium hyaluronate in 0.45-0.5% sodium chloride solution to obtain a solution with a hyaluronate concentration of 0.25-0.5%, add a 0.5-0.75% solution of cetylpyridinium chloride to the solution with stirring at an amount of cetylpyridinium chloride equimolar to the amount of hyaluronate by carboxyl groups, leave the resulting solution until the sediment matures and coagulates, after which the hyaluronate is precipitated by centrifuging the mixture and the raw precipitate of cetylpyridinium hyaluronate is collected, after which a 0.3-0.5% solution of cetylpyridinium hyaluronate in dimethyl sulfoxide, pre-cooled to a temperature of 5-7 ° C, is prepared, with stirring, after complete dissolution of the precipitate, the solution is treated with a 1.25-1.5% chloride solution on sodium at an amount of sodium chloride equimolar to the amount of hyaluronate, the resulting precipitate of hyaluronate is collected and dissolved in a 0.9% sodium chloride solution to obtain a hyaluronate solution with a concentration of 0.1-0.15%, the sodium hyaluronate solution is fed into a tangential reverse osmosis system with a membrane corresponding to the molecular weight of the original hyaluronate, while a 10-fold volume of a 0.9% sodium chloride solution is gradually poured into the system relative to the volume of the hyaluronate solution, a solution of sodium hyaluronate in a 0.9% sodium chloride solution that has passed through the system concentrated to 1-2%, filtered through a sterilizing membrane and purified hyaluronate is obtained. 2. The method according to p. 1, characterized in that the purified hyaluronate is obtained in the form of a solution that has passed through the sterilization membrane. 3. The method according to p. 1, characterized in that the purified hyaluronate is obtained by precipitating it with 96% ethanol from a solution that has passed through the sterilization membrane and drying the precipitate.