CS267079B1 - A method for preparing a pyrogen-free hyaluronic acid derivative - Google Patents

Abstract

The solution relates to the preparation of an ultrapure high molecular weight derivative of hyaluronic acid. The essence of the method of its preparation consists in that the aqueous phase after extraction of biological materials is treated with alkylating agents of the general formula X/n/-R or X/n/~ -R-P/m/, where X is chlorine, bromine or iodine, R is an organic group with a carbon number of 1 to 14, n is an integer from the interval 1 to 5, P is a polar group of anionic type such as -COOH, -OH, -COONa, -COOK, -COONH2, -SO3H and m is an integer from the interval 1 to 5, in an amount of 0.1 to 15 wt. on the weight of the dry matter of the extract for a period of 1 to 24 hours and/or with polar substances that are soluble in water and have hydrotropic properties such as urea, guanidine, or potassium iodide in an amount of 0.1 to 10% by weight on the weight of the dry matter of the extract, the resulting solution is filtered through a filter membrane with a pore size of 0.15 to 0.50 Λ™, the product is precipitated from the filtrate by adding 300% by volume of ethanol, after separation it is dissolved in water and sterilized with cetylpyridinium chloride or bromide, reprecipitated with ethanol and finally washed with acetone, dried and sterilized by heating at 105 to 150 °C for 10 to 25 minutes.

Description

2 CS 267 079 B1

The present invention relates to a process for the preparation of a pyrogen-free derivative of hyaluronic acid by extraction techniques.

Hyaluronic acid belongs to the group of naturally occurring strong polar acidic polysaccharides, referred to as glycosaminoglycans or mucopolysaccharides, and is found in all connective tissues, skin, tendons, cartilage, vitreous humor, synovial fluid of joint cases and the like. In terms of chemical structure, it is characterized by regular recurring carbohydrate units of two species, 1-3 glucuronic acid and 1-4 N-acylglucosamine, linked by glycosidic bonds to a polymeric macromolecule of 10 or more. It has long been a concern for pharmacologists to obtain very pure derivatives of hyaluronic acid (HYA) that would be useful as a substitute for or partially replacing HYA naturally occurring in glass or synovial fluid. is a defect in naturally occurring fluid characterized by its acid content. In the literature, a large number of washing machines describing so-called HYA purification and various methods of purifying naturally occurring hyaluronic acid, possibly its derivatives. Particularly purified formulations of this acid but none of these formulations are suitable for medical applications because it contains a number of accompanying proteins that cause various inflammations when the preparation is injected into the body of a mammal, particularly the eye when replacing the vitreous body. As shown by A. A. Balasz in U.S. Pat. No. 4,141,973 of 1979, the common problem of the preparations is their insufficient pyrogenicity. Therefore, these preparations have to date been applied together with anti-inflammatory drugs and antibiotics.

The first known and hitherto unique method of isolating and molding HYA, which provides material with sufficient pyrogenicity, has been described by Balasz in the above patent specification. This process does not differ from the prior art, but is supplemented by a new step, long-term (5-day continuous) stirring of the aqueous extracts of HYA with equal parts of chloroform, under conditions ensuring good contact of the two phases. Since chloroform is a water-immiscible liquid and differs from aqueous solutions by a relatively high specific gravity, relatively intensive mixing is required to ensure homogeneity. Under these conditions, the partial dopolymerization of the HYA molecules known to be supported by mechanical spraying and air oxygen, mainly under metal ion catalysis.

The method of isolation and purification of HYA according to said US patent not only is time consuming, but mixing with chloroform for several days can promote product degradation to a lower molecular weight. The accompanying protein content is also relatively high, about 0.5% by weight, and the product yield is relatively low - 0.9 g / kg of feedstock.

The above disadvantages are substantially eliminated by the process of preparing the pyrogen-free hyaluronic acid derivative by extraction of biological materials using partial deproteinization of the extract with ethanol and chlorinated hydrocarbons according to the invention, the principle being that the aqueous phase after extraction! adjusted to a pH of 5.5 to 8.5 is treated with alkylating agents of the formula X / n / R or Xh / -RP / m /, where X is chloro, bromo or iodo, R is an organic group having 1 to 14, n is an integer from 1 to 5, P is an anionic polar group such as -COOH, -OH, -COONa, -COOK, -COONH 2, -SO 4 H, and m is an integer from 1 to 5 in an amount of 0.1 to 15% wt. to the dry weight of the extract over a period of 1 to 24 hours and / or substances of a polar nature which are soluble in water and have a hydrotropicity such as urea, guanidine or potassium iodide in an amount of 0.1 to 10% by weight of the extract dry matter, filtered solution through a filter membrane having a pore size of 0.15 to 0.50 µm, the filtrate is precipitated by the addition of 300% by volume ethanol solution is dissolved in water and sterilized with cetylpyridinium chloride or bromide, precipitated with ethanol and finally washed with acetone, dried and sterilized by heating at 105 to 150 ° C for 10 to 25 minutes. The hyaluronic acid solution after filtration of the 2 + 2+ through the filter membrane can be treated with metal compounds selected from the group of Mg, Ca, CS 267 079 B1 3.

Zn 2+, Ba 2+ +, Al 2+ +, Cu 2+ +, Cr 2+ + alone or in admixture in amounts of 0.5 to 10 moles of hyaluronate. After addition of the alkylating agent, in addition to cetylpyridinium halide, a formaldehyde may be added in an amount of 0.1 to 10 g per 10 L of hyaluronate solution.

According to the invention, the decontamination of HYA from protein constituents is achieved thereby ensuring the product's pyrogenicity. An essential feature of this method is the modification of some polar and thiol groups in the contaminant protein molecule by alkylating agents, readily separable from HYA by virtue of the different solubility in ethanol. Modifying the alkylation causes disruption of non-covalently bound interhYA complexes and fibrous proteins, the formation of which causes polar and thiol groups of some amino acids of the protein.

Suitable alkylating agents are those providing the modified protein with new polar substituents of anionic nature, since the negative charging group provides the same character as HYA acidic polysaccharide and leads to reduced mutual interaction. Such an alkylating agent is, for example, a halo derivative of acetic acid which induces carboxymethylation of thiol and hydroxyl groups in cysteine, glutathione, threonine, serine and other groups, for example, quanidine and amine groups in basic amino acids. However, in addition to this group of alkylating agents, many other halogenated derivatives, including those which cannot be applied in aqueous solutions, such as methyl chloride, dichloroethane, and other homologous aliphatic and aromatic hydrocarbons, contain one or more chlorine, bromine or iodine molecules in their molecule. In such cases, methylation or other alkylation occurs in the heterogeneous system at the interface, and the excess alkylating agent can be separated in a simple manner after phase separation. Advantages of the method of the invention consist in shortening the deproteinization cycle and thereby avoiding a decrease in the molecular weight of the products obtained and in a better removal of the transient proteins, a protein content below 0.1% by weight. and a hyaluronate yield of 1.6 g / kg of raw material.

An inert atmosphere may be used in the extraction and purification operations. Example 1

The raw material used to isolate the HYA, such as the cleaned umbilical cord, the rooster crest or other binder, or the HYA-containing material, is first frozen at -20 to -40 ° C, thereby destroying the cell walls. 1 kg of frozen tissue is cut into small pieces and extracted 3 times with 90% ethanol.

Ethyl alcohol (90% v / v) was used for each extraction and the mixture was stirred for 24 hours at 18 ° C. The ethanol solution always contains 5% by volume of chloroform.

Thereafter, by adding 2 liters of redistilled water with the addition of bacteriostatic agent cetylpyridinium bromide in an amount of 0.01% by weight. induces rehydration of the substance and in three separate extraction steps, HYA is leached out with 4 1 of redistilled water with the addition of 200 ml of chloroform. Each extraction step is carried out with slow stirring at 10 ° C. Before the third extraction, it is cut to a fine grit. The extracts are combined and used for further purification.

The combined aqueous extract is treated with 1.1 kg of sodium chloride and, after complete dissolution, is stirred at 20 ° C for 4 hours with chloroform (1: 1). Thereafter, the two phases are left to separate and the aqueous layer is separated. The pH of the aqueous phase is adjusted to 4.0 with the addition of dilute hydrochloric acid and again stirred, now under a nitrogen atmosphere, with a 1: 1 by volume mixture. After phase separation, stirring with chloroform is repeated until the chloroform phase is clear. Thereafter, the pH of the separated aqueous layer is adjusted to pH 6.5-7.0 with dilute sodium hydroxide solution and 10% by weight is added. a solution of sodium monochloroacetic acid in a volume of 0.1% by volume of the solution per volume of extract. After mixing, it is extracted 24 hours in the dark, then filtered through a sintered glass filter and an auxiliary layer of diatomaceous earth and finally through a 0.25 µm pore size membrane filter. Hyaluronic acid precipitates out of the filtrate by addition of ethanol in 1.5 volumes of ethanol per volume of extract. The separated HYA was dissolved in 2 L of redistilled water containing 7.8 g of sodium chloride and 5 g of cetylpyrinium chloride. After dissolution, ophthalmic volumes of ethanol are precipitated and, after separation, the precipitate is dissolved in 2 L of 0.0 M sodium chloride. To the solution was added 1 ml of freshly precipitated aluminum hydroxide containing 10% by weight. Al (OH) 2, stir and leave for 24 hours with occasional stirring. After separation of the precipitate, the solution is precipitated with 3 volumes of ethanol. Dissolution and precipitation with ethanol was repeated three times and finally the precipitate was washed with acetone and dried under reduced pressure. The dry product is sterilized by heating at 145 ° C for 15 minutes. 1.12 g of a product with a molecular weight of 2.84 million and a protein content of 0.013% by weight were obtained. (expressed as albumin). Example 2 Rooster combs obtained immediately after slaughter, bled and surface cleaned so that the edges and tips are separated and frozen at -20 to -40 ° C. 1 kg of frozen material is crushed and purified by repeated extraction of ethanol with 3 liters of ethanol per 1 kg of grit while moving for 12 hours. The extraction is repeated four times. Thereafter, the dewatered tissue is extracted with three volumes of redistilled water under gentle stirring in a nitrogen atmosphere for 24 hours. In the first and second extractions, 0.1 g of chelatone 3 is added to the aqueous phase. Addition of 100 ml of chloroform is used in the second and third extractions. The aqueous extracts were combined and dissolved in 1.3 kg of sodium chloride. The resulting solution was mixed with trichlorethylene (1: 1) and stirred for 4 hours under ordinary conditions. After phase separation, stirring is repeated three more times. Subsequently, the aqueous phase is adjusted to a pH of 6 to 7.5 with dilute sodium hydroxide solution and 2 g of ethyl bromide and 2.5 g of 1-bromo-2-sulfoethylene sodium salt and 0.5 g of formaldehyde and 0.5 g are added. a thiylpyridinium bromide. After dissolution, the solution is left in the dark for 24 hours. After alkylation, the pH is adjusted to 3.5 to 4.0 and the solution is washed through an acidic ion exchange column with a H cycle. The column is washed with 5 volumes of water and the pH of the eluate is adjusted to neutral with 0.1 M sodium hydroxide. Thereafter, trypsin was added at 0.001 g per 10 L of solution and the free solution was stirred for 5 hours. 100 g / 10 L of urea was added to the solution and stirring was continued for 2 hours at ambient temperature. The mixture was left overnight. For the second day, HYA is separated by precipitation with three volumes of ethanol and separation. The precipitate was dissolved with 4 L of water containing 192 g of sodium chloride and filtered through a membrane filter with a pore size of 0.35 µm. HYA is precipitated from the filtrate with three volumes of ethanol and dissolved in 2 l of water containing 9.6 g of sodium chloride and precipitated with ethanol. This precipitation is repeated four times. The precipitate is then washed 3 times with acetone and dried at 40 ° C. Finally, the product is sterilized under dry nitrogen at 105 ° C for 15 minutes. 1.7 g of dry product containing 0.22 wt. protein (such as albumin) of 1.62 million. Example 267

The raw material used for the extraction was hens combs obtained after slaughter and cooking in hot water, surface cleaned, trimmed and frozen at -40 ° C. 1 kg of frozen raw material crushed to fine amounts was used for the preparation. This material was extracted three times with 4 L of ethanol for 24 hours with gentle stirring. The combined extracts are mixed to 100 g / l NaCl by addition of sodium chloride. 120 g of urea are then added and the extract is stirred for 6 hours with the same amount of a 1: 1 mixture of trichlorethylene and tetrachloromethane. The phases are separated, the separated aqueous layer is acidified to pH 4.0 with dilute hydrochloric acid and stirring is continued with trichlorethylene-carbon tetrachloride under the same conditions for 5 hours. The aqueous phase is separated and neutralized with dilute sodium carbonate solution to pH 7.0 + 0.5. Then, 1.5 g of magnesium chloride is added and the extract is left to stir for 5 hours at 5 DEG C. The solution is filtered and the filtrate is precipitated with three volumes of ethanol. The precipitate is dissolved in 1.5 L of water containing 9.2 g of sodium chloride and 1% by weight of water. cetylpyridinium bromide. After dissolution, it was precipitated with 3 volumes of HYA ethanol and separated. Dissolution in 0.1 M sodium chloride solution and precipitation of ethanol was repeated four times. Finally, the precipitate is washed with three volumes of acetone and dried under reduced pressure. In the dry state, the product is sterilized at 135 ° C for 15 minutes. 2.14 g of 1.2% by weight product were obtained. protein (such as albumin) with a molecular weight of 3.8 million. This product was suitable for surface applications. Example 4

The raw material used to prepare the hyaluronate (cleaned umbilical cord, rooster comb, ophthalmic glass or other hyaluronic acid-containing material) is mechanically ground in a frozen state and placed in an extraction vessel equipped with a stirrer. This was extracted with ethanol (90-96%) for 24 hours, the last extraction was 48 hours. The extracted raw material was used to isolate the hyaluronate. The first extraction is carried out with 10% sodium chloride solution for 24 hours at 10 L per kilogram of feedstock.

In addition, distilled water is used in the same amount for extraction, the extraction time is always 24 hours with alternating time and the melt of the extracted mixture. Combine all three extracts by addition of solid sodium chloride and adjust to 10 g / l.

The combined extracts were made slightly alkaline to pH 7.4-7.8 with sodium bicarbonate, 2.5 g / l urea and 1.5 g / l sodium chloroacetate were added. The solution is well mixed and then left to stand in a dark environment at 20 ° C for 20 hours.

The resulting homogeneous solution is filtered through a 0.5 µm pore size filter membrane and the product is precipitated with 100% v / v ethanol, separated by sedimentation and then dissolved in a 10 times smaller volume of distilled water containing 5.6 g NaCl /. 1 and cautiously purified by ethanol precipitation, now at 300 vol%. Dissolution • and precipitation is repeated 2 more times and then cetylpyridinium bromide ethanol 0.5 g / l is added to the aqueous hyaluronate solution by pre-precipitation and left to sterilize for 2 hours. -vat at ordinary temperature. Then the mixture is precipitated with ethanol and repeated twice. Finally, the solution is precipitated with 3 volumes of acetone, the precipitate is decanted three times and washed with a small amount of acetone and finally dried under reduced pressure at ambient temperature.

Claims (4)

OBJECT OF THE INVENTION A process for the preparation of a pyrogen-free derivative of hyaluronic acid by extraction of biological materials using partial deproteinization of the extract with ethanol and chlorinated hydrocarbons, characterized in that the aqueous phase after extraction adjusted to pH 5.5 to 8.5 is treated with alkylating agents. / n / -R or X / n / -RP / m /, where X is chlorine, bromine or iodine, R is an organic group with a carbon number of 1 to 14, n is an integer from 1 to 5, P is a polar group anionic type such as -COOH, -OH, -COONa, -COOK, -COONHj, -SO3H and m is an integer ranging from 1 to 5, in an amount of 0.1 to 15% by weight, based on the dry weight of the extract for 1 to 24 hours. and / or substances of polar nature which are soluble in water and have a hydrotropic capacity such as urea, guanidine or potassium iodide in an amount of 0.1 to 10% by weight, based on the dry weight of the extract, the resulting solution is filtered through a filter membrane of size pores 0.15 to 0.50 / un, the product is precipitated from the filtrate by adding 300% by volume . ethanol, after separation is dissolved in water and sterilized with cetylpyridinium chloride or bromide, reprecipitated with ethanol and finally washed with acetone, dried and sterilized by heating at 105-150 ° C for 10-25 minutes. 2. A process for the preparation of a pyrogenic hyaluronic acid derivative according to item 1, characterized in that the hyaluronic acid solution, after filtration through a filter membrane, is treated with 2+ 2+ 2+ 2+ 3+ 3+ alloys of metals selected from the group Mg, Ca, Zn, Ba, Al, Cr individually or in a mixture in amounts of 0.5 to 10 moles per mole of hyaluronate. 3. The process according to items 2 and 1, characterized in that, after the addition of the alkylating agent, formaldehyde is added in addition to cetylpyridinium halide in an amount of 0.1 to 10 g per 10 l of hyaluronate solution. 4. The process according to items 1 and 3, characterized in that after the addition of the alkylating agent, the acidified extract is washed through an acid ion exchange column in an H cycle and, after neutralization, trypsin is added in an amount of 0.001 to 0.6 g per 10 l of solution.