RU2273486C1 - Method for separating sulfated glycosoamineglycanes from biological tissue - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves comminuting soft tissues to homogeneous state and hard tissues to small pieces, washing them with 0.1 N phosphate buffer. The tissue is digested in activated 0.125-0.325% papain at 60°C during 24 h, cooled during 10 h at 4°C, filtered and the digested tissue is poured into chromatography column where bone tissue collagen is used as solid carrier having particle sizes from 0.1 to 0.5 cm³, incubated during 10 to 24 h, washing in 0.05-0.2 N hydrochloric acid and eluted with 0.5-1.5N sodium chloride, dialyzed against saturated sodium chloride, precipitated in ethanol, centrifuged during 15 min at 1500 rpm at 4°C. The precipitate is washed with ethanol, dried, packed and sterilized using radiation method.

EFFECT: increased end product output; reduced antigenic activity; high biocompatibility.

Description

The invention relates to medicine, and more specifically to biochemistry, and can be used to isolate complex polysaccharides from biological tissues of animal origin rich in these compounds, as well as the manufacture of drug substances - eye drops for the treatment of diseases and injuries of the cornea, pharmaceuticals for the treatment of diseases bones and joints, pharmaceuticals for the treatment of diseases of the cardiovascular system, as well as a number of biocomposite medical devices, in Tav which are sulfated glycosaminoglycan (sGAG).

A known method of producing sGAG from biomaterial — pig corneas that are mechanically cleaned, is hydrolyzed with activated papain, after which the hydrolyzate is boiled for 10-15 minutes, trichloroacetic acid is added, the precipitate is removed, sGAG is precipitated with ethanol and dialysed against a mixture of 1N sodium chloride solution and ethanol (RF Patent No. 2056851 of June 25, 1992), which is selected as the prototype, because it is closest in its technical solution to the proposed invention.

The disadvantages of this method is that such processing is time-consuming, costly and does not provide complete exit and release from the sGAG solution, as well as from impurities of protein and other antigenic molecules that are present in these tissues, which significantly reduces the purity of the final product.

The objective of the invention is to improve the quality of isolation of sGAG for use in biochemistry, to obtain pure complex polysaccharides, as well as pharmaceutical substances by enzymatic treatment of biological tissues and the allocation of sGAG on a solid carrier - collagen from bone tissue and, as a result, increase the yield of the final product, reduce the amount of protein in the drug, a decrease in its antigenic characteristics and an increase in its biocompatibility.

The technical result achieved by using the invention is to obtain high-quality sGAG for the production of pharmaceutical substances, which can be widely used to obtain pharmaceuticals and a number of medical devices, which include sGAG.

The technical result is achieved by using biological tissue of animals or humans containing sGAG - cornea, cartilage, bone, trachea, liver, small intestine, etc., which are first mechanically cleaned, washed with a buffer solution, treated with an enzyme, dialyzed, precipitated ethanol and centrifugation, characterized in that after mechanical cleaning, the tissue is digested in a solution of activated 0.125-0.325% palain at 60 ° C for 24 hours, the digest is cooled for 10 hours at 4 ° C, poured into a chromatographic column, where As a solid carrier is used the collagen from the bone tissue with a particle size of from 0.1 to 0.5 cm ^3, incubated for 10 to 24 hours, washed with 0.05-0.2 N hydrochloric acid and eluted with 0.5-1, 5 N sodium chloride, dialyzed against saturated sodium chloride, cGAG precipitated with ethanol, centrifuged for 15 minutes at 1500 rpm at 4 ° C, the precipitate was washed with ethanol, dried, Packed and sterilized by the radiation method.

Any tissue of farm animals or, if necessary, human donor tissue can be the raw material for the preparation of sGAG. These tissues contain proteglycans, which include sGAG, such as chondroitin sulfate, keratan sulfate, heparan sulfate, dermatan sulfate, heparin, etc. (Stacy M. and Barker S. Carbohydrates of living tissue. M .: Mir., 1965. p. -35-38).

The technology for producing sGAG requires initial mechanical processing of the tissue, when the tissue is cleansed of the remnants of soft tissues and washed from the blood.

After this treatment, the fabric is cut with a sharp tool into strips or pieces, and in the case when the fabric is denser (for example, the trachea), pass through a meat grinder. Upon receipt of bone sGAG, the bone is sawn into plates.

The cooled tissue is crushed to obtain a homogeneous mass and subjected to enzymatic hydrolysis by a known method. As an enzyme that destroys the most important antigens of the stroma - glycoproteins and proteoglycans, we used the enzyme of the melon tree papain, which destroys these compounds and other proteins at a certain temperature and time of incubation. In this case, the polysaccharide chains of sGAG are completely preserved.

The papain concentration and digestion time were determined experimentally by us in the range from 0.125 to 0.325% at a temperature of 60 ° C for 24 hours.

For most types of connective tissue - cornea, sclera, small intestine, a papain concentration of 0.125% is optimal at a temperature of digestion of 60 ° C and a time of 24 hours. However, when processing harder and denser tissues, such as trachea or bone, this concentration should be increased to 0.325% under similar digestion conditions.

The resulting digest was cooled at 4 ° C for 10 hours. At this temperature and during this time, primary deposition of tissue residues occurs.

This step is necessary for the initial removal of most of the undigested residues and fats. The quality of the enzymatic treatment of the material was determined by the yield of free sGAG spectrophotometrically by staining with 1.9-dimethylene blue at a wavelength of 535 nm according to the Farndel method.

An essential feature of the invention is that the obtained digest is poured into a chromatographic column, where collagen from bone tissue with particle sizes from 0.1 to 0.5 cm ^{3 is} used as a solid carrier.

We found that bone collagen is affinity (by affinity) able to bind sGAG and a number of other biologically active substances. It is on this principle that its use as a carrier, acting as a chromatographic substrate, is based.

Collagen from bone tissue meets all the basic requirements for carriers (matrices) used for affinity chromatography, especially for affinity separation, concentration and purification of biologically active substances. So, it contains a large number of groups capable of binding to the ligand affinity (by affinity); it does not break down upon binding to the ligand and does not lose its properties when the biomolecules are eluted from it with solutions of acids or alkalis; weakly or completely does not react with other macromolecules; provides fast and uniform course of solution.

The advantages of collagen from bone tissue as a carrier include its sufficient rigidity and resistance to the action of weak solutions of acids, alkalis and other eluents. Its porous-fibrous structure, which provides a large reaction surface and high binding ability, the ability to concentrate the ligand and, as a result, increase the yield of the final purified product.

The material for obtaining a solid carrier is, as a rule, the spongy bone of pigs or, if necessary, the human donor bone. The collagen of this tissue mainly consists of type I collagen and is characterized by very weak solubility in solutions of acids or alkalis and high resistance to collagenolytic enzymes.

The sizes of collagen pieces were established experimentally by isolating sGAG on them. In this case, the minimum size of the piece is 0.1 cm, since it provides the minimum required working volume and the binding surface.

The maximum size is set to 0.5 cm ³ , since exceeding this size makes it difficult for circulating solutions to pass into the pores of the collagen.

The technology for preparing such a carrier (matrix) is described in RF Patent No. 2161976 dated January 20, 2001. A method for producing collagen from bone tissue.

Pieces of collagen obtained using this method fill the column and pour the digest into it for 10-24 hours.

At a high concentration of sGAG in the digest (which was previously determined in the digest according to the Farndel method), the digestion solution is incubated for 10 hours, and at a lower for 24 hours. The incubation time was established by us experimentally to determine the sGAG in the digest. At a low concentration of sGAG, they are not detected after 10 hours of incubation in column washes. Similarly, with a high concentration of sGAG in washing, they are not detected after 24 hours of incubation.

Then the column is washed from non-specifically adhering residues of proteins and other substances with 0.05-0.2 N hydrochloric acid. Here, the concentration of acid is selected empirically. When the concentration of hydrochloric acid is below 0.05 N, part of the non-specifically bound proteins remains on the collagen substrate and subsequently contaminates the main product. When the concentration of hydrochloric acid is higher than 0.2 N, partial removal of sGAG from the collagen substrate occurs. Washing from non-specifically bound proteins and other substances leads to the complete disappearance of the protein in the eluate.

The protein yield in the eluate was determined by the Lowry Pharmacopoeia spectroscopically at 400 nm and the Kjeldahl method of hydroxyproline determination.

After that, the elution of sGAG is started, passing through it a 0.5-1.5 N sodium chloride solution.

The concentration of saline was established by us experimentally and is optimal for removing sGAG from various tissues from collagen. At a concentration of sodium chloride below 0.5 N, not all sGAGs leave the column and the time of their exit increases significantly. When the concentration of sodium chloride is above 1.5 N, the protein content in the eluate increases both due to the removal of proteins from the carrier and due to the partial destruction of the collagen substrate.

Elution is continued until the sGAG output from the column ceases, which is controlled by their presence in solution according to the Farndel method.

The next step in isolation is dialysis against a saturated sodium chloride solution. This stage provides the purification of sGAG from non-specifically related molecules of protein and mineral passing, as well as all other low molecular weight impurities.

Then ethanol is added to the sGAG solution and they are precipitated from the solution.

For a more complete deposition of all sGAG from the solution, it is centrifuged for 15 minutes at 1500 rpm at 4 ° C. At a given speed and given time, all sGAGs are precipitated. In this case, the best deposition and their preservation are achieved at a temperature of 4 ° C.

After centrifugation, the precipitate is again washed with ethanol to remove residual salt and moisture from it.

In order to eliminate possible bacterial contamination, sGAG powder is immediately packaged in sterile vials immediately after drying.

The thus obtained sGAG powder is sterilized by the radiation method. This sterilization method has been tested experimentally and does not lead to degradation of the drug, does not reduce its biological activity.

A comparative analysis of the quantitative and qualitative yield of the obtained product showed that the sGAG content in the samples is 99%, and the protein content is either not determined or does not exceed 1.5-2%, which significantly improves these product characteristics compared to the prototype (3-6% ) The latter is especially important in the industrial method of production of sGAG.

The biological activity of the obtained sGAGs was determined under conditions of cell cultures and in animal model experiments using the techniques described earlier (Panasyuk A.F., Larionov E.V. Chondroitin sulfates and their role in the exchange of chondrocytes and intercellular matrix of cartilage tissue. Scientific and practical Rheumatology. 2000. No. 2, pp. 46-55).

Brief technology for obtaining sGAG.

Biological tissue - 1 kg of pig’s corneas is cleaned of mechanical impurities, washed with cold water from blood, turned through a meat grinder, washed in 0.1 N phosphate buffer at pH 5.8-6.0 and placed in a solution of activated 0.125% papain at 60 ° C for 24 hours . After digestion, the solution is cooled for 10 hours at 4 ° C, filtered, poured into a column with bone collagen with particle sizes of 0.1-0.5 cm ³ , incubated for 10 hours, then washed with 0.1 N hydrochloric acid from non-specific particles and then elute with 0.5 N sodium chloride, place the eluate in dialysis bags and dialyze against saturated sodium chloride, precipitate in ethanol after dialysis, centrifuge the precipitate for 15 minutes at 1500 rpm at 4 ° C, wash with ethanol and dry, pack and sterilized by the radiation method.

Actions lead to a decrease in antigenicity, since the amount of protein is significantly reduced compared to the selected prototype.

Thus, the proposed method of tissue processing allows you to get 99% of the sGAG yield and reduce the antigenicity of the substance by removing the residual complexes of proteins and core proteins that are part of proteoglycans, which allows to increase the biocompatibility of this material and reduce the number of complications when it is used as a medicine or as part of medical devices.

For a better understanding of the essence of the invention is illustrated by examples of specific performance.

Example 1

Getting sGAG from the cornea of pigs.

1 kilogram of cornea is isolated from pig’s eyeballs, cleaned of mechanical impurities, washed from blood in 0.1 N phosphate buffer at pH 5.8–6.0 and cut into pieces of the right size. The tissue is homogenized to a homogeneous mass, then the homogenate is poured with 0.125% activated papain solution and incubated for 24 hours at 60 ° C, after digestion, the digest solution is cooled for 10 hours at 4 ° C, filtered, poured into a sponge collagen column from bone tissue pigs with particle sizes of 0.1-0.5 cm ³ , incubated for 10 hours, then washed with 0.05 N hydrochloric acid from non-specifically adhering particles and then eluted with 0.5 N sodium chloride, placed the eluate in dialysis bags and dialyzed against saturated sodium chloride, p After dialysis, precipitate in ethanol, the precipitate is centrifuged for 15 minutes at 1500 rpm at 4 ° C, washed with ethanol, dried, packaged and sterilized by the radiation method.

A quantitative analysis of sGAG, performed spectrophotometrically according to Farndel, showed that the content of sGAG in the preparation is 99.9%.

Protein according to Lowry in the modification of Bradford - less than 0.01%.

The concentration of excreted sGAG -4.9 mg / g of crude tissue.

The concentration of isolated sGAG (according to the prototype) is 4.23 mg / g of crude tissue.

Actions lead to an increase in the yield of the final product, an increase in its quality and a decrease in antigenicity, since the amount of protein is significantly reduced in comparison with the selected prototype.

Example 2. Obtaining sGAG from pig lungs.

1 kilogram of light pigs is cut into strips, cleaned of mechanical impurities, washed from blood in 0.1 N phosphate buffer at pH 5.8-6.0 and cut into pieces of the desired size, homogenized to a homogeneous mass, then the homogenate is poured with a 0.225% solution activated papain and incubated for 24 hours at 60 ° C, after digestion, the digest solution is cooled for 10 hours at 4 ° C, filtered, poured into a column with spongy collagen from pig bone tissue with a particle size of 0.3-0.5 cm ³ , incubated for 16 hours, then washed with 0.15 N salt hydrochloric acid from non-specifically adhering particles and then elute with 0.9 N sodium chloride, place the eluate in dialysis bags and dialyze against saturated sodium chloride, after dialysis precipitate in ethanol, centrifuging for 15 minutes at 1500 rpm at 4 ° C. the precipitate is washed with ethanol, dried, packaged and sterilized by a radiation method with a dose of 0.5 Mrad.

Quantitative analysis of sGAG spectrophotometrically according to Farndel - the content of sGAG in the preparation 98-99%.

Protein according to Lowry in the modification of Bradford - less than 1.5%.

Actions lead to a decrease in antigenicity, since the amount of protein is significantly reduced compared to the selected prototype.

Example 3. Obtaining sGAG from pork bones.

Bones of pigs are cleaned of muscles and cartilage, washed with cold water and cut into pieces of the right size, washed from blood in 0.1 N phosphate buffer pH 5.8-6.0 and filled with 0.325% activated papain solution, incubated for 24 hours at 60 ° C, after digestion, the digest solution is cooled for 10 hours at 4 ° C, filtered, poured into a collagen column from pig bone tissue with a particle size of 0.1-0.5 cm ³ , incubated for 24 hours, then washed 0.2 N hydrochloric acid from non-specifically adhering particles and then elute with 1.5 N sodium chloride , The eluate was placed in dialysis bags and dialyzed against a saturated sodium chloride solution, after dialysis, was precipitated in ethanol, the precipitate was centrifuged for 15 minutes at 1500 rev / min at 4 ° C, washed with ethanol, dried, packed and sterilized by radiation dose of 0.5 Mrad.

Quantitative analysis of sGAG spectrophotometrically according to Farndel - the content of sGAG in the preparation 98-99%.

The concentration of excreted sGAG -1.8 g / kg of raw tissue.

The concentration of isolated sGAG (prototype) - 1.5 g / kg of raw tissue.

Protein according to Lowry in the modification of Bradford - less than 0.02%.

Actions lead to a decrease in antigenicity, since the amount of protein is significantly reduced compared to the selected prototype.

Thus, it is seen that the proposed method of processing tissue allows you to increase the yield of sGAG, increase their purity to 99% and reduce antigenicity by removing the protein components that make up this tissue.

The latter provides high biocompatibility of sGAG and dramatically reduces the number of complications when using this drug in the form of a medicinal product or as part of medical devices.

Claims (1)

A method for isolating sulfated glycosaminoglycans from biological tissues, including mechanical cleaning of tissue, grinding, washing with a buffer solution, treatment with papain enzyme, dialysis, precipitation with ethanol and centrifugation, characterized in that, after mechanical cleaning, soft tissues are crushed to a homogeneous state, and hard ones to small pieces, washed with 0.1 N phosphate buffer, the tissue is digested in a solution of activated 0.125-0.325% papain at 60 ° C for 24 hours, cooled for 10 hours at 4 ° C, filtered, digested vayut the chromatographic column, where the solid support using collagen from the bone tissue with a particle size of from 0.1 to 0.5 cm ^3, incubated for 10 to 24 hours, washed with 0.05-0.2 N hydrochloric acid, and it is eluted with 0.5-1.5 N sodium chloride, dialyzed against saturated sodium chloride, precipitated in ethanol, centrifuged for 15 min at 1500 rpm at 4 ° C, the precipitate is washed with ethanol, dried, Packed and sterilized by the radiation method.