RU2062619C1 - Method of follicle-stimulating preparation preparing from animal adenohypophysis - Google Patents

Abstract

FIELD: biology, veterinary. SUBSTANCE: method involves ballast proteins precipitation and hormone isolation. Animal adenohypophysis were homogenized at 2-4 C in the presence of buffer solution, pH = 7.0-7.5 taken at ratio 1: (6-7). Obtained homogenate is extracted at 2-4 C for 50-60 min and centrifuged to obtain solid precipitate. Supernatant is desalted with ammonium sulfate (35-40% of saturation) and ballast proteins were removed by centrifugation at the same regime. Obtained supernatant is subjected for ultrafiltration under pressure and concentrated hormone is purified by ion-exchange chromatography followed by purification on diaphragm with saline solution at pH = 4.2-4.5 and its separation from cationite with buffer solution at pH = 8.5-9.0. EFFECT: improved method of hormone preparing. 1 tbl

Description

 The invention relates to biology, in particular to the production of drugs used in animal husbandry and veterinary medicine with the aim of stimulating sexual function, inducing superovulation for transplantation of embryos, as well as in endocrine disorders in female farm animals.

 A known method of producing follicle-stimulating hormone (FSH) from the urine of women during menopause by sorption on a mixture of kaolin and kieselguhr followed by elution with solutions of ammonium salts [1] There is also a known method for producing FSH from horse pituitary gland [2] which includes the following main steps: grinding the gland extraction with ethanol, precipitation with acetone, dialysis against sodium chloride, precipitation with metaphosphoric acid, centrifugation, gel filtration on Sephadex G-100, chromatography on carboxymethylsephadex, preparation polyacrylamide gel electrophoresis, rechromatography and drug standardization.

 However, the first method, due to the low FSH content in animal urine, is not suitable for producing a hormonal drug, and the multi-stage and complexity of the process using scarce equipment and reagents of the second method makes it long, expensive and reduces the yield of the drug.

 As a prototype, a method for producing FSH from pituitary glands of animals [3] was selected, which includes homogenization of glandular tissue, extraction and purification of the hormone by differentially dissolving it in solutions of ammonium sulfate in descending concentrations. Using for this purpose ammonium sulfate of 40, 56, 58 and 80th concentration.

 The disadvantage of this method is that the use for the purification of the hormone of differentiated dissolution in solutions of ammonium sulfate of different concentrations does not allow you to completely get rid of ballast proteins, which leads to loss of the drug and a decrease in its biological activity.

Before homogenization, the adenohypophysis, obtained from animals, is poured with a buffer solution with a pH of 7.0-7.5 in a ratio of 1: 6-7 and which includes 0.89-0.90% sodium chloride, 3.2-3.4% sulfate ammonium, 10-15% ethyl alcohol and 0.14-0.15% potassium iodide. This mixture is homogenized for 2-4 minutes at a temperature of 2-4 ^o With until a homogeneous mass. Then the homogenizate is extracted for 50-60 minutes at 2-4 ^o C. With an increase in the time of homogenization, extraction and increase in temperature, proteolytic enzymes are activated, and with a decrease in the time of homogenization, extraction and lowering the temperature, cell tissue disaggregation decreases. Both in the first and in the second case, there is a decrease in the release of the hormone from the tissues into the liquid medium. The addition of ammonium sulfate, ethyl alcohol and potassium iodide contributes to the disaggregation of cell tissue and a more complete transition of the hormone into the liquid phase.

 To release the mixture from ballast substances, centrifugation is carried out until the liquid stratifies with the formation of a dense precipitate. To precipitate concomitant proteins and other pituitary hormones, ammonium sulfate is added to the supernatant (centrate) to 35-40% of saturation and centrifuged again to form a dense precipitate. The obtained centrate in order to free from ammonium sulfate and concentrate the hormone is subjected to ultrafiltration on a UAP-200 membrane under pressure. Then the concentrated hormone is purified by ion exchange chromatography. For this, KU-2 cation exchanger is mixed with a concentrated hormone solution for 50-60 minutes until they are completely bound, transferred to a membrane and washed from ballast proteins with physiological saline with a pH of 4.2 until protein is contained in the washing liquid, the presence of which is controlled spectrophotometrically. This medium is optimal for the binding of cation exchanger to the hormone. When the pH is shifted in the direction of increasing or decreasing the interaction of the hormone with cation exchanger does not occur. The hormone is “removed” from cation exchanger with a buffer solution with a pH of 8.5–9.0, which allows the hormone to be most completely separated from cation exchanger. The result is an FSH preparation with a biological activity of 45-50 m.u. in 1 ml.

Under the conditions of a meat factory, pituitary glands were taken from cattle, their anterior lobe (adenohypophysis) was removed and frozen in liquid nitrogen. 700 ml of a buffer solution with a pH of 7.0 and containing 6.3 g of sodium chloride, 23.8 g of ammonium sulfate, 70 ml of ethyl alcohol, 1 g of potassium iodide were poured into 100 g of frozen adenohypophyses and homogenized for 3 minutes at a temperature of 2- 4 ^o C to obtain a homogeneous mass. The resulting homogenizate was extracted for 60 minutes at a temperature of 2-4 ^o C. After this, a coarse suspension of tissues was precipitated by centrifugation at a temperature of 2-4 ^o C until the liquid stratified with the formation of a dense precipitate. In order to get rid of concomitant proteins and other pituitary hormones, the obtained centrate was added with ammonium sulfate up to 40% of saturation and again centrifuged in the same mode. The obtained centrate, in order to free from ammonium sulfate and concentrate the hormone, was subjected to ultrafiltration on a UAP-200 membrane under pressure. The concentrated hormone was purified by ion exchange chromatography on KU-2 cation exchange resin. For this, cation exchanger was pretreated in a small amount of physiological saline with pH 4.2 and then a concentrated hormone solution was added to it. The mixture was stirred for 60 minutes until complete binding to the cation exchanger. After this, cation exchange resin with a bound hormone was transferred to the membrane and washed from ballast villages with physiological saline with a pH of 4.2, until a protein was found in the washing liquid. The hormone was separated from cation exchanger with a buffer solution with pH 8.5-9.0. As a result of purification, a 300 ml FSH sample with a biological activity of 45-50 m.u. in 1 ml.

 The final product was a clear, odorless and impurity liquid. After concentration of the preparation and standardization in accordance with the preparation FSH-p (Schering Corporation, USA), it was packaged in glass ampoules, which were hermetically sealed. To test the drug for toxicity, white mice and guinea pigs were used, according to existing guidelines. All experimental animals after the administration of the drug were alive.

 For the production test of the manufactured FSH preparation, scientific and production experiments were conducted. For this purpose, in the conditions of the educational and experimental farm of the Kursk Agricultural Institute, 30 clinically healthy black-motley cows were selected on the basis of analogues. Three groups of 10 animals each were formed from the selected animals. The first group was treated with the FSH preparation made, the second group was stimulated with the prototype preparation, the third group was treated with FSH-p preparation (Schering Corporation, 594M89 series). All drugs were used from the 8th day of the sexual cycle according to the 5-day regimen in combination with the prostaglandin drug estrofan (500 μg). At the same time, the dosage of both the test drug and the prototype drug corresponded to a total activity of 50 mg of American FSH-p. On 13-14 day of the sexual cycle, the selected cows were hunted using a test bull and artificially inseminated. On the 20th day of the sexual cycle, non-surgical extraction of embryos was carried out and their evaluation was carried out according to the guidelines (Moscow-Bykovo, Gosagroprom, 1989).

 The research results are presented in table 1, the analysis of which shows that the manufactured FSH preparation in its biological activity significantly exceeds the prototype drug. Thus, the presented technological process for producing FSH from animal pituitary glands is not complicated, does not require expensive equipment and scarce reagents, and the resulting product does not cause side effects and has high biological activity. TTT1

Claims (1)

A method of obtaining a follicle-stimulating hormone preparation from animal adenohypophysis, including homogenization, extraction, precipitation of ballast proteins and hormone release, characterized in that the homogenization is carried out at 2 4 ^o C in the presence of a buffer solution with a pH of 7.0 7.5 in a ratio of 1: 6 1 : 7, which includes 0.89 0.90% sodium chloride, 3.2 - 3.4% ammonium sulfate, 10 15% ethyl alcohol, 0.14 0.15% potassium iodide, the rest is distilled water, extraction is carried out over 50 - 60 min at 2 4 ^o C, the deposition of ballast proteins is carried out by centrifugation quenching the extract until the liquid separates to form a dense precipitate, then ammonium sulfate is added to 35–40% of saturation and centrifuged again, and the hormone is isolated by pressure ultrafiltration and purification by ion exchange chromatography followed by purification on the membrane with physiological saline with a pH of 4.2–4 , 5 and separation from cation exchange resin with a solution of pH 8.5 to 9.0.

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