RU2429862C1 - Method to extract gonadotropin from pregnant horses blood serum - Google Patents

Abstract

FIELD: veterinary science.

SUBSTANCE: method to extract gonadotropin from pregnant horses blood serum (PHS), including fractional deposition of ballast substances and extraction of hormonally active fraction PHS by isopropanol, besides, to deposit ballast substances at concentration of PHS gonadotropin dissolubility, and to extract hormonally active fraction of PHS at concentration of gonadotropin insolubility, the extracted preparation of PHS gonadotropin is preserved in ethanol solution with concentration of the latter that ensures preparation sterility.

EFFECT: method makes it possible to increase purity of produced preparation without loss of its hormonal activity.

1 ex

Description

The invention relates to veterinary medicine, in particular to methods for producing a ballast-free gonadotropic preparation from the blood of roe mares.

A known method for producing a gonadotropic preparation from the blood serum of roe mares (FFA) by sequentially precipitating ballast substances with rivanol solution and cooled acetone, isolating gonadotropin FFA (FFA) with acetone, keeping the mixture for 30-60 minutes at room temperature, washing and lyophilization (Auth. St. USSR No. 619188, A61K 37/38, 1975). The disadvantage of this method is the contamination of the drug with rivanol.

A known method of producing a preparation of HFA, including fractional precipitation of a hormonal substance from FFA with ethyl alcohol, mixing the precipitated fraction with 0.28-0.32% agar solution and subsequent denaturing of the drug with ethyl alcohol (Auth. Certificate. USSR No. 1012913, A61K 37 / 38, 1983).

A known method for the preparation of HFCs is provided, which involves fractional precipitation of ballast substances with ethanol in an acidic medium, washing them, precipitating a hormone-active substance from the supernatant with ethanol, lyophilization of the obtained precipitate, its subsequent purification by extraction with water, desalination by filtration through molecular sieves and reprocessing the first fraction with an organic solvent. Filtration is carried out by passing the solution through a column filled with Sephadex G-25, balanced with water (Aut. St. USSR No. 296373, A61K 35/14, 1970). The use of molecular sieves leads to an increase in the cost of the drug and limits the possibility of using the method. The high degree of purification provided by this method is unprofitable and inappropriate for agricultural production.

A known method for the isolation of gonadotropin from FFA, taken as a prototype, comprising fractional precipitation of ballast substances with metaphosphoric acid and ethanol at a concentration of 50%, precipitation of the hormone-active fraction with ethanol at a concentration of 75%, dialysis of the fraction against water and subsequent fine purification of the drug by gel filtration through Se -phadex G-100 with elution with a solution of sodium chloride buffered with phosphate buffer, and through sulfoethyl-Sephadex G-50 using acetate, phosphate and ammonium acetate buffers. (Gospodaarowicz D., Papkoff H. A simple method for isolation of pregnant mare serum gonadotropin. Endocrinology. 1967, v.80, n.1, p. 699-702).

The method allows to obtain a preparation sufficiently purified from ballast allergens with an activity of 638 (530-825) IE / mg dry matter already at the stage of rough cleaning preceding gel filtration, eliminating the manifestation of anaphylactic shock when used on farm animals. Therefore, such a preparation can be used for hormonal correction in animals and therefore the need for fine purification by gel filtration is eliminated.

However, the method requires the use of a large amount of expensive ethanol.

The technical task of the invention was to develop a method for producing gonadotropin from the blood serum of mares with replacing ethanol with a cheaper substitute having its properties in relation to HFA, that is, having a concentration threshold in the liquid fraction that separates the solubility and insolubility of gonadotropin and coagulating ballast substances at a solubility concentration gonadotropin.

In the process of isolating HFA, apply a cheaper method of preserving the finished product without losing its hormonal activity.

The technical result of the invention is achieved by the fact that the proposed method for the isolation of gonadotropin from FFA, providing for the fractional precipitation of ballast substances, the hormone-active fraction of FFA, its dialysis against water, characterized in that the deposition of ballast substances and the allocation of hormone-active fraction produce isopropanol, and the deposition of ballast substances is carried out at its solubility concentration of HFA, and to isolate the hormone-active fraction of FFA at a concentration of gonadotropin insolubility. At the same time, for preservation of the isolated preparation of HSC, the dissolution and preservation of the drug in ethanol solution are used at a concentration of the latter, which ensures sterility of the drug.

Example. At the initial stage, the bulk of the ballast substances from the FFA is removed by sedimentation by adding to the FFA a 0.5 M solution of freshly prepared metaphosphoric acid (NRA ₃ ) until a pH of 3.0 is reached.

To prepare a 0.5M solution of NRA ₃ , take 3.6 g of phosphorus pentoxide (P ₂ About ₅ ) and dissolved in 100 ml of water. Phosphorus pentoxide in air attracts water very energetically, and therefore it is difficult to accurately weigh the required amount of dry matter, so it works very quickly. When phosphorus pentoxide is combined with water, a very large amount of heat is released and the solution heats up very much, dissolution can occur in the form of an explosion, so phosphorus pentoxide must be introduced into the water in small portions. All this requires very careful handling and protective measures (safety glasses, rubber gloves, protective clothing). A 0.5M solution of NRA ₃ is prepared immediately before use and is not stored.

Due to the fact that phosphorus pentoxide vigorously attracts water from the air, it is stored in a hermetically sealed vessel (desiccator) next to concentrated sulfuric acid or other active desiccants.

The deposition of ballast substances with metaphosphoric acid is carried out at an ambient temperature of 0 ... + 5 ° C with reagents and devices cooled to this temperature. FFA with hormonal activity of at least 100 IE / ml is acidified with a 0.5M solution of NRA ₃ under constant pH control with constant stirring. Upon reaching pH 3, the addition of a 0.5M solution of NRA _{3 is} stopped, the suspension is shaken for 5 minutes. The precipitate formed is separated by filtration through a membrane filter or by centrifugation.

The reaction of the filtrate (supernatant) obtained in the previous procedure was adjusted to pH 4.5, slowly adding a 1N NaOH solution dropwise, cooled, and isopropanol (pre-cooled to -20 ° С) was added in volume to a final volume concentration of 25%. After exposure to cold (0 ... + 5 ° C) for 2 hours, the precipitate formed is separated and discarded.

Cooled isopropanol is again added to the filtrate (supernatant) to a final volume concentration of 50%, converting FFA gonadotropin to a precipitate. The mixture is defended in the cold (0 ... + 5 ° C) for two hours, the precipitate is separated, the filtrate (supernatant) is discarded. The precipitate is dissolved in water in a volume of 1/500 part (× 0.002) of the initial volume of FFA. The solution is dialyzed for 24 hours against water at 0 ... + 5 ° C. The dialysis membrane must retain polymers above 50 kD. An enzyme immunoassay (ELISA) determines the hormonal activity of the resulting drug solution.

The volume of dialysate with a 1% sodium chloride solution is adjusted to a value at which the concentration of HFA is 20,000 IE / ml. The calculation is carried out according to the following formula:

V _con = V _ref * C _ref / 20000,

where V _con is the final volume in ml, to which it is necessary to bring the solution to 1% sodium chloride;

V _ref is the initial volume in ml of the drug solution;

With _ref - HSC activity in the preparation, IE / ml.

Preservation of the drug HSC. Ethanol is added in an equal volume of 96 ° C to the resulting HFA solution. The solution of the drug is poured into vials - 0.1 ml, which is 1000 IE IHFA / vial. Bottles are closed with stoppers and rolled up with aluminum caps.

Use of the drug. Before use, 5 ml of a sterile 1% sodium chloride solution is added to the contents of the vial. For this, observing the rules of asepsis, a solution of sodium chloride is poured into the vial with a syringe through an injection needle, and shaken well. The contents are collected back into the syringe and an intramuscular injection is performed.

The invention can be used in the production of purified gonadotropin from the blood serum of roe mares at biofactories and other hormone production plants.

Claims (1)

The method of isolation of gonadotropin from the blood serum of roe mares (FFA), including fractional precipitation of ballast substances, the hormone-active fraction of FFA, dialysis against water, characterized in that the deposition of ballast substances and the allocation of the hormone-active fraction of FFA is performed with isopropanol, moreover, for the deposition of ballast substances at a concentration of the solubility of gonadotropin FFA, and for the allocation of the hormonally active fraction of FFA at a concentration of insolubility of gonadotropin, an isolated preparation of gonadotropin FFA They are diluted in ethanol at a concentration of the latter, which ensures the sterility of the preparation.