RU2100031C1 - Method of alpha-fetoprotein preparing - Google Patents

Abstract

FIELD: medicine, biotechnology. SUBSTANCE: abortive human blood is centrifuged and supernatant is purified firstly on affinity resin that contains 10 mg/ml antibodies raised to alpha-fetoprotein covalently cross-linked with BrCN-Sepharose and then on affinity resin that contains MO mg/ml antibody complex raised to human blood proteins and covalently cross-linked with BrCN-Sepharose. The obtained alpha-fetoprotein solution is desalted by dialysis against water, sterilized and dried by lyophilization. EFFECT: improved method of preparing. 3 cl

Description

 The invention relates to biological chemistry (mainly to biotechnology), in particular to the production of oncofetal protein alpha-fetoprotein, and can be used in medicine for the treatment of tumor diseases and diseases with autoimmune disorders.

 Oncofetal proteins, in particular alpha-fetoprotein (AFP), are widely used in medicine for the manufacture of various immunodiagnostics necessary for the recognition of malignant tumors, as well as the treatment of diseases with autoimmune disorders and tumor diseases, therefore, the task of improving the known methods for obtaining these proteins is relevant.

 Oncofetal proteins are obtained from human or animal embryonic or tumor tissue.

A known method of producing AFP from human tumor tissue, including the isolation and chromatographic purification of the target product sequentially on several sorbents [1]
The disadvantage of this method is the low content of the target product in the feedstock (50 to 100 μg / ml), the high complexity of the process and the low yield of the target product.

A known method of producing AFP from human embryonic tissue [2] including its grinding, washing, trypsin treatment for 1 hour at a temperature of not more than 37 ^o C. The resulting cell suspension is cultured in a nutrient medium for 7-8 days at 37 ^o C, and the target product is separated by centrifugation at 3000 rpm A clear orange-yellow liquid is obtained containing 10% oncofetal protein.

 The disadvantages of this method are the low yield and high content of impurity proteins.

A known method of producing AFP from human embryonic tissue [3] comprising treating the starting tissue with butyl alcohol, centrifuging at 6000 rpm for 45 minutes, dialysing the aqueous-protein phase against a 0.9% sodium chloride solution, centrifuging, incubating the obtained supernatant with estrogen in a final concentration of 0.005% at 0 -10 ^o C for 8 hours and the selection of the target product from the supernatant by chromatography on sepharose CL-4B with immobilized estrogen. The yield of the target product is 60-70% and the purity of the drug 90 -95%
The disadvantages of the method are the insufficient purity of the target product, the presence of an admixture of albumin and hemoglobin, as well as the high complexity of the method and the need to work with flammable sharply smelling organic reagent butyl alcohol.

 Closest to the invention is a method for producing AFP from embryonic tissue of human embryos 8-12 weeks, obtained by medical abortion, including the following stages [4] grinding and homogenization of embryonic tissue, centrifugation of the homogenate at 5000 rpm, collecting supernatant; clarification of the supernatant by centrifugation at 12,000 rpm; purification of the target product by chromatography on DE-cellulose; purification of the target product by Sephadex G-100 chromatography with cybacron blue F36-A dye immobilized on it; purification of the target product by rechromatography on Sephadex G-100 with cybacron blue dye F26-A immobilized on it; purification of the target product by chromatography on DE-cellulose.

 The disadvantages of the prototype are the duration of the method; low yield of the target product (25–30%), insufficient purity of the preparation (85–90%), high laboriousness of the method, and the need for high-speed centrifuges (which, as is known, are in great shortage in our country).

 The technical task of the invention is to simplify the known method, increasing the frequency and output of the AFP.

The task is achieved by the proposed method, which consists in the following: take abortive blood obtained from medical abortions, ballast proteins are separated by centrifugation, and the obtained supernatant is subjected to chromatographic purification first on a column with a resin containing covalently attached antibodies to AFP, and then on a column with a resin containing a complex of covalently sewn antibodies against human blood proteins. Glutaraldehyde activated Br-CN-Sepharose, containing 10 mg / ml of anti-AFP antibodies (first chromatography) and 10 mg / ml of an anti-human protein complex (second chromatography), is predominantly used as an affinity resin. The entire AFP solution passing through the second column is collected and dialyzed against distilled or deionized water. Then the drug is sterilized and freeze-dried. The resulting AFP preparation is checked for purity immunochemically and by polyacrylamide gel electrophoresis. The yield of the target product is 60 72.7% Purity of the drug 95 -96%
The defining difference of the proposed method from the prototype is that the chromatographic purification of the target product is carried out in two stages, first on an affinity resin with antibodies to AFP covalently attached to it, and then on a resin column containing a complex of covalently sewn antibodies against human blood proteins. This makes it possible to sharply increase the purity and quality of AFP, since the first sorbent has an increased affinity for AFP, and the second sorbes to the maximum the entire spectrum of impurity proteins present in the AFP solution (albumin, hemoglobin, and other proteins). The use of these highly specific sorbents can also simplify and reduce the duration of the method by reducing the stages of chromatographic purification of the target product from 4 to 2.

 Due to the fact that information on a similar method for producing alpha-fetoprotein is not found in well-known patent and scientific and technical sources, it can be concluded that the proposed technical solution meets the criteria of "novelty and inventive step".

Example 1. All procedures at +4 ^o C. 1 l of abortion blood is centrifuged at a speed of 3000 rpm for 30 minutes, the precipitate of ballast proteins is discarded, and 800 ml of the supernatant is applied to a chromatography column with a volume of 50 cm ³ with an affinity resin containing 10 mg / ml antibodies to AFP, covalently sewn to Br-CN-sepharose using glutaraldehyde (produced by NPO Vector, Russia). Application rate 100 ml / h. Next, the column was washed from unbound proteins with 200 ml of 0.1 M phosphate buffer (pH7.0). The AFP elution is carried out using a 0.1 M HCl solution (pH2.0), passing through a column of 50 ml of eluent. The elution process is controlled by the color change of the solution flowing from the brown to yellow column spectrometrically at a wavelength of 260 nm. The eluate containing the desired product is neutralized to pH7.0 with a NaOH solution and adjusted to a volume of 100 ml. Then, the purification of the target product by affinity resin chromatography containing 10 mg / ml of a balanced solution of antibodies against human blood proteins (starting from NPO Vektor, Russia) is started. For this, the eluate containing the target product after the first chromatography is applied to a 50 cm ³ chromatographic column with the above affinity resin at a rate of 50 ml / hour. The entire solution passing through the column (about 100 ml) containing AFP, purified from albumin and other ballast proteins, is collected in a container. The AFP solution from the tank is poured into a dialysis stop and dialyzed against deionized water at + 4 ^o C for 6 hours

The AFP solution desalted after dialysis is diluted with a NaCl solution (pH7.0) to an AFP concentration of 100 mg / ml, frozen in the refrigerator and stored at -18 ^° C until sterile filtration begins.

After sterilizing filtration, the AFP solution is poured into 1 ml ampoules, freeze-dried and stored at + 4 ^o C. The resulting AFP preparation is checked for purity immunochemically and by polyacrylamide gel electrophoresis. Purity of the obtained preparation 96%. Yield 72.7%. The preparation does not contain pyrogenic and toxic impurities.

Example 2. All procedures are carried out at + 4 ^o C. 1 l of abortion blood is clarified by centrifugation (3000 rpm, 15 min), the precipitate of ballast proteins is discarded, and the supernatant is applied to a chromatographic column with a volume of 50 cm ³ with an affinity resin containing 10 mg / ml of antibodies to AFP. Application rate 100 ml / hour. The column is washed from unbound proteins with 100 ml of physiological NaCl solution (pH7.0), and AFP elution is carried out using a 0.1 M HCl solution (pH2.0), passing through the column 50 ml of eluent. The eluate is collected, neutralized to pH7.0 with a NaOH solution and adjusted to a volume of 100 ml. Then the AFP solution is applied to a 50 cm ³ column with an affinity resin containing 10 mg / ml of anti-albumin antibody at a rate of 50 ml / hour. The AFP solution is collected in a container, then poured into a dialysis unit and dialyzed against distilled water for 10 hours. The resulting AFP solution is diluted with saline to an AFP concentration of 100 mg / ml, sterilized, poured into ampoules and freeze-dried. The yield of the drug is 60%. The purity of the drug is 95%.
Using the proposed method allows, in comparison with the prototype:
to simplify the method by reducing the duration and reduction of the two stages of chromatography and two stages of high-speed centrifugation;
to increase the purity and quality of the target product due to high-performance chromatographic purification of the product;
to provide the possibility of using the drug in medicine for the treatment of malignant tumors (and not just as an immunodiagnosticum).

Sources of information
1. Phil Gold et all. "Physicochemical Approach to the Purification of Human Fetoprotein from the Ascites Fluid of a Hepatoma-bearning Patient. Canser Research. 1978, No. 36, p. 6-12.

2. USSR author's certificate N 403407, class ³ A 61 K 35/48 "Method for producing a biostimulator" decl. 11/26/68. publ. 10.26.73.

3. USSR author's certificate N 1497806, MKI ⁴ A 61 K 37/02 "Method for producing alpha-fetoprotein".

 4. Klaus Huse et all "A novel purification procedure for human fetoprotein by application of immobilized Cibacron Blu F36-A as affinity ligand Clinica Chimica Acta, 133, 1983, 335-340. Prototype.

Claims (3)

 1. The method of producing alpha-fetoprotein, including clarification of the feedstock by centrifugation and chromatographic purification of the target product on an affinity sorbent, characterized in that the chromatographic purification is carried out sequentially in two stages, first on a sorbent containing antibodies to alpha-fetoprotein, and then on the sorbent, containing a complex of antibodies against human blood proteins.  2. The method according to claim 1, characterized in that Br-CN-Sepharose activated with glutaraldehyde is used as an affinity sorbent.  3. The method according to claim 1, characterized in that the affinity resin in the first stage of chromatography contains 10 mg / ml of antibodies to alpha-fetoprotein, in the second stage of 10 mg / ml of a complex of antibodies to human blood proteins.