RU2308286C1 - Method for production of alpha-fetoprotein - Google Patents

Abstract

FIELD: chemical and pharmaceutical industry, in particular method for production of alpha-fetoprotein (AFP).

SUBSTANCE: claimed method includes target preparation isolating and from blood serum and purification by double affinity chromatography, wherein between chromatography purification steps proteins are fractionated with 0.1-1 g/l aprothynine and 19-40 % ethanol at pH 5.7-5.8 and at temperature from -3°C to -5°C, and inactivation of bacteria and viruses is carried out before the first purification step on immunosorbent in presence of chloroform and Triton X-100 solutions. Blood serum is applied on affinity sorbent with preliminary blockage of non-specific sorption active site with cattle serum, and application on immunoaffinity sorbent with antibodies against AFP is carried out at pH 8.0-9.0 at 18-20°C. Impurity proteins of blood serum are removed by non-specific elution with 1 M and 2 M magnesium chloride solution, and affinity-bonded AFP is eluted with 4 M magnesium chloride solution and Gly-HCl buffer solution with pH 2.0-3.0, and as the second affinity sorbent sefarose with antibodies against human IgG is used.

EFFECT: new method for production of alpha-fetoprotein.

1 ex, 1 tbl, 1 dwg

Description

The invention relates to the pharmaceutical industry and the preparation of a lyophilized preparation of AFP, belonging to the group of immunomodulators and used as a means for the treatment of cancer, autoimmune and allergic diseases.

AFP is a fetal glycoprotein with a molecular weight of about 70 kDa. AFP is produced in large quantities by the cells of the liver and abdominal wall of the fetus. Abortive, umbilical and placental blood obtained during medical abortions from clinically healthy women who underwent a standard examination is the raw material for obtaining AFP. Obtained abortive material must be tested for the absence of antibodies to human immunodeficiency virus (HIV) and for the absence of hepatitis B virus and antibodies to hepatitis C.

AFP is used predominantly parenterally. AFP aqueous solutions are unstable during storage, so the task of obtaining highly stable dosage forms is relevant. The most effective way to stabilize drugs for long-term storage is to lyophilize them with appropriate additives [1].

Oncofetal proteins are obtained from human or animal embryonic or tumor tissue. Highly purified AFP is obtained from abortive blood by affinity chromatography.

A known method of producing AFP from human tumor tissue, including the isolation and chromatographic purification of the target product sequentially on several sorbents [2]. Its disadvantage is the high complexity of the process and the low yield of the target product.

A method has been developed for obtaining AFP from human embryonic tissue [3], including its grinding, washing, homogenization, trypsin treatment for 1 h at a temperature of no more than 37 ° C. The resulting cell suspension was cultured in a nutrient medium for 7-8 days at 37 ° C, and the target product was separated by centrifugation at 3000 rpm. A clear orange-yellow liquid is obtained containing 10% AFP.

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

There is a method for producing AFP from human embryonic tissue [2], including processing of the starting material with butyl alcohol, centrifugation, dialysis of the aqueous protein phase relative to a 0.9% sodium chloride solution, centrifugation, incubation of the obtained supernatant with estrogen at a final concentration of 0.005% at a temperature of 0-10 ° C for 8 h and the isolation 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 is 90-95%.

The disadvantages of the method are the lack of 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 a flammable sharply smelling organic reagent - butyl alcohol.

One of the closest to the invention is a method for producing AFP from human embryonic tissue for 8-12 weeks, obtained by medical abortion, which includes the stages [4]: grinding and homogenization of embryonic tissue; centrifuging the homogenate at 5000 rpm; clarification of the supernatant by centrifugation at 12,000 rpm; purification of the target product by chromatography on DEAE 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 DEAE cellulose.

The disadvantages of the described method are the duration of the procedure, low yield (25-30%) and insufficient purity (85-90%) of the target product, high complexity and the need for sophisticated equipment (high-speed centrifuges).

The closest analogue is the method for producing AFP [5]: the abortive human blood is centrifuged, the obtained supernatant is subjected to two-step chromatographic purification (affinity chromatography on Br-CN-sepharose with immobilized antibodies to AFP and affinity chromatography on Br-CN-sepharose with immobilized balanced complex human blood proteins The resulting AFP solution is desalted by dialysis against water, sterilized and lyophilized.

The disadvantages of the prototype are low yield (60-70%) and high contamination with impurity proteins (purity 90-95%) of the target product.

The technical result of the invention is to increase the yield of AFP, reducing the time of the technological cycle and achieving maximum purity.

The method is as follows.

Abortive, placental or umbilical cord blood is thawed at a temperature of 2-6 ° C and an analysis is made for the absence of viruses. An abortive, umbilical or placental blood or an extract of embryonic tissues, tested for the absence of hepatitis B virus, antibodies to HIV, hepatitis C, is added with vigorous stirring a chloroform solution to achieve 0.1% concentration in order to dissolve lipids and inactivate enveloped viruses and bacteria. Then, in order to precipitate high molecular weight (“heavy”) proteins (mainly proteins of the blood coagulation system and Ig classes A, M, G) and to block proteolysis reactions into the blood with chloroform with vigorous stirring, aprotinin is added at a rate of 0.1-1.0 g / l The blood thus treated is left for 24 hours in the refrigerator at a temperature of 2-6 ° C. After 24 hours, a significant amount of sediment is observed at the bottom of the blood vessel, which is removed by centrifugation (at 6000-16000 rpm).

Sodium chloride (0.1-1.0 mol / L) and Triton X-100 (0.01-0.5%) are added to the supernatant to block non-specific sorption and additional inactivation of viruses. Then the serum or extract is subjected to sterilizing filtration: first through three layers of cotton autoclaved and tucked into the filter holder, and then through a cascade of filters with pore sizes from 3.0 to 0.22 μm.

The supernatant is subjected to sterilizing filtration: sodium chloride (0.1-1.0 mol / L) and Triton X-100 (0.01-0.5%) are added to diluted sterile serum to block non-specific sorption and inactivation of viruses. The sterile serum thus prepared is adjusted with a sodium hydroxide solution to pH 8.0-9.0 in order to create conditions for enhancing the affinity binding of AFP to antibodies. The sorbent with immobilized antibodies to AFP before applying serum is subjected to treatment with a 1.5 M solution of magnesium chloride: sorbent / MgCl ₂ = 1: 3, exposure for 30 minutes (2 times). Then the sorbent is washed with a 0.15 M sodium chloride solution (10 volumes). After that, in order to block the active centers of non-specific sorption, the immunosorbent with antibodies against AFP is subjected to treatment with cattle serum (cattle).

Cattle serum is heated to 37 ° C in a thermostat and combined with a sorbent. Blocking lasts for 30 minutes at a temperature of 18-20 ° C (in the ratio of sorbent / serum = 1/3). After that, the sorbent is washed with a 0.15 M sodium chloride solution (10 volumes).

Non-specifically adsorbed blood components are washed from the sorbent sequentially with 4-5 volumes of 0.15 M phosphate-buffered saline with a pH of 7.0-7.5 containing 0.5 M sodium chloride and the same volume of sodium chloride solution (0.15 mol / l).

Blood serum (pH 8.0–9.0) is combined with the sorbent and the AFP is immunosorbed in the “free volume” until the serum is completely depleted. Sorption is continued for 60 minutes at a temperature of 18-20 ° C and with vigorous stirring on an overhead stirrer, followed by settling.

After settling, the serum is decanted and washed with a 1.0 M solution of magnesium chloride on a Schott filter with exposure for 30 minutes with stirring. Repeated non-specific elution is carried out with a 2.0 M solution of magnesium chloride under similar conditions. Then the sorbent is washed with 0.15 M sodium chloride solution (10 volumes).

The affinity-coupled AFP is eluted twice in the “free volume” (1.5 hours) with vigorous stirring and a temperature of 18-20 ° C with a 4.0 M solution of magnesium chloride, adjusted with 0.1 M Gly-HCl buffer to pH 2.0- 3.0 with constant monitoring of pH. The ratio of eluent to the volume of the sorbent is 1: 1. Then the sorbent is washed with a 0.15 M sodium chloride solution to a pH of 6.0-7.0.

The eluate is neutralized with a 0.5 M sodium hydroxide solution to pH 6.0-8.0 and dialyzed against 0.15 M sodium chloride in dialysis bags or on special equipment (ultrafiltration or dialysis plants).

Concentration of the eluate to the required volume is carried out according to the AFP content of 0.6-1.2 mg / ml on a concentrating cell of the Amicon type with a membrane pore of 30 μm.

Then, in order to precipitate the non-specifically adsorbed human IgG, 95% ethanol cooled in the freezer is added to the AFP concentrate with vigorous stirring until it is saturated to 19-40% by volume (formula 1), after which the pH of the concentrate is adjusted to 5.7-5, 8 Gly-HCl buffer solution (0.1 M). The exposure of the mixture at a temperature of minus 3-5 ° C lasts two days.

The formula for calculating the saturation of an AFP solution with ethanol is:

, где

where

V is the volume of 95% ethanol;

V ₁ - the required vol.% Ethanol content in the protein solution;

V ₂ - the volume of the protein solution, which must be saturated with ethanol;

V ₃ - vol.% The content of the protein solution in the mixture after adding ethanol;

1.05 - ethanol dilution factor up to 95%.

After 2 days, the precipitate formed in the AFP solution (human IgG) is separated by centrifugation in the mode of 10,000 - 26,000 rpm at a temperature of minus 3 - 5 ° C.

The supernatant is dialyzed against 0.15 M sodium chloride.

After performing the described technological process, the electrophoretic purity of the AFP protein is determined (densitometrically) and a quantitative determination of human IgG is carried out (immunoturbidimetry method).

The purity of the AFP preparation should be at least 97%.

When the content of impurity proteins is not more than 3% AFP, they are stabilized with polysaccharides (dextrans), sterilizing filtration, bottling and freeze drying of the AFP preparation are performed.

In the presence of impurity proteins (human IgG; according to two-dimensional gradient electrophoresis and mass spectrophotometry) more than 3% carry out additional purification of the AFP concentrate on an immunoaffinity sorbent with antibodies against human IgG. AFL concentrate after dialysis is applied to the sorbent with immobilized antibodies against human IgG in the “columnar” version, where affinity sorption of human immunoglobulins (negative immunosorption) occurs, which contaminate the drug after the first affinity purification and alcohol fractionation. The drug obtained after negative immunosorption is diluted with 0.9% sodium chloride solution and reopoliglyukin to a concentration of AFP of 0.6-0.9 mg / ml and dextran 4-10 mg / ml. After sterilizing filtration, the AFP substance is lyophilized.

Significant differences of the proposed method from the prototype are preliminary purification of the initial blood by fractionation of serum proteins with aprotinin, saturation of the blood with a solution of chloroform and Triton X-100 to dissolve lipids and inactivate viruses, suppress nonspecific sorption by treating the immunoaffinity sorbent with cattle serum, applying serum to the immunoaffinity sorbent pH 8.0-9.0, removal of non-specifically adsorbed proteins with 1M and 2M magnesium chloride solution, AFP elution with 4M magnesium chloride solution at H 2.0-3.0, tertiary preparation AFP ethanol fractionation method (19-40% saturation), and "negative" for the second immunosorbent chromatography, which is a Sepharose-immobilized anti-human IgG.

It should also be noted that the proposed method for the preparation of AFP reduces the time of the technological cycle from 14 to 4 days and does not require expensive chromatographic equipment.

An example of a specific implementation.

Preparation of blood serum

Defrosting 10 liters of blood. Chloroform in the amount of 100 ml per 10 l and aprotinin in the amount of 5 g are added to the blood. Blood settling in the refrigerator at a temperature of 2-6 ° C for 24 hours. Centrifugation at 6000 rpm for 40 minutes, decantation. Serum volume after centrifugation was 7680 ml. Triton X-100 is added to serum to 0.01% saturation and 156 g of sodium chloride. Filtration through 3 layers of cotton fabric and membrane filters 3.0-0.22 microns. Triton X-100 solution is added to the serum to 0.01% saturation.

II. Sorbent preparation

Sorbent (Sepharose CL-4B) with covalently sewn antibodies (4-6 mg / ml) against human AFP with a volume of 180 ml is washed with 5 volumes of a solution of 0.15 M sodium chloride.

Before applying the serum, the sorbent was treated with a 1.5 M solution of magnesium chloride: cop6ent / MgCl ₂ = 1: 3, exposure for 30 minutes (2 times). Then the sorbent is washed with a 0.15 M sodium chloride solution (10 volumes).

Cattle serum is heated to 37 ° C in a thermostat and combined with a sorbent. Blocking lasts for 30 minutes at a temperature of 18-20 ° C (in a ratio of 1: 3). After that, the sorbent is washed with a 0.15 M sodium chloride solution (10 volumes).

III. AFP sorption.

Sorption occurs at pH 9.0 for 60 minutes at room temperature and vigorously stirring with an overhead stirrer until the AFP content is completely depleted, followed by settling for 2 hours.

IV. Nonspecific elution. After settling, the serum is decanted and the sorbent is washed with a 1.0 M solution of magnesium chloride on a Schott filter with exposure for 30 minutes with stirring. Repeated non-specific elution is carried out with a 2.0 M solution of magnesium chloride under similar conditions. Then the sorbent is washed with 0.15 M sodium chloride solution (10 volumes).

V. Elution AFP.

The elution of AFP is carried out twice (for 1.5 hours) with vigorous stirring at room temperature with 4 M magnesium chloride, adjusted with 0.1 M Gly-HCl buffer to pH 2.2 with constant monitoring of pH. The ratio of eluent to the volume of the sorbent is 1: 1. Then the sorbent is washed with a 0.15 M sodium chloride solution to a pH of 7.0. The eluent is neutralized with sodium hydroxide solution to a pH of 6.8.

VI. Concentration of the eluate and alcohol precipitation.

The concentration of the eluate is carried out on a concentrating cell "Amicon" to a volume of 113 ml with the replacement of the solution with 0.15 M sodium chloride. Then, cooled 95% ethanol is added to the concentrate until it is saturated up to 32%, after which the pH of the concentrate is adjusted to 5.7-5.8 with 0.1 M Gly-HCl buffer solution. The exposure of the mixture at a temperature of minus 3-5 ° C lasts two days.

Table Sample Name Sample volume, ml AFP The concentration of human IgG (ITD), mcg / ml % contamination mcg / ml in volume, mg 44 series, after deposition by Cohn 42 501.97 21.08 0.9 (37.8) 0.18 45 series, after deposition by Cohn 43 163.56 7.03 2.6 (111.8) 1,59 Series concentrate after “negative” chromium thirty 962.87 28.88 0 0

After centrifugation in a high-speed centrifuge at 13,400 rpm for 15 minutes and a temperature of minus 3-5 ° C, the precipitate is quantitatively separated from the supernatant. The supernatant containing AFP is dialyzed against sodium chloride solution. The concentrate is subjected to analysis (see table; drawing, series 44-45). As a result of technological operations, an AFP preparation with a purity of 99.82-98.41% is obtained. After "negative" chromatography with Sepharose CL 4 B with antibodies to human IgG, the purity of the AFP preparation is about 100%.

The resulting preparation is tested for the absence of antibodies to HIV, hepatitis B virus and antibodies to hepatitis C. The drug is sterile, does not contain pyrogenic and toxic impurities.

Using the proposed method allows, in comparison with the prototype: to increase the purity of the target product due to preliminary fractionation of the proteins of the initial blood serum with aprotinin, ethanol and double affinity chromatography on sorbents of high specificity; increase the yield of AFP from an affinity sorbent with immobilized antibodies to AFP due to the use of a 4.0 M solution of magnesium chloride and Gly-HCI buffer solution; reduce the time of the full technological cycle due to the lack of gel filtration stage.

Information sources

1. Nikitin V.V., Zvyagin I.V. Freezing and drying of biological products. M .: Kolos, 1971, 344 p.

2. A.S. 1497806 USSR MKI ⁴ A61K 37/02, 48 "Method for producing alpha-fetoprotein".

3. A.S. No. 403407, class ³ A61K 35/48 "Method for producing a biostimulant"; declared 11/26/68; publ. 10.26.73.

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

5. RF patent No. 2100031 "Method for producing alpha-fetoprotein." / V.V. Starikov, S.Yu. Rodionov, Myakkohodov V.A., Pak N.A. (Russia) .- No. 95120418/113; declared 12/01/95; publ. 12/27/97; Bull. Number 36.

Claims (1)

A method of producing alpha-fetoprotein (AFP), including the isolation of blood serum and purification using double affinity chromatography, characterized in that they carry out preliminary purification of the original blood by saturating the blood with a solution of chloroform to separate lipids, fractionating serum proteins with aprotinin and saturating the blood with Triton X solution -100 for inactivation of viruses and lipid dissolution, suppression of nonspecific adsorption on a sorbent with antibodies against AFP due to the treatment of immunoaffinity sorbent with large serum about cattle (cattle), applying serum to the immunoaffinity sorbent at pH 8.0-9.0, removing non-specifically adsorbed proteins with 1M and 2M magnesium chloride solution, AFP elution is carried out with 4M magnesium chloride solution in 0.1M Gly-HCl buffer at pH 2.0-3.0 and AFP purification by ethanol fractionation of 19-40% saturation and "negative" chromatography on a second immunosorbent, which is Sepharose, with immobilized antibodies against human IgG.