RU2325171C1 - Method of trophoblastic beta-1-glycoprotein release and purification - Google Patents

Abstract

FIELD: medicine; chemistry-pharmaceutical industry.

SUBSTANCE: trophoblastic beta-1-glycoprotein is released from serosity produced as a result of differential centrifugation of retroplacental human blood. Supernatant after first centrifugation is saturated with sodium chloride (0.1-1.0 mole/l) to prevent non-specific sorption and centrifuged once more with triton X-100 (0.01-0.05%) to virus inactivation. Then it is applied to affine sorbent that is sepharose with solid-phase monoclonal antibodies to trophoblastic beta-1-glycoprotein. Protein is eluated with 0.1 M glycine-HCI buffer solution, and after neutralization and in-depth analysis second affine chromatography is carried out on sepharose with modified protein G, released from streptococcus cell wall to prevent immunoglobulin contamination. Solution passed through column and trophoblastic beta-1-glycoprotein is been concentrating and dialyzing from saline and lyophilized.

EFFECT: increased output and purity of end product.

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Description

The invention relates to the pharmaceutical industry and can be used in medicine, biotechnology to obtain a trophoblastic β-1-glycoprotein (TBH) preparation used as a component of diagnostic systems and means for treating cancer, autoimmune and allergic diseases. The method allows to increase the yield and purity of the target product.

It is known that TBH, being a specific marker of pregnancy, is synthesized by trophoblast and secreted into the bloodstream of the mother. According to most authors, TBH is a glycoprotein containing up to 28% carbohydrates and having a molecular weight of from 90,000 D (Bohn N., 1974) to 113,000 D (Tatarinov Yu.S. et al., 1974). However, there is evidence in the literature that TBH forms a family of 4 proteins with a molecular weight of 72, 64, 62, and 54 kD (Plouzek C. A. and Chou J. Y, 1991). Other authors believe that TBH is a heterogeneous protein, the heterogeneity of which is due to the presence of two variants of TBH - alpha and beta (Pola A. et al., 1992). The molecular weight of TBH-alpha is 110 kD, and TBH-beta is 75 kD (Ito M. et al., 1981). The absence of reliably confirmed data on the molecular heterogeneity of TBH and an accurate description of the structure of the molecule is largely the reason for the lack of an effective system for its isolation and purification. The source for receiving TBH is retroplacental blood.

Known multi-stage method for the allocation of TBH from human retroplacental blood [1]. Serum obtained from retro-placental blood was successively fractionated with rivanol and ammonium sulfate, and then subjected to gel filtration on Sephadex G-25. The resulting material was subjected to ion exchange chromatography on DEAE cellulose. TBH containing fractions were combined, fractionated with ammonium sulfate and gel filtration was performed on Sephadex G-200. After another ammonium sulfate fractionation and dialysis, chromatography was performed on a column of hydroxylapatite and, after clarification by centrifugation, ion-exchange chromatography on KM-32 microgranular cellulose. The final stage of purification was preparative isoelectric focusing on ampholines with a range of 3.0-10.0. The result of the entire cleaning procedure was the preparation of TBH with a degree of purification of 93%. The yield was 1.1%.

The purification procedure, which made it possible to obtain 1.8 mg of the relatively pure desired protein from a sample containing 160 mg, involves two gel filtration, two ion exchange, one hydrophobic chromatography, three ammonium sulfate fractionation and isoelectrofocusing procedures.

Thus, the obvious disadvantages of the described method are the extreme bulkiness and complexity, very high cost, especially given the extremely low yield of the target product equal to 1.1%.

There is a method of isolating TBG from waste products of gamma globulin from retro-placental blood [2], involving the suspension of a solid precipitate containing TBG in water, centrifugation and subsequent ammonium sulfate fractionation. After 4-day dialysis and centrifugation, the resulting supernatant is treated with hydroxylapatite and, after incubation for 30-60 minutes, centrifuged again. The supernatant is subjected to concentration on ultrafilters and two-day dialysis, after which it is centrifuged and lyophilized. The lyophilized preparation is dissolved in phosphate buffer and subjected to hydroxylapatite column chromatography. The eluate is dialyzed for 36 hours and lyophilized. The yield is 35.5%, the purity of the drug is 95%. The described method is superior to the previous one both in the purity of the obtained preparation and in the yield, however, it is also very laborious, takes a considerable time and leads to unjustified losses due to numerous dialysis procedures.

The closest analogue of the claimed invention is a method for producing TBG from serum of retro-placental blood or sediment obtained in the production of gamma globulin from serum of retro-placental blood [3], involving ammonium sulfate fractionation, followed by dialysis for 4 days. After centrifugation, the supernatant is subjected to ion exchange chromatography. The eluate is concentrated and dialyzed for 72 hours. The dialyzed preparation is centrifuged and passed through a sorbent containing lectin (concanavalin A). The eluted fraction containing TBH is dialyzed and lyophilized. The yield of the drug with a purity of 96% is 20%.

The procedure of the so-called bich variant of the described ion-exchange chromatography on hexyl sepharose contains high quantitative losses of protein. The duration of the method and the low yield of the target product do not allow us to consider the described method as a solution to the problem of isolation and purification of such a complex protein as trophoblastic beta-1-glycoprotein.

An object of the invention is to increase the yield and purity of released TBH. The task is achieved as follows.

Tested for the absence of hepatitis B virus antibodies to HIV, hepatitis C, retro-placental blood is centrifuged, sodium chloride (0.1-1.0 mol / L) and triton X-100 (0.01-0.05%) are added to it to block non-specific sorption and inactivation of viruses, again centrifuged. Serum thus prepared is saturated with ammonium sulfate to 30%, incubated overnight at + 4 ° C and stirring and centrifuged. The precipitate is washed three times with 30% ammonium sulfate, suspended in 0.15 M phosphate-buffered saline pH 7.0-7.5 containing 0.5 M sodium chloride, and dialyzed in an ultrafiltration cell, passing a 10-fold volume of buffer. The resulting preparation is applied to the first affinity sorbent with immobilized monoclonal antibodies to TBH. Non-specifically adsorbed serum components are washed from the sorbent sequentially with 4-5 volumes of 0.15 M phosphate-buffered saline pH 7.0-7.5 containing 0.5 M sodium chloride and the same volume of 0.15 M sodium chloride solution. Affinity-bound TBH is eluted with 0.1 M glycine-HCl buffer solution.

The eluate is quickly neutralized to a pH of 6.5-7.5, dialyzed against phosphate-buffered saline and applied to a sorbent with immobilized protein G, where selective affinity sorption (negative chromatography) of contaminating immunoglobulins occurs. The contamination of the drug with human G immunoglobulins after the first affinity purification was verified by LC-MS / MS spectroscopy. The effectiveness of the use of this sorbent is due to the ability of protein G isolated from the cell wall of streptococcus to bind extremely effectively to Fc fragments of immunoglobulins G of most mammals, and primarily to all types of human IgG.

The determining advantage of the proposed method is the possibility of a two-stage preparation of TBH with a high degree of purity and effective yield of the target product.

Monoclonal antibody affinity sorbent is prepared as follows. 200 mg of monoclonal antibodies to trophoblastic beta-1-glycoprotein) are incubated with a suspension of 50 ml BrCN-Sepharose FF, previously equilibrated in 0.1 M carbonate-bicarbonate buffer pH 8.3 with 0.5 M sodium chloride (KBB) overnight on a shaker at + 4 ° С. After washing with 15 volumes of CBB, the sorbent is incubated with a 1.0 M ethanolamine solution for 2 hours at room temperature with stirring. The sorbent synthesis is completed by five washing cycles alternately with 0.1 M acetate buffer with 0.5 M NaCl pH 3.5 and 0.1 M Tris HCl buffer with 0.5 M NaCl pH 8.5. The sorbent is stored in buffered saline with 0.1% sodium azide.

The method is illustrated by the following examples.

Example 1. All procedures are carried out at + 4 ° C. 1.0 L of retro-placental blood is centrifuged at a speed of 10,000 rpm for 1 hour, the precipitate is discarded, and sodium chloride is added to the supernatant to a final concentration of 0.5 M and Triton X-100 to a final concentration of 0.02%. Centrifugation is repeated. Serum containing 52 mg of TBH, thus prepared, is saturated with ammonium sulfate to 30%, incubated overnight with stirring, and centrifuged at 10,000 rpm for 1 hour. The precipitate is washed three times with 30% ammonium sulfate, suspended in 0.15 M phosphate-buffered saline pH 7.0-7.5 containing 0.5 M sodium chloride, and dialyzed in an ultrafiltration cell, passing a 10-fold volume of buffer. The resulting preparation is combined with 50 ml of Sepharose FF modified with monoclonal antibodies to TBH. Incubation is carried out for 12 hours with constant stirring on an orbital shaker.

Non-specifically adsorbed serum components are washed out from the sorbent sequentially with 4-5 volumes of phosphate-saline buffer solution containing 0.5 M sodium chloride and the same amount of 0.15 M sodium chloride solution. The sorbent is placed in a chromatographic column 2.5 × 8.0 cm. After washing the sorbent with a sodium chloride solution, an elution with 0.1 M glycine-HCl buffer solution with a pH of 2.55 at a rate of 80 ml / hour is carried out, controlling the process by optical density at a length of waves of 280 nm. Elution is carried out until zero optical density is reached. The eluate fractions containing the desired product are combined, immediately neutralized to a pH of 6.5-7.5, dialyzed against phosphate-buffered saline and applied to a column of Sepharose with protein G. Application rate 60 ml / h.

The resulting preparation containing TBG, purified from impurity proteins, is concentrated and dialyzed in an ultrafiltration cell relative to physiological saline and lyophilized.

The purity of the obtained preparation, analyzed by electrophoresis in a density gradient of polyacrylamide gel, is 98%. The output of the drug according to enzyme immunoassay is 35.9 mg (69%). The resulting preparation is tested for the absence of antibodies to HIV, hepatitis B virus and antibodies to hepatitis C.

Example 2. All procedures are carried out at + 4 ° C. 1.0 L of retro-placental blood is centrifuged at a speed of 10,000 rpm for 1 hour, the precipitate is discarded, and sodium chloride is added to the supernatant to a final concentration of 0.5 M and Triton X-100 to a final concentration of 0.02%. Centrifugation is repeated. Serum containing 52 mg of TBH, thus prepared, is saturated with ammonium sulfate to 30%, incubated overnight with stirring, and centrifuged at 10,000 rpm for 1 hour. The precipitate is washed three times with 30% ammonium sulfate, suspended in 0.15 M phosphate-buffered saline pH 7.0-7.5 containing 0.5 M sodium chloride, and dialyzed in an ultrafiltration cell, passing a 10-fold volume of buffer. The preparation is applied to a chromatographic column 2.5 × 8.0, filled with 50 ml of Sepharose FF with immobilized monoclonal antibodies to TBH, equilibrated with phosphate-buffered saline. Application rate 40 ml / h. Application is carried out by triple passage of the applied volume through the column.

Non-specifically adsorbed blood components are washed out of the sorbent sequentially with a phosphate-saline buffer solution containing 0.5 M sodium chloride and 0.15 M sodium chloride solution at a rate of 80 ml / h until the optical density reaches zero.

After washing the sorbent, an elution with 0.1 M glycine-HC buffer solution with a pH of 2.55 and a speed of 40 ml / h is carried out. The elution process is controlled by optical density at a wavelength of 280 nm. Elution is performed until the optical density reaches zero. The eluted fractions containing the target product are combined, immediately neutralized to a pH of 6.5-7.5, dialyzed against phosphate-buffered saline and applied to a column of Sepharose with protein G. Application rate 60 ml / h.

The entire TBH solution passing through the column is concentrated and dialyzed relative to saline and lyophilized.

The purity of the obtained preparation, analyzed by electrophoresis in a density gradient of polyacrylamide gel, is 98%. The output of the drug according to enzyme immunoassay is 39.5 mg (76%). The resulting preparation is tested for the absence of antibodies to HIV, hepatitis B virus and antibodies to hepatitis C.

Using the proposed method allows to obtain the target product with a high degree of purity (not less than 98%) and high - 69-76% yield. The results are achieved through the use of two high-affinity sorbents that exploit the unique properties of monoclonal antibodies and streptococcus G protein.

Information sources

1. A.V. Sokolov, G.A. Kozlyaev, N.V. Mesnyankin, and Yu.S. Tatarinov. Isolation and purification of specific β1-g-globulin. "Questions of medical chemistry", 1978, No. 24, pp. 240-244.

2. S.V. Moroz, S.K. Krivonosov, A.F. Pavlenko, Yu.S. Ovodov, Yu.S. Tatarinov. A method of obtaining trophoblastic beta-1-glycoprotein from waste retroplacental blood in the production of gamma globulin. A.S. No. 1341736, priority 03/28/85.

3. S.K. Krivonosov, A.A. Terentyev, I.I. Kopteva, I.V. Moskvicheva, P.P. Khokhlov, A.K. Barsukov, Yu.S. Tatarinov. A method of obtaining a trophoblastic betarglycoprotein. A.S. No. 1783644, priority 04.12.89.

Claims (1)

The method of isolation and purification of trophoblastic beta-1-glycoprotein from serum retroplacental blood by fractionation of ammonium sulfate, washing and centrifugation of the dissolved precipitate, dialysis and chromatography, characterized in that the serum after centrifugation at 10000 rpm and saturation with sodium chloride to a final concentration of 0, 1-1.0 mol / L and triton X-100 to a final concentration of 0.01-0.05%, treated with ammonium sulfate to 30% saturation and dialyzed in an ultrafiltration cell, subjected to double affinity chromatography phase, namely, it is applied to the first affinity sorbent, which is sepharose with immobilized monoclonal antibodies to trophoblastic beta-1-glycoprotein, affinity-bound TBH is eluted with 0.1 M glycine-HCl buffer solution, pH 2.55, the eluate is immediately neutralized, dialyzed against phosphate saline buffer solution and applied to the second affinity sorbent, which is sepharose, modified with protein G isolated from the cell wall of streptococcus, the solution passing through the column and containing TBH is concentrated, dialy they are called against saline and lyophilized.