Method of trophoblastic beta-1-glycoprotein release and purification

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.

2 ex

 

The invention relates to pharmaceutical industry and can be used in medicine, biotechnology for producing drug USSR β-1-glycoprotein (TBG), used as a component of diagnostic systems and tools for the treatment of cancer, autoimmune and allergic diseases. The method allows to increase the yield and purity of the target product.

It is known that TBG, as a specific marker of pregnancy, is synthesized by trophoblasts and secreted into the bloodstream of the mother. According to most authors TBG is a glycoprotein containing up to 28% carbohydrates and having a molecular weight of 90000 D (Bohn N., 1974) to 113000 D (Tatarinov US and others, 1974). However, in the literature there are data that TBG forms a family consisting of 4 proteins with a molecular mass of 72, 64, 62 and 54 KD (Plouzek CA and Chou J.Y, 1991). Other authors believe that TBG is a heterogeneous protein heterogeneity due to the presence of two options TBG - alpha and beta (Pola A. et al., 1992). When this molecular weight TBG-alpha equal to 110 KD, and TBG-beta 75 KD (M. Ito et al., 1981). The absence of well-attested data about the molecular heterogeneity of TBG and accurate description of the structure of the molecule is largely the reason for the lack of an effective system of its isolation and purification. Source for p the receipt of TBG is retroplatsentarno blood.

Known multi-step allocation method of TBG retroplatsentarno human blood [1]. Serum obtained from retroplatsentarno blood was fractionally consistently with rivanol and ammonium sulfate, and then subjected gelfiltration on Sephadex G-25. The resulting material was subjected to ion exchange chromatography on DEAE-cellulose. The fractions containing TBG, United, fractionally ammonium sulfate and were gelfiltration on Sephadex G-200. After another alpacamania fractionation and dialysis was performed chromatography on a column of hydroxylapatite and after clarification by centrifugation, ion-exchange chromatography on microgranular cellulose KM-32. The final stage of purification was the preparative isoelectrofocusing on impaling with a range of 3.0 to 10.0. The result of the entire purification procedure was to obtain the drug TBG with purification efficiency of 93%. The yield was 1.1%.

The cleaning procedure allowed to obtain 1.8 mg relatively clean of the desired protein from a sample containing 160 mg, involves two procedures gelfiltration, two ion-exchange, one hydrophobic chromatography, three procedures alpacamania fractionation, isoelectrofocusing.

Thus, the apparent disadvantages of the described method are extraordinary complexity and trudem is here, very high cost, especially considering the extremely low yield of the target product is equal to 1.1%.

There is a method of allocating TBG from waste products of gamma globulin from retroplatsentarno blood [2], providing for the suspension of a solid residue containing TBG in water, centrifugation and subsequent ammonium sulfate fractionation. After 4 days of dialysis and centrifugation of the resulting supernatant is treated with hydroxyapatite and Proskurov 30-60 minutes, and again centrifuged. The supernatant is subjected to concentration ultrafilter and the two-day dialysis, then centrifuged and lyophilizers. Dried product is dissolved in phosphate buffer and subjected to column chromatography on hydroxyapatite. The eluate cialiswhat within 36 hours and lyophilizers. The output is 35.5%, purity of the drug - 95%. The described method is superior to previous and purity of the drug and exit, but also very time-consuming, takes significant time and leads to unnecessary losses due to the numerous procedures of dialysis.

The closest analogue of the claimed invention is a method for TBG serum retroplatsentarno blood or sludge obtained in the production of gamma globulin from the serum retroplatsentarno blood [3], predusmatriva the store ammonium sulfate fractionation, followed by dialysis for 4 days. After centrifugation the supernatant is subjected to ion-exchange chromatography. The eluate concentrate and cialiswhat within 72 hours. Otvetsvennyy the preparation is centrifuged and passed through the sorbent, containing lectin (concanavalin a). Elyuirovaniya fraction containing TBG, dialist and lyophilizers. The release of the drug in the purity of 96% is 20%.

In the so-called BIC options described ion-exchange chromatography on hexylsilane laid high quantitative loss of protein. The duration of the method, and the low yield of the target product does not allow to consider the described method as a solution to the problem of isolation and purification of this protein complex as USSR beta-1-glycoprotein.

An object of the invention is to increase the yield and purity allocated TBG. This object is achieved as follows.

Proven in the absence of hepatitis b virus antibodies to HIV, hepatitis C retroplatsentarno blood is centrifuged, add sodium chloride (0.1 to 1.0 mol/l) and Triton X-100 (0.01 to 0.05%) for blocking of nonspecific adsorption and inactivation of viruses, and again centrifuged. The thus prepared serum saturated with ammonium sulfate to 30%, incubated over night at +4°and With stirring, and centrifuged. The precipitate washed three times with 3% ammonium sulfate, suspended in 0.15 M phosphate-saline buffer solution pH 7.0 and 7.5, containing 0.5 M sodium chloride, and cialiswhat in the ultrafiltration cell, missing a 10-fold volume of buffer. The resulting preparation is applied to the first affinity sorbent with immobilized monoclonal antibodies to TBG. Nonspecific sorbiruyushchiesya components of the serum wash with sorbent consistently 4-5 volumes of 0.15 M phosphate-saline buffer solution pH 7.0 and 7.5, containing 0.5 M sodium chloride, and the same volume of 0.15 M sodium chloride solution. Affine-associated TBG elute with 0.1 M glycine-HCl buffer solution.

The eluate quickly neutralized to a pH of 6.5-7.5, dialist against phosphate-saline buffer solution and applied to the sorbent with immobilized protein G, where selective affinity sorption (negative chromatography) contaminating immunoglobulins. Contamination of the drug immunoglobulins G man after the first affinity purification verified by the study of method LC-MS/MS spectroscopy. The effectiveness of the use of this sorbent due to the ability of G protein isolated from the cell wall of Streptococcus, extremely effectively communicate with Fc fragments of immunoglobulin G most mammals and in the first place with all types of human IgG.

The decisive advantage : the BA is the possibility of two-stage receiving the drug TBG with a high degree of purity and effective yield of the target product.

Affinity sorbent with monoclonal antibodies obtained as follows. 200 mg of monoclonal antibodies to the USSR beta-1-glycoprotein) is incubated with a suspension of 50 ml of Enrichment-sepharose FF, pre-equilibrated in 0.1m carbonate-bicarbonate buffer pH 8.3 with 0.5m sodium chloride (CBS) overnight on a shaker at +4°C. After washing the 15 volumes CBS sorbent incubated with 1.0 M solution of ethanolamine for 2 hours at room temperature and stirring. Completes the synthesis of the sorbent five cycles of washing successively with 0.1 M acetatis buffer with 0.5 M NaCl, pH 3.5 and 0.1 M HCl buffer with 0.5 M NaCl, pH 8.5. Stores the sorbent in buffered saline solution with 0.1% sodium azide.

The method is illustrated by the following examples.

Example 1. All procedures carried out at +4°C. 1.0 l retroplatsentarno blood centrifuged with a speed of 10,000 rpm for 1 hour, the precipitate discarded, and the supernatant add sodium chloride to a final concentration of 0.5 M and Triton X-100 to a final concentration of 0.02%. The centrifugation repeated. Serum containing 52 mg TBG, thus prepared, is saturated with ammonium sulfate to 30%, incubated overnight with stirring and centrifuged with a speed of 10,000 rpm for 1 hour. The precipitate washed three times 30% ammonium sulfate, suspended in 0.15 M phosphate-saline buffer solution pH 7.0-7.5, the content is the future of 0.5 M sodium chloride, and cialiswhat in the ultrafiltration cell, missing a 10-fold volume of buffer. The resulting preparation combined with 50 ml sepharose FF-modified monoclonal antibodies to TBG. Incubation is carried out for 12 h with constant stirring on an orbital shaker.

Nonspecific sorbiruyushchiesya components of the serum wash with sorbent consistently 4-5 volumes of phosphate-saline buffer solution containing 0.5 M sodium chloride solution and the same amount of 0.15 M solution of sodium chloride. The sorbent is placed in a chromatographic column of 2.5×8,0 see After washing the sorbent solution of sodium chloride conduct elution with 0.1 M glycine-HCl buffer solution with a pH of 2.55 at 80 ml/hour while on the optical density at a wavelength of 280 nm. The elution is carried out to achieve zero values of optical density. Fractions of the eluate containing the desired product are pooled, immediately neutralized to a pH of 6.5-7.5, dialist against phosphate-saline buffer solution and applied to a column separate with protein G. the Rate of application of 60 ml/h

The obtained product containing TBG, free from impurity protein concentrate and cialiswhat in ultrafiltration cell relative to saline and lyophilizers.

The purity of the obtained product, analyzed by electrophoresis in grad who ente density polyacrylamide gel, is 98%. The output of the preparation according to the immunoassay is only 35.9 mg (69%). The drug test for the absence of antibodies to HIV, hepatitis b virus and antibodies to hepatitis C.

Example 2. All procedures carried out at +4°C. 1.0 l retroplatsentarno blood centrifuged with a speed of 10,000 rpm for 1 hour, the precipitate discarded, and the supernatant add sodium chloride to a final concentration of 0.5 M and Triton X-100 to a final concentration of 0.02%. The centrifugation repeated. Serum containing 52 mg TBG, thus prepared, is saturated with ammonium sulfate to 30%, incubated overnight with stirring and centrifuged with a speed of 10,000 rpm for 1 hour. The precipitate washed three times 30% ammonium sulfate, suspended in 0.15 M phosphate-saline buffer solution pH 7.0 and 7.5, containing 0.5 M sodium chloride, and cialiswhat in the ultrafiltration cell, missing a 10-fold volume of buffer. The drug is applied to the chromatographic column of 2.5×8,0, filled with 50 ml sepharose FF with immobilized monoclonal antibodies to TBG, equilibriating phosphate-saline buffer solution. The rate of deposition of 40 ml/hour, a Drawing exercise three times the passage of the applied volume through the column.

Nonspecific sorbiruyushchiesya components of blood wash with sorbent consistently FOSFA the but-salt buffer solution, containing 0.5 M sodium chloride and 0.15 M sodium chloride, with a rate of 80 ml/h until reaching zero values of optical density.

After washing the sorbent is carried out the elution of 0.1 M glycine-NA buffer solution with a pH of 2.55 and a rate of 40 ml/hour elution Process control by optical density at a wavelength of 280 nm. Elution is carried out until reaching zero values of optical density. Eluruume the fractions containing the desired product are pooled, immediately neutralized to a pH of 6.5-7.5, dialist against phosphate-saline buffer solution and applied to a column separate with protein G. the Rate of application of 60 ml/h

All passed through the column a solution containing TBG, concentrate, and cialiswhat relatively saline and lyophilizers.

The purity of the obtained product, analyzed by electrophoresis in a density gradient polyacrylamide gel, is 98%. The output of the preparation according to the immunoassay is to 39.5 mg (76%). The drug test 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 purity (at least 98%) and high - 69-76% yield. The obtained results are achieved through the use of two high-affinity sorbents, exploitasaudi the unique properties of monoclonal antibodies and protein G of Streptococcus.

Sources of information

1. Aveskulov, Haasse, Nvenergy, Justiniano. Isolation and purification of specific β1-g-globulin. "Questions of medical chemistry, 1978, No. 24, SS-244.

2. Swimers, Schrivener, Appalling, Usepagov, Justiniano. The method of obtaining the USSR beta-1-glycoprotein from waste retroplatsentarno blood in the production of gamma globulin. A.S. No. 1341736, priority 28.03.85.

3. Schrivener, Art, Lev, Ivekoviceva, PKa, Ackbarali, Justiniano. The method of obtaining the USSR of betaglycomune. A.S. No. 1783644, priority 04.12.89.

The isolation and purification of USSR beta-1-glycoprotein from whey retroplatsentarno blood by fractionation with ammonium sulfate, washing and centrifugation, dissolved sediment, dialysis and chromatography, characterized in that the serum after centrifugation at 10,000 rpm and saturation with sodium chloride to a final concentration of 0.1-1.0 mol/l Triton X-100 to a final concentration of 0.01 to 0.05%, treated with ammonium sulfate to 30% saturation and utilizando in the ultrafiltration cell, subjected to double affinity chromatography, namely: will be on the first affinity sorbent, which is separate with monoclonal immobilizerturning antibodies to the USSR beta-1-CH is coprotein, ofinnovations TBG elute in 0.1m glycine-HCl buffer solution pH of 2.55, the eluate is immediately neutralized cialiswhat against phosphate-saline buffer solution and applied to the second affinity sorbent, which is separate, a modified protein G, isolated from the cell wall of Streptococcus, the solution passed through a column containing TBG, concentrate, dialist against saline and lyophilizers.



 

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4 cl

FIELD: surgery, traumatology, orthopedics.

SUBSTANCE: method of preparing platelet-rich blood autoplasma for regeneration of cortical bone consists in adding it to autogenous stabilizer blood, centrifugation, adding pre-coagulant containing 10% CaCl2 solution, and a blood coagulation factor. Centrifugation is conducted at ambient temperature for 5-8 h at a rate of 1000-2300 rpm and than 5000 units of prothrombin (blood coagulation factor II) is added.

EFFECT: increased productivity of platelet-rich blood autoplasma preparation and reduced expenses owing to exclusion of expensive equipment.

FIELD: medicine, ophthalmology.

SUBSTANCE: one should apply an autohemocomponent preparation being supernatant liquid of patient's autoblood at increased serotonin content obtained due to irreversible thrombocytic aggregation due to the impact of 0.5 mg ATP per 1.0 ml plasma followed by a 30-min-long centrifuging at the rate of 1000, 2000 and 3000 rot./min for 20, 7 and 3 min, correspondingly. In case of no exudative phenomena on patient's eye bottom the obtained preparation should introduced at the quantity of 7-10 ml once in 48 h for 1 mo (totally, 15 injections). In case of exudative-hemorrhagic phenomena it should be introduced parabulbarly at the volume of 0.5 ml and parenterally - 7.0-10.0 ml once in 48 h for 1 mo per 15 injections, correspondingly. The preparation enables to improve visual functions due to decreased tissue hypoxia and normalization of microcirculation in visual analyzer.

EFFECT: higher efficiency of therapy.

2 cl, 7 dwg, 2 ex

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