SU710504A3 - Method of purifying biological material from hepatitis b virus - Google Patents

Description

Example 1. Removal of Au antigen from plasma as a result of absorption by the conjugate. In ka4ec; TBe matrices, sepharose is used and ethylenediamine-octyl succinic acid is used as a ligand. Getting gel-derived. Sepharose activation is accomplished by thoroughly washing 100 ml of sedated Sepharose 4B with water. Then, the excess liquid is removed by filtration on a filter operating under vacuum before the gel is transferred to a solution of 4 g of cyanogen bromide in 100 ml of disintilated water. After equilibration for 15 minutes while stirring and cooling, and in an ice bath, the gel suspension is activated at pH 11 for 6-7 minutes. The pH is kept constant by the addition of 4 M sodium hydroxide solution. , To keep ethylenediamine activated: the gel is washed on a glass filter with a cold 0.5 M sodium bicarbonate-carbonate solution at pH 9.8, while it is transferred to a solution of 10 g ethylene diamine in 100 m distilled water, the pH of which is equal to 9.8 with concentrated hydrochloric acid. The reaction mixture is left under stirring; the reaction time is for 20 hours at room temperature. The resulting gel product was thoroughly washed; 1yut. 25 MP of the aqueous suspension obtained as a result of the auxiliary gel is suspended in 20 ml of dioxane. In addition, a fresh solution of 6 g of octant of succinic anhydride in 60 ml of diox is added with stirring at room temperature; the pH is maintained at 7.5-8 by addition of a pass-through solution of sodium hydroxide. The adsorbent is thoroughly washed with dioxane, a mixture of dioxane and water (2: 1), water, a 1 M solution of sodium chloride and a buffer consisting of 0.05 M tris buffer, 0.02 M cation of sodium and 0.10 M chlorine. sodium, at pH 7.5. The yield of the coupling reaction in terms of octyl succinic acid is determined by NMR metol, it is 4.8%. Ethylenediamine can be legal for 1,4-diaminobutane and 1,6-diaminohexane to give an equivalent result (5c. Test for antigen. The starting material used is: plasma with a positive reaction to an antigen with a titer of 1: b4 and 1:32. The titer values are relative to the standard antigen. 2 antigen-positive plasma and 2 m of suspension consisting of 3 are added to each of the tubes. h. carefulman of the sedimented adsorbent, equilibrium Samples were moved for 1, 2, 5, 10, 20 and 30 minutes, respectively, and the gels were separated by centrifugation. A study of the upper layers for the presence of an antigen gave a negative reaction. p 2. Removal of Hyantigen from plasma as a result of absorption by the conjugate of septarose- (hexamethylenediamine-dodecyl succinic acid). Preparation of gel-derivative. For crosslinking of sepharose, 100 ml of gel are mixed with 2 ml of epichlorohydrin and 0.5 g of sodium borohydrate. The mixture is stirred for a period of 1 hour. The gel is thoroughly heated with warm water and mixed with a solution of 0.25 g of sodium borohydride dissolved in 50 ml of 2 M sodium hydroxide. The mixture was placed in an autoclave at 120 ° C. for 1 hour. Then the gel was washed with an alkaline solution of sodium borohydrate. Concentrated acetic acid is added slowly to pH 4. Then the gel is washed with a sufficient volume of water. Preparation of an auxiliary gel from Sepharose and hexamethylenediamine was carried out according to the procedure of Example 1. 25 ml of the obtained auxiliary gel was transferred to a glass filter and washed with a mixture of dioxane and water (2: 1) before drying on the filter with vacuum and transferred to a fresh solution of 1 g / dodecyl succinic acid, 1.5 g of aqueous soluble carbodiimide, N-Cyclohexyl-N (N-methylmorpholinoethyl) -carbodiimide-p-toluyl sulfonate in 60 ml of a mixture of dioxane and water (2: 1). The mixture was stirred at room temperature for 1 hour before transferring to a glass filter and rinsing thoroughly with dioxane, a mixture of dioxane and water (2: 1), 1M sodium chloride solution, water, and test buffer. The yield of the coupling reaction, based on the fatty acid used by the NMR method, is 2.0%. Test for Au-antigen. Plasma with a positive reaction on Au-antigen, having a titer of 1: 128, is used as the starting material. The column is packed with a gel-derived, equilibrated test buffer, 7 MP plasma with a positive reaction to the Au-antigen using a pump, which is brought into contact with the gel (the antigen feed rate is 5 mp / h). The test buffer is added until the elution of the protein material is stopped, which is established by UV scanning. The collected eluate has a negative reaction. Using dialysis under pressure, the eluate is concentrated 10 times and it retains a negative reaction to hepatitis.

Example 3. Removal of AC) -antigen from adsorption to conjugate sepharose-capryl hydrazide. Getting a gel derivative,

The ethyl caprylate is transferred to the corresponding hydrazide using hydrazin lysis. 10 ml of ethyl ether is smoked with 10 ml of 98% hydrazine hydrate. Ethanol (22 ml) was added as a solvent until the reaction mixture was clarified, after which the solution was left at room temperature for 15 hours. The crystallized hydrazide was filtered and washed with ice water and aqueous methanol (1: 1) at room temperature.

Sepharose 4B is activated as in example 1.

The gel is washed with a solution of the coupling agent and dried in vacuo. The combination is carried out by transferring the activated gel to 100 ml of a saturated solution of capryl hydrazide dissolved in dimethylformamide (DMF) and 0.2 M sodium bicarbonate (1: 1). The pH value remains unchanged during the coupling reaction, which is carried out with stirring at room temperature for 15 hours. The resulting product is thoroughly heated with DMF with a mixture of DMF and water (1: 1), 1 M sodium chloride, water, and test buffer.

The yield of the coupling reaction in terms of hydrazide by the NMR method is 2.3%. Test for anti-antigen.

Plasma with a positive reaction to Au-antigen with a titer of 1: 128 is used as the initial substance. 2 ml of suspension consisting of 3 hours of thoroughly sedimented adsorb-. bent, equilibrated with the test buffer, and 1 part of this buffer, add 2 MP of Au-antigen-positive plasma. The mixture was gently mixed for 30 minutes and the gel was separated by centrifugation. The top layer has a negative NaA antigen reaction.

Example 4. Removal of Au-antigen from albumin solution by adsorption on Sepharose- (ethylenediamine-octyl succinic acid) conjugate. Getting gel-derived.

Gel conjugate receive in accordance with example 1. Test for Au-antigen.

An albumin solution with a positive reaction to an Au-antigen with a titer of 1:64 and a positive plasma antigen obtained during fractionation is used as a starting material. The adsorption is carried out periodically according to Example 3. The top layer is tested (from an antigen, a negative reaction is obtained.

Example 5. Removal of Au-antigen from a concentrate with a coagulation coefficient as a result of adsorption to the sepharose- (ethylenediamine-octyl succinic acid) conjugate. Getting gel-derived.

Gel conjugate receive according to example 1. Test for Au-antigen.

Antigen-positive plasma

is used to fractionate a concentrate comprising a coagulation coefficient. A 1% protein solution is prepared that has an antigen titer of 1:32. On a glass filter with a layer of 5 ml of gel-derivative, equilibrated with the test buffer.

10 ml of the protein test solution are added and the gel is then washed with 3 X 5 ml of test buffer. Eluates are tested for anti-antigen and receive a negative reaction.

Example b. Removal of antigen from plasma by adsorption to the sepharose-tetradecylamine conjugate. Getting gel-derived.

Sepharose activated in accordance with example 1.

For the coupling reaction, 100 ml of sepharose activated by cyanogen bromide, which is equilibrated with 95%

ethanol and dried on a filter under vacuum, added to a solution of 22 g of tetradecylamine in 100 ml of 95% ethanol. The mixture is left at room temperature with stirring for 48 hours. The gel is thoroughly washed with

warm 95% ethanol, an aqueous solution of ethanol (1: 1), 1M sodium chloride solution, water, and a test buffer. The yield of the coupling reaction by NMR is

4.3%.

Ai-antigen test,

As a source, substance. use Au-antigen-positive plasma with a titer of 1: 128. 35 ml of plasma is added to a glass filter with a layer of 10 ml of gel derivative, equilibrated in the test buffer. The material is washed with 3 x 10 ml test buffer and the eluates are used

follow on au-antigen. Eluates give a negative reaction.

Example 7. Removal of Ai-antigen from plasma by adsorption on conjugate Sepharose-2-aminododecane.

55 Preparation of gel derivative.

The 2-dodecane is converted to the corresponding amine by reaction with hydroxylamine to give the corresponding oxime, followed by catalytic 60 hydrogenation and elimination of water. Sepharose used in accordance with example 1.

For a combination of 100 ml gel, a-. washed with cyanogen bromide, washed with 95% ethanol, subjected to filter cake under vacuum and diluted with 10 kg of acid to decane, 10 ml of 95% ethanol, the mixture was left at the pen. stirring at room temperature S for h, after which the gel was tested (tagged with this NOL, water solution of ethanol (Itl) ста solution of sodium chloride, ri, water, and: test buffer. The yield of the combination reaction by NMR was 1.6%, Test on Alj-anthnen, .l l. 3 as a source material nd use Au and antigen-positive Plasma with a titer of 1: 128, the experiment was carried out according to example 3, the upper layer is given a MpMUAtal reaction „.; Example 8., Removal of the Ai-an. gene from the plasma (F1 by adsorption on conjugates of Sepharose- (ethylenediaminedehydecen-U-acid) ,, A study of gel-prismodiogu, Sepharoea-ethylenediamine system was performed according to Example 1. To a fresh solution of 1.85 g of undecyllic acid and 2.1 g of dicyclohexylcarbodiimide, dissolved in 100 ml of dioxane, 100 g of the 100% sylgate sicate and 2.1 g of dicyclohexylcarbodiimide were dissolved in 100 ml of dioxane and 100 g of the dicyclohexylcarbodiimide; dioxin and filtration in aacuum, Gelevugo suspensions left When -peromeglivanii about 15 hours at room temperature, Ind-tnshot dkoksanom, smesyu- dioxane and water (islj W sodium chloride solution and uc tatelnym buffer. The yield of the coupling reaction in terms of fatty acid in terms of NMR is 2.3%. The test for the Hyantigen. - As the initial substance, an Au-antigen-positive plasma with a titer is used, -Experience is carried out according to example 3, It was discovered that the upper layer has a negative reaction to the antigen. , Example 9. Removal of the Ai-ant gene from the plasaminogenic concentrate by adsorption to the Sepharose Kaprilhydrazide conjugate. Population of gzp-derivative,: Hepium conjugate according to Example 3. Test for Au-antigen. To 5 ml of the plasminogenic solution, Or5 ml of ylaheim with a high content of antigen is added, this mixture fliaeT positive reaction to antigay. The mixture is passed through an alumina column of 4 cm, swallowed with 10 ml of gel-gelatinate, equilibrated with the test buffer. Collect shares: aoIs are tested. Was found ; 1Tb.; The eluate containing a “plaqueminogen” gives a negative reaction. Lipstick 10, Removal of Au-enigene, from plasma by adsorption on conjugate polyacrylamide- (propylene decylne) “Preparation of gel-derivative. B-iog eE P 300 popiacrylamide, left, will swell in water and carefully & 0.05 M sodium phosphate Soupber solution with pH 7. To dissolve the process, 20 ml of gel suspension in buffer (Of 5. dry gel / 25 ml of buffer) is mixed with 5.0 m of glutaraldehyde and incubated at. within 18h Else this gel-- rinse thoroughly with dioxane, a mixture of dioxane and water (3: 2), water, and Purification buffer. . The test for Au-antigen, Antigen-positive plasma of titer 1t128 is used as the starting material, Substance-Experiment is carried out according to Example 3. When testing the upper layer it was found to be negative. .. 1 pmmer 11, Removal of Au-antigen from plasma by adsorption on Sepharose- (cysteaminoctyl succinic acid) conjugate. The resulting gel-derivative. The Sepharoecysteamine system is obtained by reducing Sepharoazgshtamine, which is obtained according to Example 1 in the presence of a 0.05 M measure of Pto ethanol in M carbonate buffer. At a pH of 8f5 for 1 h, the Gel is intensively walked with carbonate buffer, water and finally dioxane. To a solution of 1.2 g of octyl succinic acid -3 40 ml of dioxane is added a solution. 1.84 g of dicyclohexylcarbodiimide in 10 ml of dioxane and 25 ml of sepharose cysteine after 2 h at. stirring at room temperature. After 2 h, the gel is washed with dioxane. The reaction procedure is repeated and then thoroughly washed with dioxane, a mixture of dioxane and water, water and 0.1 M carbonate at pH 8.5. The remaining thiol groups are blocked by treating the gel with .60 mg of iodoacetamide for 45 minutes. Finally, the gel is washed with a test buomer. Test for Au-antigen ,. Young-positive plasma with Is 32 titer is used1 as a starting material. Adsorption is carried out periodically according to the example of Zo. The top layer is tested for antigen and it gives. impersonate. the reaction. Example 12, Removal of A; antigen from plasma by adsorption to the sepharose-conjugate (reaggleti dianiminiaftil-l-cyclic acid). A sepharose-hexamethylene diamine system is obtained according to example i, 25 ml of the obtained adjuvant gel is transferred to GLPP "shir and prodded". @ @ X1 YV and SOItbt (3s2) n-before suox "9rv" 4 MmMM

Claims (2)

Claim 1. The method of purification of biological material from hepatitis B virus by sorption of the source material, mainly blood, on a sorbent, which consists in the fact that, in order to increase the degree of purification and simplify the method, a water-insoluble matrix covalently bonded through such groups is used as a sorbent , as - О - С —ΊΝΗ—. - 0 - С— ΝΗ-ΊΝΗ—, II ’B’ PN UH o -in- (cn ₂ ) ₂ _ _b -in - c-in-or-uh-co, with a hydrophobic ligand. 2. The method according to claim 1, characterized in that sepharose 4B is used as a water-insoluble matrix.