Preparation method of ulinastatin adsorption resin

Abstract

The invention discloses a preparation method of ulinastatin adsorption resin, and belongs to the technical field of adsorption resin. Comprising the following steps: (1) preparing a water phase: adding pure water, gelatin, cellulose, sodium chloride and lignin into a reaction kettle until the materials are fully dissolved; (2) preparing an oil phase, wherein the oil phase contains divinylbenzene, methyl acrylate, benzoyl peroxide, tert-butyl peroxy-2-ethylhexanoate, triallyl isocyanurate, tripropanol alkyl trimethacrylate and methylbenzene, and the oil phase is uniformly stirred and mixed with the methylbenzene and the trivinylbenzene, the methyl acrylate, the benzoyl peroxide, the tert-butyl peroxy-2-ethylhexanoate, the triallyl isocyanurate, the tripropanol alkyl trimethacrylate and the methylbenzene; (3) suspension polymerization: putting the oil phase into the water phase, and carrying out staged heating reaction; (4) amination reaction; (5) alkylation reaction; and (6) acid-base alcohol treatment. Compared with the existing resin, the resin disclosed by the invention has the characteristics of high adsorption exchange rate, high desorption rate and high desorption concentration on ulinastatin, and the yield of ulinastatin is increased by 1.17 times compared with that of D215 resin.

Description

Technical field

[0001] The present invention belongs to the technical field of adsorption resins, and particularly relates to a ulinastatin exchange adsorption resin. Background technique

[0002] The chemical formula of ulinastatin is 3-(furan-2-yl)-2,4-dioxaspiro[5.5]undec-9-ene. It can be clinically used to treat acute pancreatitis, chronic relapsing pancreatitis, and can also be used as an adjuvant drug for the rescue of acute circulatory failure.

[0003] Ulinastatin is a glycoprotein isolated and purified from the urine of healthy people that can inhibit the activities of a variety of proteolytic enzymes. It belongs to protease inhibitors, and the activity of ulinastatin contained in every 1 mg of protein shall not be less than 3500 units.

[0004] The following problems exist in the production process of ulinastatin: Reduction of product yield: The yield of ulinastatin is related to factors such as the quality of urine, the cleaning of various containers, the concentration and pH of various solutions, and the adsorbent used. In particular, the quality and the added amount of the adsorbent chitosan seriously affect the yield.

[0005] Low titer per gram of crude ulinastatin: It is mainly related to factors such as rinsing after adsorption and the amount collected after diatomite filtration. The degree of cleaning after chitosan adsorption largely determines the titer per gram of the product.

[0006] Links such as the recycling, transportation, treatment, and storage of urine will all affect the production efficiency. Summary of the invention

[0007] In order to solve the above problems, the present invention provides a preparation method of a ulinastatin adsorption resin. The prepared resin has a high adsorption rate, a high desorption rate, and a high desorption concentration for ulinastatin.

[0008] A preparation method of a ulinastatin adsorption resin includes the following steps: (1) Prepare the aqueous phase: Add pure water to the reaction kettle, add gelatin and cellulose, and then heat up to 50°C to dissolve. After fully dissolving, add sodium chloride and lignin until fully dissolved; add 2-4 g of cellulose, 2-4 g of gelatin, 100-120 g of sodium chloride, and 0.5-0.8 g of lignin to each liter of pure water.

[0009] (2) Preparation of the oil phase: Add divinylbenzene and methyl acrylate into a mixing kettle, then add benzoyl peroxide, tert-butyl peroxy-2-ethylhexanoate, triallyl isocyanurate, tripropyleneglycol trimethacrylate, and toluene, and stir to mix evenly; the mass ratio of divinylbenzene, methyl acrylate, benzoyl peroxide, tert-butyl peroxy-2-ethylhexanoate, triallyl isocyanurate, tripropyleneglycol trimethacrylate, and toluene is 10-57:243-290:3:3:3-15:3-9:120-360.

[0010] (3) Suspension polymerization: Put the oil phase into the water phase, feed at 50 °C, and heat up to 68 °C at a heating rate of 5 °C every 10 minutes for heat preservation and shaping. After the white balls are shaped, keep them at 68 °C for 6 hours, then heat up to 75 °C for 2 hours, and then heat up to 85 °C for 4 hours. After the heat preservation is completed, wash the resin white balls with 80 °C hot water until the effluent is clear, distill off toluene, and dry and screen the particle size; the mass ratio of the water phase to the oil phase is 1.5-3.5:1.

[0011] (4) Amination reaction: Put the dried white balls above into a high-temperature reaction kettle, add dimethylpropylenediamine for swelling at room temperature. After complete swelling, slowly heat up to 175 °C and keep it for 18 hours. After the heat preservation is completed, wash with water until neutral to obtain amine balls; the mass ratio of the white balls to dimethylpropylenediamine is 1:5-7.

[0012] (5) Alkylation reaction: Put the amine balls into the reaction kettle, add chloroethanol, and dropwise add sodium hydroxide to adjust the pH value to 14, then slowly heat up to 85 °C and keep it for 12 hours. After the heat preservation is completed, wash the resin with water until the effluent is clear; the mass ratio of the amine balls to chloroethanol is 1:5-7.

[0013] (6) Acid-base-alcohol treatment: Add the resin obtained in step (5) to 5% hydrochloric acid and soak for 2 hours, then wash with water until neutral; then add it to 3% sodium hydroxide and soak for 2 hours, then wash with water until neutral; then wash the resin with ethanol, and finally wash with water until there is no alcohol.

[0014] Preferably, add 4 g of cellulose, 4 g of gelatin, 120 g of sodium chloride, and 0.8 g of lignin to every 1.2 L of pure water in the water phase.

[0015] Preferably, the mass ratio of divinylbenzene, methyl acrylate, benzoyl peroxide, tert-butyl peroxy-2-ethylhexanoate, triallyl isocyanurate, tripropyleneglycol trimethacrylate, and toluene in the oil phase is 10-57:243-290:3:3:3-15:3-9:120-360.

[0016] Preferably, the mass ratio of the water phase to the oil phase in step (3) is 1.5-3.5:1.

[0017] Preferably, in step (4), the mass ratio of the white balls to dimethylpropylenediamine is 1:6.

[0018] Preferably, in step (5), the mass ratio of the amine balls to chloroethanol is 1:6.

[0019] The present invention also provides the application of the resin prepared by the above preparation method in the adsorption of ulinastatin.

[0020] In the oil phase: Divinylbenzene: crosslinking agent. When methyl acrylate and divinylbenzene are copolymerized, there is non-uniformity in the crosslinking structure due to different monomer activities. In the later stage of copolymerization, relatively long polyacrylate segments may be formed, and even linear acrylate copolymers may be produced. Therefore, other crosslinking agents need to be added to change the non-uniformity of the crosslinking structure.

[0021] Methyl acrylate: monomer. The anti-pollution ability of methyl acrylate is better than that of styrene. The carboxyl group of methyl acrylate forms hydrogen bonds with ulinastatin, which is beneficial to the exchange adsorption of ulinastatin.

[0022] Benzoyl peroxide: initiator tert-Butyl peroxy-2-ethylhexanoate: initiator Triallyl isocyanurate: crosslinking agent. Its free radical polymerization activity is relatively low. When mixed with divinylbenzene with higher activity, resins with higher exchange capacity and better mechanical strength can be obtained.

[0023] Tripropyleneglycol trimethacrylate: crosslinking agent. Each molecule of it has three alkenyl groups, and its purity is also higher than that of divinylbenzene. Therefore, the crosslinking efficiency can be improved, and when mixed with divinylbenzene, the non-uniformity of the crosslinking structure can be changed.

[0024] Toluene; pore-forming agent Compared with the prior art, the present invention has the following advantages: The resin of the present invention has a high adsorption rate, desorption rate, and desorption concentration for ulinastatin.

[0025] When the resin of the present invention adsorbs ulinastatin and passes 10 times the urine through the column, the color of the effluent from the column is significantly lighter than that of the control D215 resin, indicating a high adsorption and exchange rate. When eluting with 7% sodium chloride solution, the total protein concentration in the desorbed solution is significantly higher than that of the D215 resin, indicating a high desorption rate and desorption concentration of the resin of the present invention. The ulinastatin recovery rate can be increased by up to 1.17 times compared with the D215 resin. Specific embodiments

[0026] The following further illustrates the present invention through specific examples. However, those skilled in the art should know that the specific examples of the present invention do not limit the present invention in any way, and any equivalent substitution made on the basis of the present invention falls within the protection scope of the present invention. Example 1

[0027] Aqueous phase preparation: Add 1200 ml of pure water into a three-necked flask, add 4 g of cellulose and 4 g of gelatin, heat up to 50 °C and dissolve for 30 minutes, then add 120 g of sodium chloride and 0.8 g of lignin and dissolve for 30 minutes.

[0028] Oil phase preparation: Add 30 g of divinylbenzene with inhibitor removed and 270 g of methyl acrylate into a beaker, then add 3 g of benzoyl peroxide and 3 g of tert-butyl peroxy-2-ethylhexanoate, then add 9 g of tripropylolpropane trimethacrylate and 3 g of triallyl isocyanurate, and then add 180 g of toluene and stir to dissolve.

[0029] Suspension polymerization: Put the above oil phase into a three-necked flask, feed at 50 °C, heat up at a rate of 5 °C every 10 minutes, heat up to 68 °C and keep for shaping, keep at 68 °C for 6 hours after the white balls are shaped, then keep at 75 °C for 2 hours and at 85 °C for 4 hours. After the insulation is over, wash the resin white balls with hot water at 80 °C until the effluent is clear and turbid-free, add them to a distillation kettle to distill toluene, keep at 100 °C for 1 hour after the toluene is distilled, cool down to below 80 °C and discharge. Then dry until the water content is 3% and screen the particle size.

[0030] Amination reaction: Put 100 g of the extracted and dried white balls into a dry high-temperature reaction kettle, add 500 g of dimethylpropylenediamine, swell at room temperature for 2 hours, then slowly heat up to 175 °C and keep for 18 hours. Then wash with water until neutral.

[0031] Alkylation reaction: Put 100 g of the above amine balls and 500 mL of chloroethanol into a three-necked flask, add sodium hydroxide dropwise to adjust the pH to 14, then slowly heat up to 85 °C and keep for 12 hours. Then wash the resin with water until the effluent is clear.

[0032] Acid-base-alcohol treatment: Add the resin with clear water wash into 5% hydrochloric acid and soak for 2 hours, wash with water until neutral, then add it into 3% sodium hydroxide and soak for 2 hours, wash with water until neutral. Then add ethanol and wash the resin according to the ratio of resin to ethanol = 1:4, and finally wash with water until there is no ethanol.

[0033] Detection data of the resin in this example: Table 1

[0034] Column adsorption experiment: Take 100 ml of the resin prepared in this example and load it into a resin column wetly, regenerate with sodium hydroxide, and then wash with pure water until neutral.

[0035] Pass 10 times the column volume of urine through the resin layer at a flow rate of 8 - 10 ml, and collect the effluent.

[0036] After the upper column is completed, wash the impurities with pure water.

[0037] Elute ulinastatin with a 5-10% sodium chloride aqueous solution at an elution flow rate of 5-8 ml, and collect the eluate to measure the content and titer of ulinastatin.

[0038] Select commercially available D215 resin and repeat the above experimental process.

[0039] Table 2

[0040] It can be seen from the data in Table 2 that the total protein concentration of the resin analytical solution prepared in this example is higher than that of the D215 analytical solution, indicating that the content and titer of ulinastatin obtained by resin analysis in this example are high. Example 2

[0041] Aqueous phase preparation: Add 2000 ml of pure water to a three-necked flask, add 8 g of cellulose and 4 g of gelatin, heat to 50 °C and dissolve for 30 minutes, then add 200 g of sodium chloride and 1.6 g of lignin and dissolve for 30 minutes.

[0042] Oil phase preparation: Add 57 g of divinylbenzene from which the polymerization inhibitor has been removed and 250 g of methyl acrylate to a beaker, then add 3 g of benzoyl peroxide and 3 g of tert-butyl peroxy-2-ethylhexanoate, then add 3 g of tripropylolpropane trimethacrylate and 3 g of triallyl isocyanurate, and then add 321 g of toluene and stir to dissolve.

[0043] Suspension polymerization: Put the above oil phase into a three-necked flask, feed at 50 °C, raise the temperature at a rate of 5 °C every 10 minutes, raise the temperature to 68 °C and keep it for shaping, keep the white balls at 68 °C for 6 hours after shaping, then keep them at 75 °C for 2 hours and at 85 °C for 4 hours. After the heat preservation is completed, wash the resin white balls with hot water at 80 °C until the effluent is clear and turbid-free, add them to the distillation kettle to distill toluene, keep it at 100 °C for 1 hour after the toluene distillation, and discharge after cooling below 80 °C. Then dry to 3% moisture and screen the particle size.

[0044] Amination reaction: Put 100 g of the extracted and dried white balls into a dry high-temperature reaction kettle, add 700 g of dimethylpropylenediamine, swell at room temperature for 2 hours, then slowly raise the temperature to 175 °C and keep it for 18 hours. Then wash with water until neutral.

[0045] Alkylation reaction: Put 100 g of the above amine balls and 410 mL of chloroethanol into a three-necked flask, adjust the pH to 14 by dropping sodium hydroxide, then slowly raise the temperature to 85 °C and keep it for 12 hours. Then wash the resin with water until the effluent is clear.

[0046] Acid-base-alcohol treatment: Add the resin that has been washed clear with water to 5% hydrochloric acid and soak for 2 hours, wash with water until neutral, then add to 3% sodium hydroxide and soak for 2 hours, wash with water until neutral. Then add ethanol and wash the resin according to the ratio of resin to ethanol = 1:4, and finally wash with water until there is no ethanol left. Example 3

[0047] Aqueous phase preparation: Add 2000 ml of pure water to a three-necked flask, add 4 g of cellulose and 8 g of gelatin, heat up to 50 °C and dissolve for 30 minutes, then add 240 g of sodium chloride and 1 g of lignin and dissolve for 30 minutes.

[0048] Oil phase preparation: Add 20 g of divinylbenzene from which the polymerization inhibitor has been removed and 380 g of methyl acrylate to a beaker, then add 6 g of benzoyl peroxide and 6 g of tert-butyl peroxy-2-ethylhexanoate, then add 18 g of tripropyleneglycol trimethacrylate and 15 g of triallyl isocyanurate, and then add 650 g of toluene and stir to dissolve.

[0049] Suspension polymerization: Put the above oil phase into a three-necked flask, feed at 50 °C, heat up at a rate of 5 °C every 10 minutes, heat up to 68 °C and keep it for shaping. After the white balls are shaped, keep them at 68 °C for 6 hours, then keep them at 75 °C for 2 hours and at 85 °C for 4 hours. After the heat preservation is over, wash the resin white balls with hot water at 80 °C until the effluent is clear and turbid-free, add them to a distillation kettle to distill toluene. Keep it at 100 °C for 1 hour after the toluene is distilled, and discharge after cooling below 80 °C. Then dry until the moisture content is 3% and screen the particle size.

[0050] Amination reaction: Put 100 g of the extracted and dried white balls into a dry high-temperature reaction kettle, add 600 g of dimethylpropylenediamine, swell at room temperature for 2 hours, then slowly heat up to 175 °C and keep it for 18 hours. Then wash with water until neutral.

[0051] Alkylation reaction: Put 100 g of the above amine balls and 580 mL of chloroethanol into a three-necked flask, dropwise add sodium hydroxide to adjust the pH to 14, then slowly heat up to 85 °C and keep it for 12 hours. Then wash the resin with water until the effluent is clear.

[0052] Acid-base-alcohol treatment: Add the resin that has been washed clear with water to 5% hydrochloric acid and soak for 2 hours, wash with water until neutral, then add to 3% sodium hydroxide and soak for 2 hours, wash with water until neutral. Then add ethanol and wash the resin according to the ratio of resin to ethanol = 1:4, and finally wash with water until there is no ethanol left.

Claims

1. A preparation method of ulinastatin adsorption resin, characterized in that It includes the following steps: (1) Preparation of the aqueous phase: Add pure water into a reaction kettle, add gelatin and cellulose, heat up to 50 °C and dissolve, after complete dissolution, add sodium chloride and lignin until completely dissolved; (2) Preparation of the oil phase: Add divinylbenzene and methyl acrylate into a mixing kettle, then add benzoyl peroxide, tert-butyl peroxy-2-ethylhexanoate, triallyl isocyanurate, tripropylolpropane trimethacrylate and toluene, stir and mix evenly; (3) Suspension polymerization: Put the oil phase into the aqueous phase, feed at 50 °C, heat up to 68 °C at a heating rate of 5 °C every 10 minutes for heat preservation and shaping, keep the white balls at 68 °C for 6 hours after shaping, then heat up to 75 °C for 2 hours, heat up to 85 °C for 4 hours, after the heat preservation is completed, wash the resin white balls with 80 °C hot water until the effluent is clear, distill off toluene, and dry and screen the particle size; (4) Amination reaction: Put the dried white balls obtained above into a high-temperature reaction kettle, add dimethylpropylenediamine for swelling at room temperature, slowly heat up to 175 °C after complete swelling and keep warm for 18 hours, after the heat preservation is completed, wash with water until neutral to obtain amine balls; (5) Alkylation reaction: Put the amine balls into a reaction kettle, add chloroethanol, dropwise add sodium hydroxide to adjust the pH value to 14, then slowly heat up to 85 °C and keep warm for 12 hours, after the heat preservation is completed, wash the resin with water until the effluent is clear; (6) Acid-base-alcohol treatment: Immerse the resin obtained in step (5) in 5% hydrochloric acid for 2 hours, wash with water until neutral; then add it to 3% sodium hydroxide and immerse for 2 hours, wash with water until neutral; then wash the resin with ethanol, and finally wash with water until there is no alcohol.

2. The preparation method of the ulinastatin adsorption resin according to claim 1, characterized in that: Add 2-4 g of cellulose, 2-4 g of gelatin, 100-120 g of sodium chloride and 0.5-0.8 g of lignin into each liter of pure water in the aqueous phase.

3. The preparation method of the ulinastatin adsorption resin according to claim 2, characterized in that: The mass ratio of divinylbenzene, methyl acrylate, benzoyl peroxide, tert-butyl peroxy-2-ethylhexanoate, triallyl isocyanurate, tripropylolpropane trimethacrylate and toluene in the oil phase is 10-57:243-290:3:3:3-15:3-9:120-360.

4. The preparation method of the ulinastatin adsorption resin according to any one of claim 3, characterized in that: In step (3), the mass ratio of the aqueous phase to the oil phase is 1.5-3.5:

1.

5. The preparation method of the ulinastatin adsorption resin according to claim 4, characterized in that: In step (4), the mass ratio of the white balls to dimethylpropylenediamine is 1:5-7.

6. The preparation method of the ulinastatin adsorption resin according to claim 4, characterized in that: In step (5), the mass ratio of the amine balls to chloroethanol is 1:5-7.

7. Application of the resin prepared by the preparation method of claim 5 or 6 in the adsorption of ulinastatin.

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