A kind of purification method of protamine sulfate

Abstract

The invention discloses a purification method of protamine sulfate. The purification method comprises the following steps: (1) dissolving a crude protamine sulfate extract into water to obtain a solution, adding acid, and regulating the pH value of the solution to 1-2; (2) adding an adsorbent, stirring, and filtering to obtain filter liquor; (3) adding alkali, regulating the pH value of the filter liquor to 10-12, then adding the adsorbent, stirring, and filtering; and (4) adding an organic solvent, centrifugalizing to remove a supernatant, and carrying out vacuum drying on precipitates, wherein the adsorbent added in the step (2) and the step (3) is one or more of diatomite, activated bauxite, bentonite, bone coal, active carbon and silica gel. The refining method for directly adsorbing and purifying the protamine sulfate by adopting the adsorbent is easy to operate, far lower than a chromatography method in cost, far higher than the chromatography method in production scale, equivalent to the chromatography method in product purity and very suitable for carrying out industrialized production.

Description

technical field

[0001] The invention relates to the technical field of protein purification, in particular to a method for purifying protamine sulfate. Background technique

[0002] Protamine is a polycationic peptide sulfate extracted from fish sperm cells. Clinically, it is the only drug that can be used to antagonize excessive heparin in cardiac surgery. At the same time, protamine sulfate can also be used to prepare Long-acting insulin and long-acting corticotropin. Because protamine sulfate has antibacterial effect, in the food industry, protamine sulfate can also be used to make food preservatives.

[0003] The production of protamine sulfate mainly adopts three steps of extraction, separation and refinement. Usually, a crude protamine extract can be obtained from milt after extraction and separation. But no matter which extraction method is adopted, the final crude protamine extract needs to go through the final refining step.

[0004] The extraction of protamine ...

Examples

Embodiment 1

[0036] Purification of Protamine Sulfate Crude Extract

[0037] (1) Protamine sulfate crude extract dissolved

[0038] Accurately weigh 500 g of protamine sulfate crude extract (provided by Shanghai First Biochemical Pharmaceutical Co., Ltd.), add it to 5 L of purified water, and bathe in 35°C water bath for 30 minutes. After it is completely dissolved, adjust the pH to 1.8 with sulfuric acid;

[0039] (2) The first adsorption

[0040] Weigh 150 g of diatomite, add it to the above protamine sulfate solution, stir in a water bath at 30°C for about 2 hours, filter through filter paper, remove the diatomite, and adjust the pH of the filtrate to 10.0 with sodium hydroxide;

[0041] (3) The second adsorption

[0042] Weigh 100 g of diatomite, add it to the protamine sulfate solution whose pH has been adjusted, put it in a water bath at 80°C, stir and adsorb for 3 hours, and remove the diatomite by filtration;

[0043] (4) Organic solvent precipitation

[0044] Take the filtrate...

Embodiment 2

[0100] Purification of Protamine Sulfate Crude Extract

[0101] (1) Protamine sulfate crude extract dissolved

[0102] Accurately weigh 500 g of protamine sulfate crude extract (provided by Shanghai No. 1 Biochemical Pharmaceutical Co., Ltd.), add it to 10 L of purified water, and bathe in 30 °C water for 30 min. After it is completely dissolved, adjust the pH to 1.2 with sulfuric acid.

[0103] (2) The first adsorption

[0104] Weigh 150 g of bentonite, add it to the protamine sulfate solution, stir in a water bath at 30°C for about 2 hours, filter through filter paper, remove the bentonite, and adjust the pH of the filtrate to 11.0 with sodium hydroxide.

[0105] (3) The second adsorption

[0106] Weigh 100 g of bentonite, add it into the protamine sulfate solution whose pH has been adjusted, put it in a water bath at 85° C., stir and adsorb for 3 hours, and remove the bentonite by filtration.

[0107] (4) Organic solvent precipitation

[0108] Take the filtrate, add an ...

Embodiment 3

[0132] Purification of Protamine Sulfate Crude Extract

[0133] (1) Protamine sulfate crude extract dissolved

[0134] Accurately weigh 500 g of protamine sulfate crude extract (provided by Shanghai No. 1 Biochemical Pharmaceutical Co., Ltd.), add it to 10 L of purified water, and bathe in 35 ° C water for 30 min. After it is completely dissolved, adjust the pH to 1.4 with sulfuric acid.

[0135] (2) The first adsorption

[0136] Weigh 100 g of activated carbon, add it to the protamine sulfate solution, stir in a water bath at 30°C for about 2 hours, filter through filter paper, remove the activated carbon, and adjust the pH of the filtrate to 12.0 with sodium hydroxide.

[0137] (3) The second adsorption

[0138] Weigh 50 g of activated carbon, add it into the protamine sulfate solution whose pH has been adjusted, put it in a water bath at 85° C., stir and adsorb for 3 hours, and remove the activated carbon by filtration.

[0139] (4) Organic solvent precipitation

[0140] ...

technical field

[0001] The invention relates to the technical field of protein purification, in particular to a method for purifying protamine sulfate. Background technique

[0002] Protamine is a polycationic peptide sulfate extracted from fish sperm cells. Clinically, it is the only drug that can be used to antagonize excessive heparin in cardiac surgery. At the same time, protamine sulfate can also be used to prepare Long-acting insulin and long-acting corticotropin. Because protamine sulfate has antibacterial effect, in the food industry, protamine sulfate can also be used to make food preservatives.

[0003] The production of protamine sulfate mainly adopts three steps of extraction, separation and refinement. Usually, a crude protamine extract can be obtained from milt after extraction and separation. But no matter which extraction method is adopted, the final crude protamine extract needs to go through the final refining step.

[0004] The extraction of protamine ...

Examples

Embodiment 1

[0036] Purification of Protamine Sulfate Crude Extract

[0037] (1) Protamine sulfate crude extract dissolved

[0038] Accurately weigh 500 g of protamine sulfate crude extract (provided by Shanghai First Biochemical Pharmaceutical Co., Ltd.), add it to 5 L of purified water, and bathe in 35°C water bath for 30 minutes. After it is completely dissolved, adjust the pH to 1.8 with sulfuric acid;

[0039] (2) The first adsorption

[0040] Weigh 150 g of diatomite, add it to the above protamine sulfate solution, stir in a water bath at 30°C for about 2 hours, filter through filter paper, remove the diatomite, and adjust the pH of the filtrate to 10.0 with sodium hydroxide;

[0041] (3) The second adsorption

[0042] Weigh 100 g of diatomite, add it to the protamine sulfate solution whose pH has been adjusted, put it in a water bath at 80°C, stir and adsorb for 3 hours, and remove the diatomite by filtration;

[0043] (4) Organic solvent precipitation

[0044] Take the filtrate...

Embodiment 2

[0100] Purification of Protamine Sulfate Crude Extract

[0101] (1) Protamine sulfate crude extract dissolved

[0102] Accurately weigh 500 g of protamine sulfate crude extract (provided by Shanghai No. 1 Biochemical Pharmaceutical Co., Ltd.), add it to 10 L of purified water, and bathe in 30 °C water for 30 min. After it is completely dissolved, adjust the pH to 1.2 with sulfuric acid.

[0103] (2) The first adsorption

[0104] Weigh 150 g of bentonite, add it to the protamine sulfate solution, stir in a water bath at 30°C for about 2 hours, filter through filter paper, remove the bentonite, and adjust the pH of the filtrate to 11.0 with sodium hydroxide.

[0105] (3) The second adsorption

[0106] Weigh 100 g of bentonite, add it into the protamine sulfate solution whose pH has been adjusted, put it in a water bath at 85° C., stir and adsorb for 3 hours, and remove the bentonite by filtration.

[0107] (4) Organic solvent precipitation

[0108] Take the filtrate, add an ...

Embodiment 3

[0132] Purification of Protamine Sulfate Crude Extract

[0133] (1) Protamine sulfate crude extract dissolved

[0134] Accurately weigh 500 g of protamine sulfate crude extract (provided by Shanghai No. 1 Biochemical Pharmaceutical Co., Ltd.), add it to 10 L of purified water, and bathe in 35 ° C water for 30 min. After it is completely dissolved, adjust the pH to 1.4 with sulfuric acid.

[0135] (2) The first adsorption

[0136] Weigh 100 g of activated carbon, add it to the protamine sulfate solution, stir in a water bath at 30°C for about 2 hours, filter through filter paper, remove the activated carbon, and adjust the pH of the filtrate to 12.0 with sodium hydroxide.

[0137] (3) The second adsorption

[0138] Weigh 50 g of activated carbon, add it into the protamine sulfate solution whose pH has been adjusted, put it in a water bath at 85° C., stir and adsorb for 3 hours, and remove the activated carbon by filtration.

[0139] (4) Organic solvent precipitation

[0140] ...

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