Preparation method of high-purity protamine sulfate

Abstract

The invention discloses a preparation method of high-purity protamine sulfate, and belongs to the field of biological medicine. Comprising the following steps: S1, preparing a crushing buffer solution; s2, adding the fish white into the crushing buffer solution, uniformly stirring to obtain a homogeneous solution, and adjusting the pH value of the homogeneous solution; s3, nuclease is added into the homogenized liquid, stirring and enzymolysis are conducted, enzymatic hydrolysate is obtained, the pH is adjusted, and acid extract is obtained; s4, filtering the acid extract, performing ultrafiltration with an ultrafiltration membrane, and concentrating to obtain a concentrated solution; and S5, adjusting the pH value of the concentrated solution, adding a decolorizing adsorbent for treatment, carrying out hot filtration to obtain a filtrate, standing, pouring supernatant, and carrying out vacuum drying on a lower-layer oily substance to obtain protamine sulfate. According to the method, impurities are not mixed, the protamine can be simply and selectively extracted from the fish white, the condition that miscellaneous proteins such as nucleic acid and histone are mixed in the protamine sulfate is changed, the absorbance of the product at 260-280 nm is not greater than 0.1, and the total impurity content of the protamine is less than 5.0%.

Description

Technical Field

[0001] The present application relates to a method for preparing highly pure protamine sulfate, belonging to the field of biomedicine. Background Art

[0002] Protamine is a small and simple spherical polycationic peptide with a molecular weight of about 5 kDa, generally composed of 30 - 50 amino acids, and mainly exists in the mature testis tissues of various male animals. Since protamine is a cationic protein, it binds tightly to negatively charged DNA, and the nucleoprotein in fish sperm is formed by the combination of DNA and protamine in a ratio of 2:1. Protamine has a small relative molecular weight, its isoelectric point is at pH 10 - 12, it is soluble in water and dilute acids, and does not coagulate when heated. Research results show that protamine can either delay or prevent insulin release, or act as an antidote for heparin. And the latest research results show that it can be used as a carrier for nucleic acid drugs. It has important applications in the fields of medicine and health care.

[0003] An enzyme that can cleave the phosphodiester bond of a polynucleotide chain is called a nuclease. Nucleases belong to hydrolases and act on the P - O position of the phosphodiester bond. Nucleases from different sources have different specificities and modes of action. Some nucleases have low specificity and can act on both RNA and DNA, and are called omnipotent nucleases. According to the different positions where nucleases act, nucleases can be further divided into exonucleases and endonucleases.

[0004] The industrial preparation method of protamine is generally to grind the mature testis tissue (fish milt) of aquatic organisms, and extract protamine from the ground material with inorganic acids such as sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, etc. Usually, the blood, mucus and other miscellaneous proteins attached to the fish milt, as well as the nucleic acids separated from protamine, are extracted together with protamine to obtain a mixed inorganic acid extract. Therefore, various purification methods for the inorganic acid extract have been proposed to obtain highly pure protamine. For example, methods such as fractional salting - out, precipitation by adjusting pH, precipitation with organic solvents such as ethanol, dialysis, chromatography, resin or electrodialysis after protein liberation have been proposed. In addition, there is also a method of directly extracting protamine with inorganic acid without grinding the fish milt. However, even with these methods, it is difficult to effectively purify protamine. Considerable time and cost must be spent to produce highly pure protamine. Summary of the Invention

[0005] The present invention provides a production method for highly pure purification of protamine, which can simply and selectively extract protamine from fish milt without mixing in impurities, changing the situation that protamine sulfate is mixed with miscellaneous proteins such as nucleic acids and histones, and the absorbance of the product between 260 - 280 nm is not greater than 0.1, and the total impurities of protamine are < 5.0%.

[0006] Under specific conditions, nuclease digests protamine DNA in fish milt into acid-soluble oligonucleotides and mononucleotides. After filtration through a Buchner funnel, insoluble fats, attached blood, mucus, and other impurities are removed. The filtrate is ultrafiltered through a 10 kDa flat-sheet ultrafiltration module to remove macromolecular impurities such as contaminating proteins and nuclease. Then, the protamine is ultrafiltered using a 1 kDa flat-sheet ultrafiltration membrane to remove all low-molecular-weight contaminants such as mononucleotides, oligonucleotides, and small-molecule polypeptides.

[0007] A method for preparing high-purity protamine sulfate, the preparation method comprising:

[0008] S1 Prepare a lysis buffer;

[0009] S2 Add fish milt to the lysis buffer, stir evenly to obtain a homogeneous solution, and adjust the pH of the homogeneous solution;

[0010] S3 Add nuclease to the homogeneous solution, stir and enzymatically digest to obtain an enzymatically digested solution, adjust the pH to obtain an acid extraction solution;

[0011] S4 After filtering the acid extraction solution, perform ultrafiltration through an ultrafiltration membrane, and concentrate to obtain a concentrated solution;

[0012] S5 Adjust the pH of the concentrated solution, add a decolorizing adsorbent for treatment, perform hot filtration to obtain a filtrate, let it stand, pour off the supernatant, and vacuum-dry the lower layer of oily substance to obtain protamine sulfate.

[0013] Optionally, in step S1, the composition of the lysis buffer is: 20 mM Tris-HCl, 50 mM NaCl, 3 mM magnesium chloride, pH = 7.4 - 7.6.

[0014] Optionally, dissolve tris(hydroxymethyl)aminomethane and sodium chloride in water with stirring, adjust the pH value with hydrochloric acid, add magnesium chloride, stir to dissolve, and make up the volume with water to obtain the lysis buffer.

[0015] Optionally, in step S2, the m / V ratio of the fish milt to the lysis buffer is 1 g : 4 - 6 mL.

[0016] Optionally, in step S2, use a Tris-HCl solution to adjust the pH value of the homogeneous solution to 7.4 - 7.6.

[0017] Optionally, the composition of the Tris-HCl solution is: 1 M Tris-HCl, pH = 7.9 - 8.1.

[0018] Optionally, in step S3, the nuclease is a pan-endonuclease.

[0019] Optionally, in step S3, the amount of nuclease added is such that the final concentration of nuclease is 20 - 30 U / mL.

[0020] Optionally, in step S3, the temperature for stirring enzymatic hydrolysis is 36 - 38 °C, and the time is 0.5 - 2 hours. In this application, the temperature with the highest activity of the enzyme is 37 °C, and the temperature range for enzyme activity is 0 - 42 °C. If the temperature is too high, the enzyme will be inactivated. As the temperature of enzymatic hydrolysis decreases, a longer enzymatic hydrolysis time is required.

[0021] Optionally, in step S3, when the temperature of the enzymatic hydrolysate is 20 - 30 °C, use sulfuric acid solution to adjust the pH value of the enzymatic hydrolysate to 3 - 4.

[0022] Optionally, the mass concentration of the sulfuric acid solution is 3 - 8%.

[0023] Optionally, in step S4, the mass of the concentrated solution obtained is 0.7 - 0.9 times the mass of the fish milt.

[0024] Optionally, in step S4, the temperature for concentration is 70 - 80 °C.

[0025] Optionally, in step S4, the ultrafiltration with the ultrafiltration membrane is as follows: the filtrate is ultrafiltered through a 10 kDa flat - type ultrafiltration module, and then ultrafiltered through a 1 kDa flat - type ultrafiltration membrane.

[0026] Optionally, in step S5, use sodium hydroxide solution to adjust the pH value of the concentrated solution to 6 - 8.

[0027] Optionally, the mass concentration of the sodium hydroxide solution is 5 - 15%.

[0028] Optionally, in step S5, the dosage of the decolorizing adsorbent is 0.5 - 1% of the mass of the fish milt.

[0029] Optionally, in step S5, the temperature for treating with the decolorizing adsorbent is 70 - 80 °C, and the time is 1 - 2 hours.

[0030] Optionally, in step S5, the temperature for standing is 0 - 5 °C, and the standing time is 15 - 20 hours.

[0031] Optionally, in step S5, the decolorizing adsorbent is activated carbon.

[0032] In this application, fish milt is the mature testis tissue of aquatic organisms, and specifically can be salmon fish milt, etc.

[0033] As a preferred embodiment, a method for preparing high - purity protamine sulfate includes the following steps:

[0034] (1) Prepare the lysis buffer: Weigh tris(hydroxymethyl)aminomethane (Tris), sodium chloride (NaCl), add water and stir to dissolve. Adjust the pH value with hydrochloric acid, add magnesium chloride, stir to dissolve, and make up the volume with water;

[0035] (2) Homogenize: Weigh the fish sperm, add the lysis buffer, stir evenly, and homogenize to ensure complete release of the product. After homogenization, check the pH of the homogenate and adjust the pH value of the homogenate with Tris-HCl solution;

[0036] (3) Enzymatic hydrolysis: Add nuclease, stir for enzymatic hydrolysis, and add sulfuric acid solution dropwise to adjust the pH;

[0037] (4) Ultrafiltration with flat-sheet ultrafiltration membrane: After filtration with a Buchner funnel, the filtrate is ultrafiltered through a 10 kDa flat-sheet ultrafiltration module; then ultrafilter the protamine with a 1 kDa flat-sheet ultrafiltration membrane and concentrate;

[0038] (5) Add sodium hydroxide solution to the concentrate to adjust the pH, add activated carbon, heat up for decolorization and stirring, after hot filtration, let it stand, pour out the upper layer liquid, and vacuum dry to obtain protamine sulfate;

[0039] Among them, the nuclease described in step (3) is a pan-endonuclease.

[0040] The final composition of the lysis buffer solution described in step (1): 20 mM Tris-HCl, 50 mM NaCl, 3 mM magnesium chloride, pH = 7.5 ± 0.1.

[0041] The m / V ratio of the fish sperm and the lysis buffer described in step (2) is 1 / 4 - 6. The Tris-HCl solution is 1 M Tris-HCl, pH = 8.0 ± 0.1, and the homogenization time is 10 - 60 minutes; the pH value of the homogenate is 7.5 ± 0.1.

[0042] The stirring temperature described in step (3) is 37 ± 1 °C, the enzymatic hydrolysis time is 0.5 - 2 hours, and the amount of nuclease added is a final concentration of 25 U / mL of nuclease. The mass concentration of the sulfuric acid is 5%, and the pH value adjusted by adding sulfuric acid is 3 - 4. The dropping temperature of the sulfuric acid is 25 ± 5 °C.

[0043] The weight of the final concentrate described in step (4) is 0.7 - 0.9 times the weight of the fish sperm. The concentration temperature described in step (4) is 75 ± 5 °C.

[0044] In step (5), the mass concentration of the sodium hydroxide solution is 10%, the pH value adjusted by adding sodium hydroxide is 6 - 8, the amount of activated carbon added is 0.5% - 1% of the weight of the fish sperm, the decolorization temperature is 75 ± 5 °C, the decolorization time is 1 - 2 hours, the standing temperature is 0 - 5 °C, and the standing time is 15 - 20 hours.

[0045] The beneficial effects that can be produced by this application include:

[0046] 1) The preparation method of high-purity protamine sulfate provided by this application uses nuclease to decompose nucleic acid and disrupt the electrostatic force between nucleic acid and protamine. Without the use of a large amount of high-concentration inorganic acid, only a low-concentration inorganic acid is needed to dissolve the oligonucleotides and mononucleotides decomposed from nucleic acid and form a salt with protamine, which not only prevents the denaturation of protamine under high-acidity conditions but also improves the disadvantages of traditional methods: impurity proteins such as histone and components such as blood and mucus components are mixed in the inorganic acid extraction solution, and a high-purity inorganic acid extraction solution is obtained.

[0047] 2) The preparation method of high-purity protamine sulfate provided by this application, after obtaining a high-purity inorganic acid extraction solution, uses a flat-sheet ultrafiltration module, which can not only remove small-molecule impurities such as ribose, bases, and nucleotides and some inorganic salts but also remove macromolecular impurity proteins, avoiding the time-consuming chromatographic procedures and / or the use of organic solvents such as alcohol, acetone, or acetonitrile and hazardous substances such as picric acid involved in most purification methods, and thus bringing problems of waste liquid and wastewater treatment.

[0048] 3) The preparation method of high-purity protamine sulfate provided by this application is simpler and more practical compared with enzymatic hydrolysis, chromatography, dialysis, and anion exchange, etc., which are not applicable to large-scale commercial production processes; the process flow is more efficient and reliable, the product quality is higher, and the safety is stronger. Description of the Drawings

[0049] Figure 1 It is the absorption spectrum diagram of protamine sulfate prepared in Example 1;

[0050] Figure 2 It is the absorption spectrum diagram of protamine sulfate prepared in Example 2;

[0051] Figure 3 It is the absorption spectrum diagram of protamine sulfate prepared in Example 4;

[0052] Figure 4 It is the absorption spectrum diagram of protamine sulfate prepared in Example 5;

[0053] Figure 5 It is the liquid chromatography diagram of protamine sulfate prepared in Example 1;

[0054] Figure 6 The liquid chromatogram of protamine sulfate prepared in Example 2;

[0055] Figure 7 The liquid chromatogram of protamine sulfate prepared in Example 3;

[0056] Figure 8 The liquid chromatogram of protamine sulfate prepared in Example 4;

[0057] Figure 9 The liquid chromatogram of protamine sulfate prepared in Example 5. Detailed implementation mode

[0058] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the described examples. For the experimental methods without specific conditions noted in the following examples, conventional methods and conditions are selected.

[0059] In the present invention, unless otherwise specified, the reagents used are all commercially available reagents.

[0060] Endonuclease: Intelli nuclease, purchased from Shanghai Duoning Biotechnology Co., Ltd., is an all-round nuclease expressed by recombinant Escherichia coli, about 27.5KDa, with an enzyme activity > 250U / μL, and is a non-specific nuclease.

[0061] Example 1

[0062] An industrial preparation method of protamine sulfate, comprising the following steps:

[0063] (1) Prepare the lysis buffer

[0064] Weigh 0.61 g of Tris and 0.73 g of NaCl, add 200 mL of water and stir to dissolve. Adjust the pH to 7.52 with hydrochloric acid, add 0.07 g of magnesium chloride, stir to dissolve, and make up the volume to 250 mL with water. The final composition of the lysis buffer solution: 20 mM Tris-HCl, 50 mM NaCl, 3 mM magnesium chloride, pH = 7.54;

[0065] (2) Homogenize

[0066] Weigh 50 g of salmon milt, add 250 mL of lysis buffer, and stir for 10 min. Adjust the pH of the homogenate to 7.53 with 1 M Tris-HCl, pH = 8.01 solution (12.1 g of Tris is dissolved in 90 mL of water, adjusted to pH = 8.01 with hydrochloric acid, and made up the volume to 100 mL);

[0067] (3) Enzymatic hydrolysis

[0068] Add 25 μL of nuclease according to the amount of 25 U / mL in the final concentration, and stir and enzymatically hydrolyze at 37.0 °C for 0.5 h. It is observed that the viscosity of the solution decreases significantly. Cool the enzymatic hydrolysate to 25.3 °C, and adjust the pH of the enzymatic hydrolysate to 3.53 with a sulfuric acid solution with a mass concentration of 5%, and stir for 30 min;

[0069] (4) Ultrafiltration with flat ultrafiltration membrane

[0070] After the acid extract is filtered through a Buchner funnel, the filtrate is first ultrafiltered through a 10 kDa flat ultrafiltration module; then the protamine acid extract is ultrafiltered with a 1 kDa flat ultrafiltration membrane, and concentrated to a concentrated solution weight of 41.3 g at 75 °C;

[0071] (5) Add a sodium hydroxide solution with a mass concentration of 10% dropwise to the concentrated solution to adjust the pH to 7.08; add 0.5 g of activated carbon to the concentrated solution, heat up to 70 °C, stir for one hour, and perform hot filtration to obtain a filtrate. Let it stand at 2 °C for 16.5 h, pour out the supernatant, and vacuum dry the lower layer of oily substance to obtain protamine.

[0072] Example 2

[0073] An industrial preparation method of protamine sulfate includes the following steps:

[0074] (1) Prepare the lysis buffer

[0075] Weigh 1.21 g of Tris and 1.46 g of NaCl, add 450 mL of water and stir to dissolve. Adjust the pH to 7.58 with hydrochloric acid, add 0.14 g of magnesium chloride, stir to dissolve, and make up the volume to 500 mL with water. The final composition of the lysis buffer solution: 20 mM Tris-HCl, 50 mM NaCl, 3 mM magnesium chloride, pH = 7.58;

[0076] (2) Homogenize

[0077] Weigh 100 g of salmon milt, add 500 mL of lysis buffer, and stir for 30 min. Adjust the pH of the homogenate to 7.47 with a 1 M Tris-HCl, pH = 8.05 solution (12.1 g of Tris is dissolved in 90 mL of water, adjusted to pH = 8.05 with hydrochloric acid, and made up the volume to 100 mL);

[0078] (3) Enzymatic hydrolysis

[0079] Add 50 μL of nuclease according to the amount of 25 U / mL in the final concentration, and stir and enzymatically hydrolyze at 37.2 °C for 1 h. It is observed that the viscosity of the solution decreases significantly. Cool the enzymatic hydrolysate to 24.6 °C, and adjust the pH of the enzymatic hydrolysate to 3.03 with a sulfuric acid solution with a mass concentration of 5%, and stir for 30 min;

[0080] (4) Ultrafiltration with flat-sheet ultrafiltration membrane

[0081] After the acid extract was filtered through a Buchner funnel, the filtrate was first ultrafiltered through a 10 kDa flat-sheet ultrafiltration module; then, the protamine acid extract was ultrafiltered with a 1 kDa flat-sheet ultrafiltration membrane and concentrated at 75 °C until the weight of the concentrated solution was 82.6 g.

[0082] (5) Add a 10% sodium hydroxide solution by mass concentration dropwise to the concentrated solution to adjust the pH to 6.22; add 0.8 g of activated carbon to the concentrated solution, raise the temperature to 70 °C, stir for one hour, filter while hot to obtain a filtrate, let it stand at 2 °C for 18 hours, pour off the supernatant, and vacuum dry the lower oily substance to obtain protamine.

[0083] Example 3

[0084] An industrial preparation method of protamine sulfate, comprising the following steps:

[0085] (1) Prepare the lysis buffer

[0086] Weigh 2.42 g of Tris and 2.93 g of NaCl, add 900 mL of water and stir to dissolve. Adjust the pH = 7.48 with hydrochloric acid, add 0.29 g of magnesium chloride, stir to dissolve, and make up the volume to 1 L with water. The final composition of the lysis buffer solution: 20 mM Tris-HCl, 50 mM NaCl, 3 mM magnesium chloride, pH = 7.48;

[0087] (2) Homogenize

[0088] Weigh 200 g of salmon testis, add it to the lysis buffer, and stir for 60 min. Adjust the pH of the homogenate to 7.50 with a 1 M Tris-HCl, pH = 8.00 solution (12.1 g of Tris is dissolved in 90 mL of water, adjusted to pH = 8.00 with hydrochloric acid, and made up the volume to 100 mL);

[0089] (3) Enzymatic hydrolysis

[0090] Add 100 μL of nuclease according to the final concentration of 25 U / mL, stir and enzymatically hydrolyze at 37.5 °C for 2 hours. After the viscosity of the solution decreased significantly, cool the enzymatic hydrolysate to 20.4 °C, and adjust the pH of the enzymatic hydrolysate to 3.97 with a 5% sulfuric acid solution by mass concentration, and stir for 30 min;

[0091] (4) Ultrafiltration with flat-sheet ultrafiltration membrane

[0092] After the acid extract was filtered through a Buchner funnel, the filtrate was first ultrafiltered through a 10 kDa flat-sheet ultrafiltration module; then, the protamine acid extract was ultrafiltered with a 1 kDa flat-sheet ultrafiltration membrane and concentrated at 75 °C until the weight of the concentrated solution was 141.1 g;

[0093] (5) Add sodium hydroxide solution with a mass concentration of 10% dropwise to the concentrated solution to adjust the pH to 8.00; add 1.0 g of activated carbon to the concentrated solution, raise the temperature to 72 °C, stir for one hour, perform hot filtration to obtain a filtrate, let it stand at 2 °C for 20 hours, pour off the supernatant, and vacuum-dry the lower-layer oily substance to obtain protamine.

[0094] Example 4

[0095] An industrial preparation method of protamine sulfate includes the following steps:

[0096] (1) Prepare the lysis buffer

[0097] Weigh 2.41 g of Tris and 2.93 g of NaCl, add 900 mL of water and stir to dissolve, adjust the pH = 7.45 with hydrochloric acid, add 0.30 g of magnesium chloride, stir to dissolve, and make up the volume to 1 L with water. The final composition of the lysis buffer solution: 20 mM Tris-HCl, 50 mM NaCl, 3 mM magnesium chloride, pH = 7.45;

[0098] (2) Homogenize

[0099] Weigh 200 g of salmon milt, add it to the lysis buffer, and stir for 60 min. Adjust the pH of the homogenate to 7.50 with 1 M Tris-HCl, pH = 8.00 solution (12.1 g of Tris is dissolved in 90 mL of water, adjusted to pH = 8.00 with hydrochloric acid, and made up the volume to 100 mL);

[0100] (3) Enzymatic hydrolysis

[0101] Add 100 μL of nuclease according to the amount of a final concentration of 25 U / mL, stir and enzymatically hydrolyze at 35.0 °C for 6 hours. After the viscosity of the solution significantly decreases, cool the enzymatic hydrolysate to 20.0 °C, and adjust the pH of the enzymatic hydrolysate to 3.92 with sulfuric acid solution with a mass concentration of 5%, and stir for 30 min;

[0102] (4) Ultrafiltration with a flat-sheet ultrafiltration membrane

[0103] After the acid extract is filtered through a Buchner funnel, the filtrate is first ultrafiltered through a 10 kDa flat-sheet ultrafiltration module; then the protamine acid extract is ultrafiltered with a 1 kDa flat-sheet ultrafiltration membrane and concentrated to 140.9 g of concentrated solution at 75 °C;

[0104] (5) Add sodium hydroxide solution with a mass concentration of 10% dropwise to the concentrated solution to adjust the pH to 7.17; add 1.0 g of activated carbon to the concentrated solution, raise the temperature to 72 °C, stir for one hour, perform hot filtration to obtain a filtrate, let it stand at 2 °C for 20 hours, pour off the supernatant, and vacuum-dry the lower-layer oily substance to obtain protamine.

[0105] Example 5

[0106] An industrial preparation method of protamine sulfate, comprising the following steps:

[0107] (1) Prepare the lysis buffer

[0108] Weigh 10.65 g of Tris and 13.00 g of NaCl, add 3600 mL of water and stir to dissolve. Adjust the pH to 7.53 with hydrochloric acid, add 1.35 g of magnesium chloride, stir to dissolve, and make up the volume to 4 L with water. The final composition of the lysis buffer solution: 20 mM Tris-HCl, 50 mM NaCl, 3 mM magnesium chloride, pH = 7.53;

[0109] (2) Homogenize

[0110] Weigh 1 Kg of salmon milt, add it to the lysis buffer, and stir for 60 min. Adjust the pH of the homogenate to 7.55 with 1 M Tris-HCl, pH = 8.00 solution (12.1 g of Tris is dissolved in 90 mL of water, adjusted to pH = 8.00 with hydrochloric acid, and made up the volume to 100 mL);

[0111] (3) Enzymatic hydrolysis

[0112] Add 400 μL of nuclease according to the final concentration of 25 U / mL, stir and enzymatically hydrolyze at 37.6 °C for 2 hours. After the viscosity of the solution decreases significantly, cool the enzymatic hydrolysate to 20.0 °C, and adjust the pH of the enzymatic hydrolysate to 3.23 with 5% sulfuric acid solution by mass, and stir for 30 min;

[0113] (4) Ultrafiltration with a flat-sheet ultrafiltration membrane

[0114] After the acid extract is filtered through a Buchner funnel, the filtrate is first ultrafiltered through a 10 kDa flat-sheet ultrafiltration module; then the protamine sulfate extract is ultrafiltered with a 1 kDa flat-sheet ultrafiltration membrane and concentrated to 819.4 g of the concentrate at 75 °C;

[0115] (5) Dropwise add 10% sodium hydroxide solution by mass to the concentrate to adjust the pH to 6.68; add 7.09 g of activated carbon to the concentrate, heat up to 72 °C, stir for one hour, perform hot filtration to obtain the filtrate, let it stand at 2 °C for 20 hours, pour out the supernatant, and vacuum dry the lower oily substance to obtain protamine sulfate.

[0116] Determination of the absorbance of protamine sulfate

[0117] Respectively take 0.2 g of the protamine sulfate products prepared in Examples 1-5 above, dissolve them in water and dilute to 10.0 mL. Take 2.5 mL of this solution, dilute it to 5 mL with water, scan its absorption spectrum in the range of 260-280 nm, and the test instrument is an ultraviolet-visible spectrophotometer. Figure 1Absorption spectrum of protamine sulfate prepared in Example 1; Figure 2 Absorption spectrum of protamine sulfate prepared in Example 2; Figure 3 Absorption spectrum of protamine sulfate prepared in Example 4; Figure 4 Absorption spectrum of protamine sulfate prepared in Example 5. The absorbances at 260 nm and 280 nm are shown in Table 2.

[0118] Identification of related substances of protamine sulfate

[0119] The related substances of protamine sulfate were identified by high performance liquid chromatography. The specific chromatographic conditions are as follows:

[0120] Chromatographic column: Packed with end-capped solid octadecylsilyl silica gel; 4.0×150 mm; 3.6 μm (Phenomenex Aerls TM 3.6 μm WIDEPORE XB-C18)

[0121] Column temperature: 50 °C

[0122] Injection volume: 30 μL

[0123] Flow rate: 1.5 mL / min

[0124] Wavelength: 210 nm

[0125] Solvent: 618 g / L hydrochloric acid solution; 1.03 g / L hydrochloric acid solution (0.4:99.6)

[0126] Test solution: Take 30 mg of this product and place it in a 100 mL volumetric flask. Dissolve it with 400 μL of 618 g / L hydrochloric acid solution, and dilute it to the mark with 1.03 g / L hydrochloric acid solution. Shake well.

[0127] Reference solution a: Take 1 vial of USP reference standard of protamine sulfate, dissolve it with 40 μl of 618 g / L hydrochloric acid solution, dilute it with 1.03 g / L hydrochloric acid solution and make up to 10 mL. Shake well.

[0128] Reference solution b: Accurately measure 1 mL of reference solution a and place it in a 50 mL volumetric flask. Dilute it to the mark with 1.03 g / L hydrochloric acid solution. Shake well.

[0129] Mobile phase: Using trifluoroacetic acid-acetonitrile-water (0.1:5:95) as mobile phase A, and trifluoroacetic acid-acetonitrile-water (0.1:52:48) as mobile phase B.

[0130] The gradient elution conditions are shown in Table 1:

[0131] Table 1

[0132] Time (min) Mobile phase A (%) Mobile phase B (%) 0 95 5 2 95 5 17 80 20 17.2 20 80 20 20 80 20.1 95 5 28 95 5

[0133] Determination method: Precisely measure 30 μL each of the solvent, reference solution a, reference solution b, and test solution, and inject them into the high-performance liquid chromatograph respectively, and record the chromatogram.

[0134] Calculation method: Area normalization method, calculation formula:

[0135]

[0136] r A-D : The peak areas of the chromatographic peaks of protamine peptide A to protamine peptide D in the test solution;

[0137] r r : The sum of the chromatographic peak areas in the test solution.

[0138] Identification of protamine sulfate content

[0139] The content of protamine sulfate is identified by high-performance liquid chromatography. The specific chromatographic conditions are as follows:

[0140] Chromatographic conditions: Identification of related substances of protamine sulfate

[0141] Solution preparation: Identification of related substances of protamine sulfate

[0142] Determination method: Precisely measure 30 μL each of the solvent, reference solution, and test solution, and inject them into the high-performance liquid chromatograph respectively, and record the chromatogram.

[0143] Calculation method: Calculated by the external standard method based on the peak area

[0144]

[0145] F: Absolute correction factor;

[0146] The average value of the absolute correction factors of two reference solutions;

[0147] C 对 : The concentration of the reference solution;

[0148] A 样 : The sum of the peak areas of all chromatographic peaks with an RRT greater than the reporting limit within the range of 0.80 - 1.30 with protamine peptide A as the reference in the test solution;

[0149] A 对 : The sum of the peak areas of all chromatographic peaks with an RRT greater than the reporting limit within the range of 0.80 - 1.30 with protamine peptide A as the reference in the reference solution;

[0150] V s样: Dilution factor of the test sample solution;

[0151] m 样 : Sampling amount of the test sample (mg)

[0152] D: Data of loss on drying, % (determined according to the method for the determination of loss on drying in the Chinese Pharmacopoeia 2020 Edition, Appendix 0831)

[0153] S: Data of sulfate, % (determined according to the method for the determination of sulfate in the Chinese Pharmacopoeia 2020 Edition, Appendix 0802)

[0154] The protamine sulfate products prepared in Examples 1-5 above were identified for their content according to the above method. Figure 5 、 Figure 6 、 Figure 7 、 Figure 8 、 Figure 9 They are the liquid chromatograms of the protamine sulfate prepared in Example 1, Example 2, Example 3, Example 4, and Example 5 respectively.

[0155] The results are statistically shown in Table 2 below:

[0156] Table 2

[0157]

[0158] As can be seen from the above table: The absorbance of the protamine sulfate prepared by the present invention exceeds the absorbance between 260 and 280 nm specified in the United States Pharmacopeia (USP) and the Japanese Pharmacopeia (JP), which is not more than 0.1, and the total impurities of protamine are <5.0%. It can be seen from Example 4 that reducing the enzymatic hydrolysis temperature requires a longer enzymatic hydrolysis time, increasing the time cost. It can be seen from Example 5 that the method of the present application can achieve the extraction of high-purity protamine sulfate from kilogram-level fish milt, and it is a commercial production process suitable for large-scale production.

[0159] The above are only several embodiments of the present application, and do not impose any form of limitation on the present application. Although the present application is disclosed above with preferred embodiments, it is not intended to limit the present application. Any person skilled in the art, without departing from the scope of the technical solution of the present application, makes some changes or modifications using the technical content disclosed above, which are equivalent to equivalent embodiments and all fall within the scope of the technical solution.

Claims

1. A method for preparing high-purity protamine sulfate, characterized in that, The preparation method includes: S1 Prepare a lysis buffer; S2 Add fish milt into the lysis buffer, stir evenly to obtain a homogenate, and adjust the pH of the homogenate; S3 Add nuclease into the homogenate, stir for enzymatic digestion to obtain an enzymatic hydrolysate, adjust the pH to obtain an acid extract; S4 Filter the acid extract, perform ultrafiltration through an ultrafiltration membrane, and concentrate to obtain a concentrate; S5 Adjust the pH of the concentrate, add a decolorizing adsorbent for treatment, perform hot filtration to obtain a filtrate, let it stand, pour out the supernatant, and the lower layer of oily substance is vacuum dried to obtain protamine sulfate.

2. The preparation method according to claim 1, characterized in that, In step S1, the composition of the lysis buffer is: 20 mM Tris-HCl, 50 mM NaCl, 3 mM magnesium chloride, pH = 7.4 - 7.6; Preferably, dissolve tris(hydroxymethyl)aminomethane and sodium chloride in water with stirring, adjust the pH value with hydrochloric acid, add magnesium chloride, stir for dissolution, and make up the volume with water to obtain the lysis buffer.

3. The preparation method according to claim 1, wherein In step S2, the m / V ratio of the fish milt to the lysis buffer is 1 g:4 - 6 mL.

4. The preparation method according to claim 1, characterized in that, In step S2, use a Tris-HCl solution to adjust the pH value of the homogenate to 7.4 - 7.6; The composition of the Tris-HCl solution is: 1 M Tris-HCl, pH = 7.9 - 8.

1.

5. The preparation method according to claim 1, characterized in that, In step S3, the nuclease is a universal endonuclease.

6. The preparation method according to claim 1, wherein, In step S3, the amount of nuclease added is such that the final concentration of nuclease is 20 - 30 U / mL.

7. The preparation method according to claim 1, characterized in that, In step S3, the temperature for stirring enzymatic digestion is 36 - 38 °C, and the time is 0.5 - 2 hours.

8. The preparation method according to claim 1, characterized in that, In step S3, when the temperature of the enzymatic hydrolysate is 20 - 30 °C, use a sulfuric acid solution to adjust the pH value of the enzymatic hydrolysate to 3 - 4; The mass concentration of the sulfuric acid solution is 3 - 8%.

9. The preparation method according to claim 1, characterized in that, In step S4, the mass of the obtained concentrate is 0.7 - 0.9 times the mass of the fish milt; Preferably, in step S4, the temperature for concentration is 70 - 80 °C; Preferably, in step S4, the ultrafiltration through the ultrafiltration membrane is as follows: the filtrate is ultrafiltered through a 10 kDa flat-sheet ultrafiltration module, and then ultrafiltered through a 1 kDa flat-sheet ultrafiltration membrane.

10. The preparation method according to claim 1, characterized in that, In step S5, use a sodium hydroxide solution to adjust the pH value of the concentrate to 6 - 8; The mass concentration of the sodium hydroxide solution is 5 - 15%; Preferably, in step S5, the dosage of the decolorizing adsorbent is 0.5 - 1% of the mass of the fish milt; Preferably, in step S5, the temperature for treatment with the decolorizing adsorbent is 70 - 80 °C, and the time is 1 - 2 hours; Preferably, in step S5, the temperature for standing is 0 - 5 °C, and the standing time is 15 - 20 hours; Preferably, in step S5, the decolorizing adsorbent is activated carbon.

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