阅读说明：本技术 一种精氨加压素的纯化方法 (Method for purifying argirelin ) 是由 李海贵 黄伟 钟南旦 周亚清 于 2021-05-31 设计创作，主要内容包括：本发明提供了一种精氨加压素的纯化方法,将粗品溶解于乙腈水溶液中,经氧化得环肽粗品,过滤；将滤液用一种反相填料,两步纯化,一种转盐方法即可得到高纯度、高质量、高稳定性的精氨加压素原料药。通过优化纯化方法,控制纯化过程中产品组分液的pH值,和优化转盐过程中的乙酸比例、降低有机试剂比例进行等度洗脱,即可将产品中易降解杂质的含量稳定在≤0.10％范围内。本发明的纯化方法操作简单易行,纯化轮数少,试剂使用成本低,提高了生产效率,又有效地解决了精氨加压素稳定性差、纯度低的问题。(The invention provides a purification method of argininol, which comprises the steps of dissolving a crude product in acetonitrile aqueous solution, oxidizing to obtain a cyclic peptide crude product, and filtering; purifying the filtrate with a reverse phase filler in two steps, and obtaining the high-purity, high-quality and high-stability argininol bulk drug by a salt conversion method. By optimizing the purification method, controlling the pH value of the product component liquid in the purification process, optimizing the acetic acid proportion in the salt conversion process and reducing the organic reagent proportion for isocratic elution, the content of easily degradable impurities in the product can be stabilized within the range of less than or equal to 0.10 percent. The purification method provided by the invention is simple and feasible to operate, has few purification rounds and low reagent use cost, improves the production efficiency, and effectively solves the problems of poor stability and low purity of the argininol.)

1. A method for purifying argininol, comprising: the method comprises the following steps:

s1, preparation of a crude cyclic peptide solution: dissolving the linear arginine vasopressin crude product in an acetonitrile aqueous solution with the volume fraction of 5-10%, wherein the mass-to-volume ratio g/L of the linear arginine vasopressin crude product to the acetonitrile aqueous solution is 3-22: 1.5-11, adjusting the pH value to 6.0-9.0, adding an oxidant, and filtering to obtain a filtrate, namely a cyclic peptide crude product solution;

s2, first-step purification: the crude cyclopeptide solution was subjected to a first purification step:

mobile phase A: 0.05 to 1.0 mass percent of phosphoric acid aqueous solution, adjusting the pH value to 2.0 to 4.5,

mobile phase B: a mixed solution of organic reagents or an organic reagent,

flow rate: 20 to 100mL/min of the reaction solution,

gradient elution: eluting 10-50 wt% of the mobile phase B for 30-80 min, and collecting the peptide solution A with the target peak value in a segmented manner;

s3, second-step purification: the peptide solution a collected in S2 was diluted with water and subjected to a second purification step:

mobile phase A: 10 to 100mmol/L acetate buffer solution, adjusting the pH to 4.0 to 6.0,

mobile phase B: a mixed solution of organic reagents or an organic reagent,

flow rate: 20 to 100mL/min of the reaction solution,

gradient elution: eluting for 30-80 min by 15-60 wt% of mobile phase B, collecting peptide solution B with a target peak value in a segmented manner, and adjusting the pH value to 2.5-4.5 to obtain a high-purity peptide solution;

s4, converting acetic acid into salt:

the high-purity peptide solution obtained in S3 is subjected to salt conversion by an acetic acid method:

mobile phase A: 0.1-1.0% by mass of an acetic acid aqueous solution,

mobile phase B: the acetic acid-acetonitrile solution is prepared by dissolving acetic acid-acetonitrile,

flow rate: 50 to 100mL/min of the reaction solution,

isocratic elution: eluting the mobile phase B for 25-40 min, and freeze-drying to obtain a pure freeze-dried product of the argininyl vasopressin.

2. A method of purifying argireline as claimed in claim 1, wherein: the oxidant in the S1 is one or more selected from hydrogen peroxide, dimethyl sulfoxide, elemental iodine and a metal ion oxidant, and the metal ion oxidant is an iron ion oxidant, a copper ion oxidant or a silver ion oxidant.

3. A method of purifying argireline as claimed in claim 1, wherein: the organic reagent mixed liquid of the mobile phase B in the S2 and S3 is organic reagent-aqueous solution or organic reagent-mobile phase A mixed liquid.

4. A method of purifying argireline as claimed in claim 3, wherein: the volume ratio of the organic reagent to the aqueous solution in the organic reagent-aqueous solution is 50-100: 0-50; in the mixed liquid of the organic reagent and the mobile phase A, the volume ratio of the organic reagent to the mobile phase A is 50-100: 0-50.

5. A method of purifying argireline as claimed in claim 3 or 4, wherein: the organic reagent is one or a mixture of acetonitrile, methanol, isopropanol, ethanol and tetrahydrofuran.

6. A method of purifying argireline as claimed in claim 5, wherein: the organic reagent is one or a mixture of acetonitrile and methanol.

7. A method of purifying argireline as claimed in claim 1, wherein: the mass percent of the mobile phase B in the gradient elution in the S2 and S3 is 10-60%.

8. A method of purifying argireline as claimed in claim 1, wherein: the volume ratio of the peptide solution A to the water in the S3 is 1: 1.

9. A method of purifying argireline as claimed in claim 1, wherein: the acetate buffer solution in the S3 is one or a mixture of ammonium acetate, sodium acetate and potassium acetate.

10. A method of purifying argireline as claimed in claim 1, wherein: the mobile phase B in the S4 is a mixture of a solvent and a solvent, wherein the volume ratio of the mobile phase B in the S4 is 0.5: 99.5 acetic acid and acetonitrile, isocratic elution being 15 wt% mobile phase B.

Technical Field

The invention relates to the technical field of purification in polypeptide synthesis, in particular to a purification method of argininol.

Background

Argininol is a synthetic polypeptide consisting of nine amino acids, has a theoretical molecular weight of 1084.24, and is an antidiuretic hormone. Is an important neurotransmitter in the central nervous system. It is a substance secreted by hypothalamic cells, and is closely related to body fluid metabolism, blood volume, cardiovascular function, body temperature balance, learning and memory, secretion of adrenocorticotropic hormone, etc. Has effects in increasing blood pressure, promoting urination, improving memory, inhibiting fever, and relieving fever. Meanwhile, the compound also has wide cardiovascular effects, such as the functions of regulating water and sodium retention and vasoconstriction, directly regulating myocardial cells, participating in myocardial hypertrophy, myocardial fibrosis, arrhythmia and the like. Has been successfully used for treating vasodilative shock, cardio-pulmonary-cerebral resuscitation, diabetes insipidus and hemorrhagic diseases. Diabetes insipidus can be caused by insufficient secretion of antidiuretic hormone, symptoms of headache, dizziness and memory deterioration can be easily caused if the antidiuretic hormone cannot be treated in time, serious patients can suffer from water poisoning, visual deterioration, coma and even death, and the harm is great. Therefore, the method has important significance for the research of the purification of the argininopressin.

The patent "a purification method of desmopressin acetate", publication no: CN105131079B, the purity is low when the method is used for one-time purification, and the proportion of organic reagents used in the salt conversion step is higher and reaches 5% -18%, so that the impurity removal effect is poor;

the patent "a method for purifying terlipressin acetate", publication No.: CN102775475A, the purification effect is poor and the purification is unstable. It is known that the stability of the arginine vasopressin in the prior art is poor, the purity is low, the impurity content is high, and the like.

Disclosure of Invention

Accordingly, the present invention provides a method for purifying argininol, which solves the above problems.

The technical scheme of the invention is realized as follows: a method for purifying argininol, comprising the steps of:

s1, preparation of a crude cyclic peptide solution: dissolving the linear arginine vasopressin crude product in 5-10% (V/V) acetonitrile water solution, wherein the mass volume ratio g/L of the linear arginine vasopressin crude product to the acetonitrile water solution is 3-22: 1.5-11, adjusting the pH value to 6.0-9.0 by using an alkaline regulator, preferably the pH value to 7.0-8.0, adding an oxidant for oxidation, filtering by using a 0.22 mu m organic micron membrane, and obtaining a filtrate which is a cyclopeptide crude product solution;

s2, first-step purification: the crude cyclopeptide solution was subjected to a first purification step:

the chromatographic column diameter and length are: 30 x 250mm, 50 x 250mm, 80 x 250 mm.

Stationary phase: octadecylsilane chemically bonded silica filler,

mobile phase A: 0.05 to 1.0 mass percent of phosphoric acid aqueous solution, preferably 0.5 percent (m/V), and the pH is adjusted to 2.0 to 4.5, preferably 2.5 to 4.0,

mobile phase B: a mixed solution of organic reagents or an organic reagent,

flow rate: 20 to 100mL/min of the reaction solution,

gradient elution: 10-50 wt% of mobile phase B, preferably 30 wt%, eluting for 30-80 min, preferably 60min, detecting the wavelength to be 220nm, and collecting the peptide solution A of the target peak value in a segmented manner;

s3, second-step purification: the peptide solution a collected in S2 was diluted with purified water and subjected to a second purification step:

stationary phase: octadecylsilane chemically bonded silica filler,

mobile phase A: 10 to 100mmol/L acetate buffer solution, adjusting the pH to 4.0 to 6.0,

mobile phase B: a mixed solution of organic reagents or an organic reagent,

flow rate: 20 to 100mL/min of the reaction solution,

gradient elution: eluting the mobile phase B by 15-60 wt% for 30-80 min, preferably 60min, detecting the wavelength of 220nm, collecting the peptide solution B by stages, and adjusting the pH value to 2.5-4.5, preferably 3.0-4.0 to obtain a high-purity peptide solution;

s4, converting acetic acid into salt: the high-purity peptide solution obtained in S3 is subjected to salt conversion by an acetic acid method:

stationary phase: octadecylsilane chemically bonded silica filler,

mobile phase A: an aqueous acetic acid solution having a mass fraction of 0.1 to 1.0%, preferably 0.5% (m/V),

mobile phase B: the acetic acid-acetonitrile solution is prepared by dissolving acetic acid-acetonitrile,

flow rate: 50 to 100mL/min of the reaction solution,

isocratic elution: eluting the mobile phase B for 25-40 min, preferably 30min, and freeze-drying to obtain a pure freeze-dried product of the argininyl vasopressin.

Further, the oxidant in S1 is one or more selected from hydrogen peroxide, dimethyl sulfoxide, elemental iodine, and a metal ion oxidant, and the metal ion oxidant is an iron ion oxidant, a copper ion oxidant, or a silver ion oxidant.

In addition, the organic reagent mixed solution of the mobile phase B in S2 and S3 is an organic reagent-aqueous solution or an organic reagent-mobile phase A mixed solution or an organic reagent.

Further, the volume ratio of the organic reagent to the aqueous solution in the organic reagent-aqueous solution is 50-100: 0-50; in the mixed liquid of the organic reagent and the mobile phase A, the volume ratio of the organic reagent to the mobile phase A is 50-100: 0-50.

Further, the organic reagent is one or more of acetonitrile, methanol, isopropanol, ethanol and tetrahydrofuran, and preferably one or two of acetonitrile and methanol.

Further, the mass percent of the mobile phase B in the gradient elution in the S2 and S3 is 10-60%.

Further, the volume ratio of the peptide solution A to the water in the S3 is 1: 1.

Further, the acetate buffer solution in S3 is one or more of ammonium acetate, sodium acetate, and potassium acetate.

Further, the mobile phase B in S4 is a mixture of two or more of 0.5: 99.5 acetic acid and acetonitrile, isocratic elution being 15 wt% mobile phase B.

Compared with the prior art, the invention has the beneficial effects that:

(1) according to the invention, the arginine vasopressin bulk drug with high purity, high quality and high stability can be obtained by only one kind of reverse phase filler, two-step purification and one kind of salt conversion method, the purification method is simple and easy to operate, the purity can reach 98% in one step, the high-purity product with the purity of more than 99.5% and the single impurity content of less than or equal to 0.10% can be obtained by two-step purification, the reagent use cost is low, the number of purification rounds is few, the production efficiency is improved, and the problems of poor stability, low purity and high impurity content of the arginine vasopressin are effectively solved.

(2) According to the invention, by optimizing the purification method, controlling the pH value of the product component liquid in the purification process to be 2.5-4.5, optimizing the acetic acid ratio in the salt conversion process, reducing the organic reagent ratio, and carrying out isocratic elution, the easily degradable impurities in the product can be obtained: the contents of Dimer, Ac 1-9, Trisulfide, Glu4, Gly9-OH and the like are stabilized within the range of less than or equal to 0.10 percent, and finally, a pure product with good stability and high purity can be obtained. The acetic acid salt conversion method has the advantages that the organic reagent with low proportion is used for isocratic elution, and the use cost of the organic reagent is effectively reduced.

Drawings

FIG. 1 is an HPLC chart of the crude argininopressin cyclopeptide in example 3;

FIG. 2 is a HPLC chart of the product of the first purification step of argininol in example 3;

FIG. 3 is a HPLC chart of the product of the second purification step of argininol in example 3;

FIG. 4 is a MS picture of the product of argininol in example 3.

Detailed Description

In order to better understand the technical content of the invention, specific examples are provided below to further illustrate the invention.

The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.

The materials, reagents and the like used in the examples of the present invention can be obtained commercially without specific description.

Example 1

A method for purifying argininol, comprising the steps of:

s1, preparation of a crude cyclic peptide solution:

dissolving 3 g of the arginine vasopressin linear peptide crude product in 1.5L of 5% (V/V) acetonitrile water solution, adjusting the pH of the linear peptide crude product to 7.0 by using sodium hydroxide, oxidizing by using 0.3% (V/V) hydrogen peroxide, filtering by using a 0.22 micron membrane after the oxidation is finished, and collecting the filtered crude product solution for later use;

s2, first-step purification:

a chromatographic column: stationary phase: octadecylsilane chemically bonded silica packing, the chromatographic column diameter and length are: 30 x 250 mm.

Mobile phase A: 0.5 percent phosphoric acid aqueous solution by mass fraction, adjusting the pH value to 4.0 by using sodium hydroxide,

mobile phase B: the volume ratio is 80%: 20% acetonitrile-mobile phase a solution,

flow rate: 20mL/min, wavelength: 220nm, the loading amount is 3 g,

gradient elution: eluting by 10 wt% of mobile phase B for 60min, and collecting the target peak peptide solution A of the gradient section in a segmented manner to obtain a target peak peptide solution with the purity of more than 98%, wherein the purification yield of the first step is more than 85%.

S3, second-step purification:

diluting the peptide solution A collected in the first purification step with purified water according to the volume ratio of 1:1, and performing second purification:

stationary phase: octadecylsilane chemically bonded silica filler

Mobile phase A: 80mmol/L ammonium acetate (pH adjusted to 6.0 with phosphoric acid),

mobile phase B: the volume ratio is 80%: a 20% aqueous solution of acetonitrile in water,

flow rate: 20mL/min, wavelength: 220 nm.

Gradient elution: 15 wt% mobile phase B, elution 60 min.

And (3) collecting the peptide solution B of the target peak value of the gradient section in a segmented manner, adjusting the pH value to 3.0-4.0 by using phosphoric acid to obtain the target peak value peptide solution with the purity of more than or equal to 99.5 percent and the single impurity of less than or equal to 0.10 percent, wherein the purification yield of the second step is more than 85 percent, and the purification yield of the two steps is more than 72 percent.

S4, converting acetic acid into salt:

the high-purity peptide solution obtained in S3 is subjected to salt conversion by an acetic acid method:

stationary phase: octadecylsilane chemically bonded silica filler,

mobile phase A: 0.5 percent of acetic acid aqueous solution by mass fraction,

mobile phase B: acetic acid-acetonitrile solution with mass fraction of 0.5 percent,

flow rate: 20mL/min, wavelength: 220 nm.

And (3) elution: and (3) isocratically eluting the 15 wt% mobile phase B for 30min to obtain the peptide solution after salt conversion.

And (3) putting the peptide solution after the salt conversion into a freeze-drying tray, and freeze-drying to obtain the high-purity and high-stability argininyl vasopressin freeze-dried powder with the purity of more than or equal to 99.5 percent and the single impurity of less than or equal to 0.10 percent, wherein the total purification yield is 78 percent.

Example 2

A method for purifying argininol, comprising the steps of:

s1, preparation of a crude cyclic peptide solution:

dissolving 8 g of the argininol linear peptide crude product in 4L of 10% (V/V) acetonitrile aqueous solution, adjusting the pH of the linear peptide crude product to 7.0 by using sodium hydroxide, oxidizing by using 0.3% (V/V) hydrogen peroxide, filtering by using a 0.22 micron membrane after the oxidation is finished, and collecting the filtered crude product solution for later use.

S2, first-step purification:

a chromatographic column: stationary phase: octadecylsilane chemically bonded silica packing, the chromatographic column diameter and length are: 50 x 250 mm.

Mobile phase A: phosphoric acid aqueous solution with mass fraction of 0.5 percent is adjusted to pH value of 4.0 by sodium hydroxide,

mobile phase B: the volume ratio is 80%: 20% acetonitrile-mobile phase a solution,

flow rate: 50mL/min, wavelength: 220nm, the sample loading amount is 8 g,

gradient elution: eluting by 50 wt% of mobile phase B for 60min, and collecting the target peak peptide solution A of the gradient section in a segmented manner to obtain a target peak peptide solution with the purity of more than 98%, wherein the purification yield of the first step is more than 85%.

S3, second-step purification:

diluting the peptide solution A collected in the first purification step with purified water according to the volume ratio of 1:1, and performing second purification:

stationary phase: octadecylsilane chemically bonded silica filler

Mobile phase A: 80mmol/L ammonium acetate (pH adjusted to 6.0 with phosphoric acid),

mobile phase B: the volume ratio is 80%: 20% acetonitrile in water.

Flow rate: 50mL/min, wavelength: 220 nm.

Gradient elution: 60 wt% mobile phase B, eluting for 60 min.

Collecting the target peak peptide solution B of the gradient section in a segmented manner, adjusting the pH value to 3.0-4.0 by using phosphoric acid to obtain the target peak peptide solution with the purity of more than or equal to 99.5 percent and the single impurity of less than or equal to 0.10 percent, and the purification yield of the second step is more than 85 percent. The yield of the two-step purification is more than 72 percent.

S4, converting acetic acid into salt:

the high-purity peptide solution obtained in S3 is subjected to salt conversion by an acetic acid method:

stationary phase: octadecylsilane chemically bonded silica filler,

mobile phase A: 0.5% (m/V) of an aqueous acetic acid solution,

mobile phase B: 0.5 percent of acetic acid-acetonitrile solution by mass fraction,

flow rate: 50mL/min, wavelength: 220 nm.

And (3) elution: and (3) isocratically eluting the 15 wt% mobile phase B for 30min to obtain the peptide solution after salt conversion.

And (3) putting the peptide solution after the salt conversion into a freeze-drying tray, and freeze-drying to obtain the high-purity and high-stability argininyl vasopressin freeze-dried powder with the purity of more than or equal to 99.5 percent and the single impurity of less than or equal to 0.10 percent, wherein the total purification yield is 75 percent.

Example 3

A method for purifying argininol, comprising the steps of:

s1, preparation of a crude cyclic peptide solution:

dissolving 22 g of the argininol linear peptide crude product in 11L of 7% (V/V) acetonitrile aqueous solution, adjusting the pH of the linear peptide crude product to 7.0 by using sodium hydroxide, oxidizing by using 0.3% (V/V) hydrogen peroxide, filtering by using a 0.22 micron membrane after the oxidation is finished, and collecting the filtered crude product solution for later use. The chromatogram of the crude product of the cyclic peptide of argininopressin is shown in FIG. 1, and the purity is 75.2%

S2, first-step purification:

a chromatographic column: stationary phase: octadecylsilane chemically bonded silica packing, the chromatographic column diameter and length are: 80 x 250 mm.

Mobile phase A: phosphoric acid aqueous solution with mass fraction of 0.5 percent is adjusted to pH value of 4.0 by sodium hydroxide,

mobile phase B: the volume ratio is 80%: 20% acetonitrile-mobile phase a solution,

flow rate: 100mL/min, wavelength: 220nm, the loading amount is 22 g,

gradient elution: eluting 30 wt% of mobile phase B for 60min, collecting the gradient section target peak peptide solution A in a segmented manner to obtain a target peak peptide solution with the purity of more than 98%, and taking the target peak peptide solution as the peptide solution purified in the second step, wherein the purification yield in the first step is more than 85%. The chromatogram of the product after the first purification step is shown in FIG. 2, and the purity is 98.5%.

S3, second-step purification:

diluting the peptide solution A collected in the first purification step with purified water according to the volume ratio of 1:1, and performing second purification:

stationary phase: octadecylsilane chemically bonded silica filler,

mobile phase A: 80mmol/L ammonium acetate (pH adjusted to 6.0 with phosphoric acid),

mobile phase B: the volume ratio is 80%: a 20% aqueous solution of acetonitrile in water,

flow rate: 100mL/min, wavelength: 220 nm.

Gradient elution: 40 wt% mobile phase B, elution 60 min.

And collecting the peptide solution B of the target peak value of the gradient section in a segmented manner, adjusting the pH value of the program to 3.0-4.0 by using phosphoric acid to obtain the target peak value peptide solution with the purity of more than or equal to 99.5 percent and the single impurity of less than or equal to 0.10 percent, and the purification yield of the second step is more than 80 percent. The yield of the two-step purification is more than 68 percent. The chromatogram of the product after the second purification step is shown in FIG. 3, the purity is 99.8, and the single impurity content is less than or equal to 0.04%.

S4, converting acetic acid into salt:

the high-purity peptide solution obtained in S3 is subjected to salt conversion by an acetic acid method:

stationary phase: octadecylsilane chemically bonded silica filler,

mobile phase A: acetic acid water solution with the mass fraction of 0.5 percent,

mobile phase B: 0.5% by mass acetic acid-acetonitrile solution.

Flow rate: 100mL/min, wavelength: 220 nm.

And (3) elution: and (3) isocratically eluting the 15 wt% mobile phase B for 30min to obtain the peptide solution after salt conversion.

And (3) putting the peptide solution after the salt conversion into a freeze-drying tray, and freeze-drying to obtain the high-purity and high-stability argininyl vasopressin freeze-dried powder with the purity of more than or equal to 99.5 percent and the single impurity of less than or equal to 0.10 percent, wherein the total purification yield is 72 percent. MS profile of the final lyophilized product is shown in fig. 4.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.