METHOD FOR REFINING VASOPRESSIN

Abstract

A method for refining vasopressin, including: subjecting a crude vasopressin solution to reversed-phase enrichment, reversed-phase salt conversion and reversed-phase purification sequentially using reversed-phase high performance liquid chromatography. The crude vasopressin solution is obtained by oxidizing a crude reduced vasopressin prepared by solid phase synthesis. A super water-resistant packing material is used in the reversed-phase high performance liquid chromatography.

Claims

1. A method for refining vasopressin, comprising: subjecting a crude vasopressin solution to reversed-phase enrichment, reversed-phase salt conversion and reversed-phase purification sequentially using reversed-phase high performance liquid chromatography (RP-HPLC); wherein a packing material used in the RP-HPLC is a water-resistant packing material; the reversed-phase enrichment, the reversed-phase salt conversion and the reversed-phase purification are all completed in one reversed-phase elution, and conditions of the reversed-phase elution are listed as follows: TABLE-US-00006 Steps Time Eluent 1 0-50 min 100% sample C1 2 51-71 min 100% mobile phase C2 3 72-90 min 100% mobile phase A 4 90-95 min 100% mobile phase A.fwdarw.90% mobile phase A + 10% mobile phase B 5 95-125 min 90% mobile phase A + 10% mobile phase B.fwdarw.80% mobile phase A + 20% mobile phase B wherein the mobile phase A consists of 0.005-0.1% by volume of acetic acid and water; the mobile phase B consists of 0.005-0.1% by volume of acetic acid and acetonitrile; the sample C1 is the crude vasopressin solution; the mobile phase C2 is a 5-50 mM aqueous NH.sub.4AcNH.sub.4OH solution at pH 7.0-9.0; and a flow rate of the eluent is 80-100 mL/min; and collecting an eluate within a retention time of 105-115 min to obtain a pure vasopressin solution.

2. The method of claim 1, wherein the crude vasopressin solution is obtained by dissolving, diluting, and oxidizing a crude reduced vasopressin product prepared by solid phase synthesis.

3. The method of claim 2, wherein the crude reduced vasopressin product is dissolved and diluted to produce a solution of the crude reduced vasopressin product; a concentration of the solution of the crude reduced vasopressin product is 0.1-4 mg/mL; and a solvent for dissolving the crude reduced vasopressin product is a 50% aqueous acetic acid solution.

4. The method of claim 3, wherein the concentration of the solution of the crude reduced vasopressin product is 0.5-2 mg/mL.

5. The method of claim 1, wherein a formula of vasopressin in the crude vasopressin solution is ##STR00006## and a solvent of the crude vasopressin solution is an aqueous solution containing trifluoroacetic acid and acetic acid.

6. The method of claim 1, wherein the water-resistant packing material is UniSil ODS-AQ material.

7. The method of claim 1, wherein the water-resistant packing material has a pore diameter of 7-10 nm and a particle size of 10 m.

8. The method of claim 1, wherein a detection wavelength of the RP-HPLC is 220 nm.

9. The method of claim 1, wherein the mobile phase A consists of 0.02-0.05% by volume of acetic acid and water; and/or the mobile phase B consists of 0.02-0.05% by volume of acetic acid and acetonitrile; and/or the mobile phase C2 is a 10-20 mM aqueous NH.sub.4AcNH.sub.4OH solution; and/or the pH of the mobile phase C2 is 7.5-8.5; and/or a HPLC purity of crude vasopressin in the crude vasopressin solution is 60%-85%.

10. The method of claim 9, wherein the HPLC purity of the crude vasopressin in the crude vasopressin solution is 70%-85%.

11. The method of claim 10, wherein the HPLC purity of the crude vasopressin in the crude vasopressin solution is 70%-80%.

12. The method of claim 1, wherein during a period of 50-51 min, the eluent is changed from the sample C1 to the mobile phase C2; and during a period of 71-72 min, the eluent is changed from the mobile phase C2 to the mobile phase A.

13. The method of claim 1, wherein in steps (4) and (5) of the reversed-phase elution, a proportion of the mobile phase A in the eluent decreases at a uniform rate and a proportion of the mobile phase B in the eluent increases at a uniform rate.

Description

DETAILED DESCRIPTION OF EMBODIMENTS

[0041] The invention will be further described below in detail with reference to the embodiments, but the invention is not limited to these embodiments. Unless otherwise specified, the experiments in the following embodiments are performed according to conventional methods and conditions, or according to the instruction of the manufacturer.

[0042] The UniSil ODS-AQ super water-resistant packing material (pore size: 10 nm; particle size: 10 m) used in the embodiments is purchased from Suzhou NanoMicro Technology Co., Ltd.

[0043] The purity of vasopressin in the crude and purified vasopressin solutions is detected by HPLC, where the HPLC parameters are listed as follows.

[0044] Instrument: Agilent 1260 High Performance Liquid Chromatograph;

[0045] Chromatographic column: Waters)(Bridge C18 (4.6250 mm, 5 m);

[0046] Mobile phase: A: an aqueous solution containing 0.1% by volume of trifluoroacetic acid; B: an aqueous solution containing 0.1% by volume of trifluoroacetic acid and 50% by volume of acetonitrile;

[0047] Flow rate: 1.0 mL/min;

[0048] Detection wavelength: 210 nm;

[0049] Column temperature: 25 C.

[0050] The elution gradient is shown in Table 1, where the percentage is calculated by volume.

TABLE-US-00002 TABLE 1 Elution program for HPLC determination of vasopressin Steps Time Eluent 1 0-2 min 95% A + 5% B 2 2-12 min 95% A + 5% B.fwdarw.85% A + 15% B 3 12-22 min 85% A + 15% B 4 22-30 min 85% A + 15% B.fwdarw.77% A + 23% B 5 30-30.1 min 77% A + 23% B.fwdarw. 50% A + 50% B 6 30.1-35 min 50% A + 50% B

[0051] The crude vasopressin solution is obtained by dissolving, diluting, and oxidizing a crude reduced vasopressin synthesized by solid phase synthesis. Specifically, Rink Amide MBHA resin is used as solid support, and Fmoc-protected amino acids are coupled one by one in the presence of HOBt/DIC (condensing agent) to form a peptide. The peptide is cleaved from the resin under the action of a cleaving agent and precipitated with methyl tert-butyl ether to obtain crude reduced vasopressin, where the cleaving agent is a mixture of TFA, TIS and H.sub.2O in a volume ratio of 90:7.5:2.5. Then the crude reduced vasopressin is dissolved with a 50% aqueous acetic acid solution, and diluted with water to obtain the crude reduced vasopressin solution, which is subsequently adjusted to pH 7.0-9.0 with a basic substance and added with 30% hydrogen peroxide (0.5 mL per gram of crude reduced vasopressin) for oxidation to obtain a crude oxidized vasopressin solution, where the basic substance is NaOH.

Example 1 Packing of L&L4002 Preparation Column with an Inner Diameter of 50 mm

[0052] UniSil ODS-AQ material with a pore size of 10 nm and a particle size of 10 m was used as packing material, and the packing was performed by a Load&Lock dynamic axial compression and static locking technology at a pressure of 1000 psi using a Varian chromatography column packing station. Specifically, 300 g of powdered UniSil ODS-AQ material was mixed evenly with 600 mL of isopropanol under stirring, and then the mixture was poured into the Load&Lock4002 preparation column with an inner diameter of 50 mm, where the compression ratio was 1.5:1; the carrier gas was N.sub.2, which was adjusted such that a pressure displayed on a oil pressure gauge was 1500 psi; and the dynamic axial compression is performed to enable that the column bed length was 25 cm. The obtained preparation column was used in the reversed-phase enrichment, salt conversion and purification.

Example 2 Reversed-Phase Enrichment, Reversed-Phase Salt Conversion and Reversed-Phase Purification of Crude Vasopressin Solution

[0053] Instrument: Varian SD-1 preparative high-pressure liquid chromatograph system. Column: self-prepared preparation column Load&Lock4002 (50250 mm, UniSil ODS-AQ (particle size: 10 m; pore size: 10 nm)).

[0054] The vasopressin had a structural formula of

##STR00003##

The concentration of the crude reduced vasopressin in the crude reduced vasopressin solution was 0.1 mg/mL, and the solvent of the crude vasopressin solution was an aqueous solution containing trifluoroacetic acid and acetic acid.

[0055] The mobile phase A was a 0.02% aqueous acetic acid solution; the mobile phase B consisted of 0.02% by volume of acetic acid and acetonitrile; the sample C1 was the crude vasopressin solution, in which the vasopressin had a HPLC purity of 73.63%; and the mobile phase C2 was a 10 mM aqueous NH.sub.4AcNH.sub.4OH solution (pH 7.5).

[0056] The conditions for reversed-phase enrichment, reversed-phase salt conversion and reversed-phase purification were listed as follows: flow rate: 1.0 mL/min; detection wavelength: 220 nm; and elution gradient was shown in Table 2 (% referred to percentage by volume).

TABLE-US-00003 TABLE 2 Elution program for reversed-phase enrichment, salt conversion and purification Steps Time Eluent 1 0-50 min 100% sample C1 2 51-71 min 100% mobile phase C2 3 72-90 min 100% mobile phase A 4 90-95 min 100% mobile phase A.fwdarw.90% mobile phase A + 10% mobile phase B 5 95-125 min 90% mobile phase A + 10% mobile phase B.fwdarw.80% mobile phase A + 20% mobile phase B

[0057] During an elution period of 125-126 min after the step (5), a proportion of mobile phase A in the eluent uniformly decreased from 80% to 50%, and a proportion of the mobile phase B in the eluent uniformly increased to 50% correspondingly. During the period of 126-135 min, the eluent was kept constant in the composition (50% mobile phase A and 50% mobile phase B) to clean the chromatographic column. The eluate with a retention time of 105-115 min was collected as the purified vasopressin solution, which was measured by HPLC to have a vasopressin purity of 99.56%.

[0058] The removal rate of impurities in the crude vasopressin solution was 25.93%. The eluents used in the steps (1) to (3) were all aqueous solutions, which can be directly treated and recycled after use and will not pollute the environment. Compared with the traditional preparation process, the method provided herein greatly reduced the generation of hazardous waste liquid, lowering the treatment cost and avoiding environmental pollution.

Example 3 Reversed-Phase Enrichment, Reversed-Phase Salt Conversion and Reversed-Phase Purification of Crude Vasopressin Solution

[0059] Instrument: Varian SD-1 preparative high-pressure liquid chromatograph system.

[0060] Column: self-prepared preparation column Load&Lock4002 (50250 mm, UniSil ODS-AQ (particle size: 10 m; pore size: 10 nm)).

[0061] The vasopressin had a structural formula of

##STR00004##

The concentration of the crude reduced vasopressin in the crude reduced vasopressin solution was 1.5 mg/mL, and the solvent of the crude vasopressin solution was an aqueous solution containing trifluoroacetic acid and acetic acid.

[0062] The mobile phase A was a 0.05% aqueous acetic acid solution; the mobile phase B consisted of 0.05% by volume of acetic acid and acetonitrile; the sample C1 was the crude vasopressin solution, in which the vasopressin had a HPLC purity of 75.23%; and the mobile phase C2 was a 20 mM aqueous NH.sub.4AcNH.sub.4OH solution (pH 8.5).

[0063] The conditions for reversed-phase enrichment, reversed-phase salt conversion and reversed-phase purification were listed as follows: flow rate: 100 mL/min; detection wavelength: 220 nm; and elution gradient was shown in Table 3 (% referred to percentage by volume)

TABLE-US-00004 TABLE 3 Elution program for reversed-phase enrichment, salt conversion and purification Steps Time Eluent 1 0-50 min 100% sample C1 2 51-71 min 100% mobile phase C2 3 72-90 min 100% mobile phase A 4 90-95 min 100% mobile phase A.fwdarw.90% mobile phase A + 10% mobile phase B 5 95-125 min 90% mobile phase A + 10% mobile phase B.fwdarw.80% mobile phase A + 20% mobile phase B

[0064] During an elution period of 125-126 min after the step (5), a proportion of mobile phase A in the eluent uniformly decreased from 80% to 50% mobile phase A, and a proportion of the mobile phase B in the eluent uniformly increased to 50% correspondingly. During the period of 126-135 min, the eluent was kept constant in the composition (50% mobile phase A and 50% mobile phase B) to clean the chromatographic column. The eluate with a retention time of 105-115 min was collected as the purified vasopressin solution, which was measured by HPLC to have a vasopressin purity of 99.62%. In this example, the removal rate of impurities in the crude vasopressin solution was 24.39%.

Example 4 Reversed-Phase Enrichment, Reversed-Phase Salt Conversion and Reversed-Phase Purification of Crude Vasopressin Solution

[0065] Instrument: Varian SD-1 preparative high-pressure liquid chromatograph system

[0066] Column: self-prepared preparation column Load&Lock4002 (50250 mm, ODS-AQ (particle size: 10 m; pore size: 10 nm)).

[0067] The vasopressin had a structural formula of

##STR00005##

The concentration of the crude reduced vasopressin in the crude reduced vasopressin solution was 0.8 mg/mL, and the solvent of the crude vasopressin solution was an aqueous solution containing trifluoroacetic acid and acetic acid.

[0068] The mobile phase A was a 0.05% aqueous acetic acid solution; the mobile phase B consisted of 0.05% by volume of acetic acid and acetonitrile; the sample C1 was the crude vasopressin solution, in which the vasopressin had a HPLC purity of 75.66%; and the mobile phase C2 was a 20 mM aqueous NH.sub.4AcNH.sub.4OH solution (pH 7.5).

[0069] The conditions for reversed-phase enrichment, reversed-phase salt conversion and reversed-phase purification were listed as follows: flow rate: 100 mL/min; detection wavelength: 220 nm; and elution gradient was shown in the Table 4 (% referred to percentage by volume).

TABLE-US-00005 TABLE 4 Elution program for reversed-phase enrichment, salt conversion and purification Steps Time Eluent 1 0-50 min 100% sample C1 2 51-71 min 100% mobile phase C2 3 72-90 min 100% mobile phase A 4 90-95 min 100% mobile phase A.fwdarw.90% mobile phase A + 10% mobile phase B 5 95-125 min 90% mobile phase A + 10% mobile phase B.fwdarw.80% mobile phase A + 20% mobile phase B

[0070] During an elution period of 125-126 min after the step (5), a proportion of mobile phase A in the eluent uniformly decreased from 80% mobile phase A to 50%, and a proportion of the mobile phase B in the eluent uniformly increased to 50% correspondingly. During the period of 126-135 min, the eluent was kept constant in the composition (50% mobile phase A and 50% mobile phase B) to clean the chromatographic column. The eluate with a retention time of 105-115 min was collected as the purified vasopressin solution, which was measured by HPLC to have a vasopressin purity of 99.52%. The removal rate of impurities in the crude vasopressin solution was 23.86%.

Example 5 Mass Spectrometry (MS) Detection of Vasopressin

[0071] Waters Micromass ZQ single quadrupole electrospray ionization mass spectrometry (ESI-MS) was used to determine the purified vasopressin obtained in Examples 2, 3 and 4, where the MS analysis was carried out under the following conditions:

[0072] ion source: electron spray ion source (ESI+);

[0073] capillary ionization voltage: 3.0 kV;

[0074] cone voltage: 35 kV;

[0075] ion source temperature: 115 C.;

[0076] desolvation temperature: 350 C.;

[0077] desolvation nitrogen flow rate: 700 L/h;

[0078] cone counter-blow nitrogen: 50 L/h; and

[0079] scan range (m/z): 50.0-1500.

[0080] It can be seen from the detection results that a mass-to-charge ratio (m/z) of the molecular ion peak [M+H].sup.+ was 1084.41, and a primary ion fragment peak [M+2H].sup.2+ had a mass-to-charge ratio (m/z) of 542.71, which were consistent with the theoretical molecular weight (1084.24) of vasopressin.