CN106749541B - Preparation method of vasopressin [5-Asp ] - Google Patents
Preparation method of vasopressin [5-Asp ] Download PDFInfo
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- CN106749541B CN106749541B CN201710002366.6A CN201710002366A CN106749541B CN 106749541 B CN106749541 B CN 106749541B CN 201710002366 A CN201710002366 A CN 201710002366A CN 106749541 B CN106749541 B CN 106749541B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/16—Oxytocins; Vasopressins; Related peptides
Abstract
The invention discloses a method for preparing vasopressin [5-Asp ]. The preparation method comprises the following steps: performing reverse phase cyclization, reverse phase purification and reverse phase desalting on the crude solution of the vasopressin [5-Asp ] precursor by adopting a high performance liquid reverse phase chromatography in turn; the filler of the high performance liquid phase reverse phase chromatography is styrene-divinylbenzene copolymer; the crude vasopressin [5-Asp ] precursor is crude vasopressin [5-Asp ] precursor containing two free sulfydryl groups. The invention innovatively applies the cyclization, purification and desalination by a reversed-phase adsorption method, solves the problems of cyclization, purification and desalination at one time, optimizes the production process and is suitable for industrial continuous production.
Description
Technical Field
The invention relates to the field of biological pharmacy. More specifically, the invention relates to a method for preparing vasopressin [5-Asp ].
Background
Vasopressin is a synthetic polypeptide consisting of nine amino acid residues and has the chemical structure
Pro-Arg-Gly-NH2The theoretical molecular weight of vasopressin is 1084.24. The vasopressin tannate is an antidiuretic hormone drug, can promote reabsorption of water by distal renal tubules and collecting ducts, has an antidiuretic effect, and is clinically used for treating diabetes insipidus.
At present, vasopressin [5-Asp ] is used domestically]Protection of vasopressin [5-Asp ] mostly by solid-phase synthesis]Cracking and drying the resin to obtain the vasopressin [5-Asp ]]Precursor crude product (Cys-Tyr-Phe-Gln-Asp-Cys-Pro-Arg-Gly-NH2Trifluoroacetate salt of (b), highly diluted cyclization, purification, salt conversion and other steps to finally obtain the vasopressin [5-Asp ]]. The high dilution cyclization is very unfavorable for the later purification due to the dilute concentration and large volume of the sample. An efficient method for preparing disulfide bond-containing polypeptide drug substances is still lacking, and therefore, development of a novel method for preparing disulfide bond-containing polypeptides is still urgently needed.
In the case of a drug, the small amount of impurities contained therein is the most important cause for the side effects of the drug, so that the purity inspection is one of the important bases for ensuring the safety and effectiveness of the drug, and the content of the purity inspection is somewhat different according to the properties and characteristics of each drug, but basically involves respective inspection research on "related substances". Although the purification process of the synthesized polypeptide has been greatly improved at present, the process impurities are still important sources of the synthesized polypeptide-related substances, mainly because some process impurities (such as deletion peptides, broken peptides, oxidized peptides, products of disulfide bond exchange and the like) of the synthesized polypeptide may be very similar to the properties of the drug per se, thereby causing certain difficulty in purification. Studies have shown that the most common degradation products in the synthesis of polypeptides are deamidates, oxygenates, and hydrolysates. Among the various amino acids that make up a polypeptide, asparagine, glutamine and peptide chain C-segment amide are susceptible to deamidation reactions (especially at elevated pH and elevated temperatures).
Because the properties of some impurities in the synthesized polypeptide are very close to those of a target product, establishing a proper method to fully detect the impurities is a great difficulty in researching substances related to the synthesized polypeptide drugs.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for preparing vasopressin [5-Asp ] in order to overcome the defects of complex preparation process, low yield, dilute sample concentration and large volume of vasopressin [5-Asp ] in the prior art. The preparation method of the vasopressin [5-Asp ] takes a polypeptide crude product containing a pair of free sulfydryl (-SH), namely a vasopressin [5-Asp ] precursor crude product, as a starting material, and prepares the high-purity vasopressin [5-Asp ] through the steps of high pressure liquid phase reverse phase chromatography cyclization, purification and desalination. The polypeptide of the invention is 1 key impurity in the preparation process of the vasopressin, so that the polypeptide can be used as a standard reference substance in the detection process of the vasopressin to carry out qualitative and quantitative analysis on the vasopressin and the impurities, and has important significance for improving the quality standard of the vasopressin and controlling the product quality.
The invention solves the technical problems through the following technical scheme:
the invention provides a preparation method of vasopressin [5-Asp ], which comprises the following steps: performing reverse phase cyclization, reverse phase purification and reverse phase desalting on the crude solution of the vasopressin [5-Asp ] precursor by adopting a high performance liquid reverse phase chromatography in turn; the filler of the high performance liquid phase reverse phase chromatography is styrene-divinylbenzene (PS-DVB) copolymer;
the crude vasopressin [5-Asp ] precursor is a crude vasopressin [5-Asp ] precursor containing two free sulfydryl groups;
the described vasopressin [5-Asp ]]Is composed of-Pro-Arg-Gly-NH2。
Wherein, the crude vasopressin [5-Asp ] precursor is preferably prepared into the crude vasopressin [5-Asp ] precursor by a solid-phase synthesis method through cracking and drying, and the HPLC purity is 60-90%; the structural formula of the crude vasopressin [5-Asp ] precursor is trifluoroacetate of Cys-Tyr-Phe-Gln-Asp-Cys-Pro-Arg-Gly-NH 2.
Wherein, the solution of the crude vasopressin [5-Asp ] precursor is preferably 5g/L solution of the crude vasopressin [5-Asp ] precursor dissolved in 5% by volume of acetonitrile water solution.
In the invention, the reverse phase cyclization, the reverse phase purification and the reverse phase desalination are all completed in a one-step reverse phase elution process.
Wherein, the conditions of reverse phase cyclization, reverse phase purification and reverse phase desalting are preferably as follows: the mobile phase A1 is pure water, A2 is H with a volume percentage of 0.01-0.05% (preferably 0.02-0.03%)2O2NaOH aqueous solution with pH of 7.5-9.0, wherein the mobile phase B is acetonitrile, and the mobile phase C1 is vasopressin [5-Asp ]]The precursor crude product solution is a 0.1mol/L NaOH solution as a mobile phase C2, the flow rate is 180-220 mL/min (preferably 200mL/min), and the detection wavelength is 220 nm;
carrying out on-line sample loading and elution according to the conditions of the following table, wherein the percentage is volume percentage;
| step of elution | Elution time | Eluent |
| 1 | 0～10min | 100％C1 |
| 2 | 10.1～25min | 75％A1+5％B+20％C2 |
| 3 | 25.1～40min | 95％A1+5％B |
| 4 | 40.1～45min | 95％A2+5％B |
| 5 | 45.1～60min | 95％A1+5％B |
| 6 | 60～75min | 95％A1+5％B→80％A1+20％B |
| 7 | 75～90min | 80％A1+20％B→73％A1+27％B |
| 8 | 90～105min | 50％A1+50％B |
| 9 | 105～115min | 95％A1+5％B |
Collecting the eluent with the retention time of 75-90 min to obtain the vasopressin [5-Asp ] solution.
Wherein, the packing conditions of the high performance liquid reverse phase chromatography are preferably as follows: the filler is Agilent PLRP-S styrene-divinylbenzene (PS-DVB) copolymer, the pore diameter is 10nm, and the particle diameter is 10 mu m.
Vasopressin [5-Asp ] is a polypeptide substance which is unstable and easy to degrade under alkaline conditions, and particularly under a strong alkaline environment, the concentration and time of alkaline elution are comprehensively considered, so that the damage and loss of a sample in the desalting process are reduced.
The innovation point of the invention is that the polypeptide pure product is obtained by one-step phase reversal of cyclization, purification and desalination, vasopressin [5-Asp ] contains two sulfydryl groups and basic amino acid, the traditional process is performed in steps of cyclization and purification, the cyclization volume is large, the purification difficulty is increased, and the latest application of the polymer filler PLRP-S styrene-divinylbenzene is designed for the rapid and efficient cyclization and preparation process. The invention innovatively applies the reverse phase adsorption method for cyclization, purification and desalination, and solves the problems of cyclization, purification and desalination at one time.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows:
(1) the invention adopts an on-line cyclization method, firstly, a reduced polypeptide crude product is adsorbed on a stationary phase, strongly bound acid radical ions are eluted on a chromatographic column in an alkaline manner and then free hydroxyl is eluted in a neutral manner by utilizing the hydrophobic combination of the polypeptide and a reverse phase filler, and two sulfydryl groups are promoted to form disulfide bonds by adopting a mobile phase containing H2O2 with alkaline pH value to wash, so that a target polypeptide crude product is obtained, and a sample is retained on the chromatographic column.
(2) The method adopts on-line cyclization, the sample subjected to cyclization avoids elution, and gradient elution purification can be carried out after mobile phase conversion directly to obtain a final pure product, so that the method is suitable for continuous production.
(3) The invention creatively uses the one-step method of reversed phase adsorption cyclization, purification and desalination to prepare the pure polypeptide product, optimizes the production process and is suitable for industrial continuous production.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
In the following examples, vasopressin [5-Asp ]]Is composed of
-Pro-Arg-Gly-NH2。
Example 1
EXAMPLE 1 HPLC method for determining the purity of crude vasopressin [5-Asp ] precursor and purified product solution
The instrument comprises the following steps: waters 2695/2489 high performance liquid chromatograph
Separating the column: agilent XDB-C184.6X 250mm, 5 μm
Mobile phase: a is 0.1% by volume aqueous TFA, B is 0.1% by volume TFA-50% aqueous acetonitrile (TFA is trifluoroacetic acid)
The flow rate was 1.0mL/min, the detection wavelength was 214nm, the detection was performed at room temperature, the elution gradient is shown in the table below, and the percentages are volume percentages.
| Step of elution | Elution time | Eluent |
| 1 | 0～5min | 95％A+5％B |
| 2 | 5～25min | 95％A+5％B→50％A+50％B |
| 3 | 25～30min | 100％B |
| 4 | 30～35min | 95％A+5％B |
Example 2(75mm inner diameter L & L4003 preparation column packing)
Application of Load&The Lock dynamic axial compression and static locking technology is characterized in that a filler is styrene-divinylbenzene copolymer (reverse phase filler Agilent PLRP-S), the pore diameter is 10nm, the particle size is 10 mu m, the packing density is 0.33g/mL, the pressure of a packed column bed is 650psi, a Varian chromatography packing system, 370g of dry powder filler and 2L of methanol are adopted for stirring and homogenizing, and then the mixture is poured into a L with the inner diameter of 75mm&L4003 column preparation, compression ratio of 3:1, carrier gas of N2The carrier gas pressure was adjusted to 2000psi oil pressure and dynamic axial compression to 26cm bed height as a preparative column for reverse phase cyclization, reverse phase purification and reverse phase desalination protocols.
EXAMPLE 3 reverse phase cyclization, reverse phase purification and reverse phase desalting of crude vasopressin [5-Asp ] precursor starting Material
The instrument comprises the following steps: varian SD-1 high-pressure liquid phase preparation system
A chromatographic column: example 2 self-contained preparative column Load & Lock 400375X 260mm, PLRP-S10 μm 10nm
Vasopressin [5-Asp ]]The precursor crude product is vasopressin [5-Asp ] prepared by cracking and drying by adopting a solid-phase synthesis method]The precursor crude product has a structural formula of Cys-Tyr-Phe-Gln-Asp-Cys-Pro-Arg-Gly-NH2The trifluoroacetic acid salt of (1).
The solution of the crude vasopressin [5-Asp ] precursor is a 5g/L solution of the crude vasopressin [5-Asp ] precursor in a 5% strength by volume aqueous solution of acetonitrile.
The mobile phase A1 is purified water, A2 is 0.03 percent by volume of H2O2NaOH aqueous solution with pH of 7.5, mobile phase B of acetonitrile, and mobile phase C1 of vasopressin [5-Asp ]]Precursor crude solution (vasopressin [5-Asp ] prepared by solid phase synthesis method]Crude precursor) having an HPLC purity of 84.23% as determined by the method of example 1, a retention time of 12.6min and a mobile phase of 0.1M NaOH solution in C2.
The reverse phase cyclization, reverse phase purification and reverse phase desalting conditions of this example are as follows: the flow rate was 200mL/min, the detection was at 220nm, and the purification elution gradient is shown in the following table, with percentages being by volume.
| Step of elution | Elution time | Eluent |
| 1 | 0～10min | 100％C1 |
| 2 | 10.1～25min | 75％A1+5％B+20％C2 |
| 3 | 25.1～40min | 95％A1+5％B |
| 4 | 40.1～45min | 95％A2+5％B |
| 5 | 45.1～60min | 95％A1+5％B |
| 6 | 60～75min | 95％A1+5％B→80％A1+20％B |
| 7 | 75～90min | 80％A1+20％B→73％A1+27％B |
| 8 | 90～105min | 50％A1+50％B |
| 9 | 105～115min | 95％A1+5％B |
Collecting the eluent with the retention time of 75-90 min to obtain a vasopressin [5-Asp ] solution. Its HPLC purity was 99.61% as determined in example 1, with a retention time of 9.40 min.
EXAMPLE 4 reverse phase cyclization, reverse phase purification and reverse phase desalting of crude vasopressin [5-Asp ] precursor starting material
The instrument comprises the following steps: varian SD-1 high-pressure liquid phase preparation system
A chromatographic column: example 2 self-contained preparative column Load & Lock 400375X 260mm, PLRP-S10 μm 10nm
Vasopressin [5-Asp ]]The precursor crude product is vasopressin [5-Asp ] prepared by cracking and drying by adopting a solid-phase synthesis method]The precursor crude product has a structural formula of Cys-Tyr-Phe-Gln-Asp-Cys-Pro-Arg-Gly-NH2The trifluoroacetic acid salt of (1).
The solution of the crude vasopressin [5-Asp ] precursor is a 5g/L solution of the crude vasopressin [5-Asp ] precursor in a 5% strength by volume aqueous solution of acetonitrile.
The mobile phase A1 is purified water, A2 is 0.02 volume percent of H2O2NaOH aqueous solution with pH of 9.0, mobile phase B of acetonitrile, and mobile phase C1 of vasopressin [5-Asp ]]Precursor crude solution (vasopressin [5-Asp ] prepared by solid phase synthesis method]Crude precursor) having an HPLC purity of 84.23% as determined by the method of example 1, a retention time of 12.6min and a mobile phase of 0.1M NaOH solution in C2.
The reverse phase cyclization, reverse phase purification and reverse phase desalting conditions of this example are as follows: the flow rate was 200mL/min, the detection was at 220nm, and the purification elution gradient is shown in the following table, with percentages being by volume.
| Step of elution | Elution time | Eluent |
| 1 | 0～10min | 100％C1 |
| 2 | 10.1～25min | 75％A1+5％B+20％C2 |
| 3 | 25.1～40min | 95％A1+5％B |
| 4 | 40.1～45min | 95％A2+5％B |
| 5 | 45.1～60min | 95％A1+5％B |
| 6 | 60～75min | 95％A1+5％B→80％A1+20％B |
| 7 | 75～90min | 80％A1+20％B→73％A1+27％B |
| 8 | 90～105min | 50％A1+50％B |
| 9 | 105～115min | 95％A1+5％B |
Collecting the eluent with the retention time of 75-90 min to obtain a vasopressin [5-Asp ] solution. Its HPLC purity was 99.52% as determined in example 1, with a retention time of 9.41 min.
Example 5 Mass spectrometric detection of vasopressin [5-Asp ]
Single-weight quadrupole electronic injection adopting waters micromass ZQDetermination of vasopressin [5-Asp ] obtained in examples 3 and 4 by mist Mass Spectrometry (ESI-MS)]Molecular mass peak of (1) [ M +1 ]]+Found 1086.39, main ion fragment Peak [ M +2 ]]2+The measured values 543.71 all met the theoretical value 1085.24.
Claims (8)
1. A process for the preparation of vasopressin [5-Asp ], which comprises the steps of: performing reverse phase cyclization, reverse phase purification and reverse phase desalting on the crude solution of the vasopressin [5-Asp ] precursor by adopting a high performance liquid reverse phase chromatography in turn; the filler of the high performance liquid phase reverse phase chromatography is styrene-divinylbenzene copolymer;
the crude vasopressin [5-Asp ] precursor is a crude vasopressin [5-Asp ] precursor containing two free sulfydryl groups;
the described vasopressin [5-Asp ]]Is composed of
The conditions of reverse phase cyclization, reverse phase purification and reverse phase desalting are as follows: the mobile phase A1 is pure water, A2 is H with the volume percentage of 0.01-0.05%2O2The pH of the NaOH aqueous solution is 7.5-9.0, the mobile phase B is acetonitrile, the mobile phase C1 is the vasopressin [5-Asp ]]The precursor crude product solution is 0.1mol/L NaOH solution as a mobile phase C2, the flow rate is 180-220 mL/min, and the detection wavelength is 220 nm;
carrying out on-line sample loading and elution according to the conditions of the following table, wherein the percentage is volume percentage;
Collecting the eluent with the retention time of 75-90 min to obtain the vasopressin [5-Asp ] solution.
2. The method according to claim 1, wherein the crude vasopressin [5-Asp ] precursor is prepared by a solid-phase synthesis method through cracking and drying, and the HPLC purity is 60-90%.
3. The method of claim 1, wherein said vasopressin [5-Asp ]]The structural formula of the precursor crude product is Cys-Tyr-Phe-Gln-Asp-Cys-Pro-Arg-Gly-NH2The trifluoroacetic acid salt of (1).
4. The method of claim 1, wherein the solution of the crude vasopressin [5-Asp ] precursor is a 5g/L solution of the crude vasopressin [5-Asp ] precursor in a 5% strength by volume aqueous acetonitrile.
5. The method of claim 1, wherein the reverse phase cyclization, the reverse phase purification and the reverse phase desalting are all completed in a one-step reverse phase elution process.
6. The method according to claim 1, wherein A2 is H0.02-0.03 vol%2O2The pH of the NaOH aqueous solution is 7.5-9.0.
7. The method of claim 1, wherein the flow rate is 200 mL/min.
8. The method of claim 1, wherein the high performance liquid reverse phase chromatography is carried out under the following packing conditions: the filler is Agilent PLRP-S styrene-divinylbenzene copolymer, the aperture is 10nm, and the particle size is 10 mu m.
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| CN109929011B (en) * | 2019-05-06 | 2021-04-06 | 上海上药第一生化药业有限公司 | Method for refining vasopressin [5-Asp ] impurity |
| CN109942686B (en) * | 2019-05-06 | 2021-06-25 | 上海上药第一生化药业有限公司 | Purification method of vasopressin acetylated impurities |
| CN110003313B (en) * | 2019-05-06 | 2021-03-02 | 上海上药第一生化药业有限公司 | Vasopressin [ -NH ]2]Method for purifying impurities |
| CN109929010B (en) * | 2019-05-06 | 2021-05-14 | 上海上药第一生化药业有限公司 | Method for refining vasopressin |
Citations (4)
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|---|---|---|---|---|
| CN103374054A (en) * | 2012-04-28 | 2013-10-30 | 上海第一生化药业有限公司 | One-step method based solid-phase polypeptide synthesis method |
| CN103980351A (en) * | 2014-05-27 | 2014-08-13 | 上海第一生化药业有限公司 | Method for preparing vasopressin and vasopressin tannate |
| CN104530198A (en) * | 2014-12-09 | 2015-04-22 | 兰州大学 | Method for preparing desmopressin acetate through fragment condensation |
| CN104672308A (en) * | 2014-12-23 | 2015-06-03 | 青岛康原药业有限公司 | Method for preparing vasopressin tannate |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103374054A (en) * | 2012-04-28 | 2013-10-30 | 上海第一生化药业有限公司 | One-step method based solid-phase polypeptide synthesis method |
| CN103980351A (en) * | 2014-05-27 | 2014-08-13 | 上海第一生化药业有限公司 | Method for preparing vasopressin and vasopressin tannate |
| CN104530198A (en) * | 2014-12-09 | 2015-04-22 | 兰州大学 | Method for preparing desmopressin acetate through fragment condensation |
| CN104672308A (en) * | 2014-12-23 | 2015-06-03 | 青岛康原药业有限公司 | Method for preparing vasopressin tannate |
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