CN110845599A - Preparation and purification method of polypeptide - Google Patents
Preparation and purification method of polypeptide Download PDFInfo
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- CN110845599A CN110845599A CN201810959066.1A CN201810959066A CN110845599A CN 110845599 A CN110845599 A CN 110845599A CN 201810959066 A CN201810959066 A CN 201810959066A CN 110845599 A CN110845599 A CN 110845599A
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Abstract
The invention belongs to the technical field of medicines, and particularly discloses a method for preparing and purifying polypeptide. The method for inhibiting methionine oxidation is used in the process of preparing and purifying the ularitide, so that the purity of the purified ularitide refined product is up to 99.90 percent, and the methionine oxidation impurity peptide is controlled within 0.1 percent.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a preparation and purification method of polypeptide.
Background
The structure of the ularitide is a polypeptide which is composed of 32 amino acids and contains a pair of intramolecular disulfide bonds, the polypeptide is generally prepared by chemical synthesis, and the sequence structure is as follows:
H-Thr-Ala-Pro--Arg-Arg-Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr-OH(S-S)。
urapide (Ulatride) is a natriuretic peptide isolated from human urine, and originally a renal natriuretic peptide belonging to the Atrial Natriuretic Peptide (ANP) family was isolated from urine by SchulzKnappe et al in 1988. Endogenous ularitide is synthesized in renal distal tubular cells, secreted behind the lumen, bound to a downstream natriuretic peptide A type receptor in an internal marrow collecting duct, and can regulate the excretion of renal sodium and water, so that the ularitide has the effects of vasodilation and natriuretic effect, and the ularitide is proved to reduce the reabsorption of urine by the kidney. Clinical treatment of Decompensated Heart Failure (DHF) aims at relieving symptoms and stabilizing hemodynamics of patients, and currently used therapeutic drugs include diuretics, vasodilators, and inotropic drugs, which all have clinical limitations. The second phase clinical research shows that the ulipriside can lower cardiac filling pressure and improve dyspnea, has no obvious adverse effect on the kidney function of DHF patients, and has wide prospect in treating DHF.
At present, two synthesis methods for the ularitide are mainly used, one is to complete the coupling of linear peptide resin in a solid phase and then adopt iodine solid phase oxidation to form a corresponding disulfide bond; another method is to synthesize linear peptide precursors in solid phase and then oxidize in liquid phase in very low concentration organic solvent/water solution to form the corresponding disulfide bonds.
The former synthesis method adopts Fmoc solid phase peptide synthesis technology, uses Wang resin as starting material, sequentially couples each amino acid residue to obtain linear peptide resin, then adds solid iodine simple substance, oxidizes to form disulfide bond to obtain oxidized cyclized peptide resin, and cracks to obtain crude product. Although the synthetic steps are simple, in the process of coupling peptide resin, partial residues are difficult to couple, and the purity of the obtained crude peptide is low. Meanwhile, iodine is oxidized on the peptide resin, and the iodine is difficult to completely elute in the whole process, so that the final pure product, namely the raw material medicine contains trace iodine, and the raw material medicine is yellow. In addition, the peptide is oxidized by a strong oxidant, so that the secondary structure of the peptide sequence is easily damaged, and the bioactivity and the drug effect of the peptide are influenced finally. The latter method is therefore generally used for the synthesis of ularitide.
The Fmoc solid phase peptide synthesis technology is also adopted in the synthesis method of the latter, Wang resin is taken as a starting material, and amino acid residues are sequentially coupled to obtain linear peptide resin, and the linear peptide resin is cracked to obtain a crude linear peptide product. The crude peptide was purified by HPLC to give the linear peptide fine peptide. Dissolving the linear peptide fine peptide by using methanol or acetonitrile, adding water to dilute, and oxidizing for 72 hours under the air oxidation condition to obtain an oxidized crude cyclic peptide product. And purifying the crude cyclopeptide product by HPLC to obtain refined peptide. This method has the same disadvantages as the former synthetic scheme, i.e., difficult coupling of partial residues and low purity of crude linear peptides. Compared with the former, the space secondary structure of the final product can be completely reserved, and the bioactivity of the final refined peptide is better than that of the former. However, when the scheme is adopted for oxidation, the operation is very difficult and the reaction time is long due to the difficulty in dissolving the ularitide and the difficulty in carrying out final oxidation at a very dilute concentration, so that the large-scale production is not facilitated.
The ularitide is a white or off-white powder, is easily soluble in water, is insoluble in common single organic solvents such as methanol, ethanol, acetonitrile, isopropanol, acetone and tetrahydrofuran, but can be dissolved by using an organic solvent/water mixed solution. Acetonitrile/water has higher solubility to samples than ethanol/water and methanol/water, has higher clarity, and is often used as a mobile phase for purifying the ularitide sample. Domestic and foreign patents mainly focus on polypeptide synthesis, product yield and purity, production cost, production period and the like, and the disclosed purification method is less.
Patent CN103145827 discloses a solid phase synthesis method of ularitide without further purification. The Mezueli et al (modern medicine and clinic, 2013(28)) use a reversed-phase high-performance liquid phase further purification method to obtain the ularitide with the purity of 98%. Patent CN106519009 mentions the use of purification and salt conversion to obtain ularitide with a purity of 98% or more, and does not describe the process. The patent CN106554406 and the patent CN106554407 use ionic liquid carrier synthesis and oxidation processes to improve the purity of crude peptide, and prepare the uracride with the purity of more than 99% through one-step reversed phase purification, however, the ionic liquid carrier is high in cost and difficult to realize industrialization. Through analysis, the impurities in the oxidation process are not further controlled in the prior art, and the conventional detection method of the polypeptide cannot effectively detect the impurities in the oxidation process, so that the HPLC purity in the prior art has no guiding significance.
Disulfide bond formation in peptide chains plays an important role in maintaining spatial conformation of polypeptides and biological activity determined thereby, and many methods for disulfide bond formation are used, and oxidation with an oxidizing agent, air oxidation, dimethyl sulfoxide (DMSO), N-iodosuccinimide (NIS), iodine hydride (ICN), and the like are used in most cases to obtain thermodynamically controlled products. Using stronger oxidizing agentsE.g. iodine, potassium ferricyanide (K)3Fe(CN)6) And thallium trifluoroacetate ((CF)3COO)3Tl), etc., to give a kinetically controlled product. Potassium ferricyanide (K)3Fe(CN)6) And thallium trifluoroacetate ((CF)3COO)3Tl) and the like, which are complicated in reaction and difficult to remove by purification, are not recommended.
The oxidation step in the preparation process of the ularitide is to oxidize sulfydryl in two cysteines in the ularitide sequence into ring through a disulfide bond. However, the ularitide also contains methionine, and the methylthio group in the methionine is oxidized to generate impurities in the oxidation process, so that the structure and the function of the ularitide are changed. Through sample tracking and monitoring, the ularitide methionine oxide runs through the whole purification process, becomes a main impurity component at the later stage of ularitide purification, and can reach 2.0-5.0% after sample freeze-drying. The oxidation reaction of methionine is represented as follows:
methionine is oxidized by active oxygen to methionine sulfoxide and can be further irreversibly oxidized to methionine sulfone. In the purification process of the polypeptide, the higher the purity of the ularitide, the more easily methionine is oxidized. However, this impurity cannot be detected effectively by conventional polypeptide detection methods, and thus methionine oxidation impurities have not been studied in a targeted manner. At present, polypeptide medicine preparation literatures and patents have no detailed research on polypeptide oxides, and the polypeptide amino acid oxides are not listed as impurities in quality standards.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a polypeptide preparation and purification method, in particular to a polypeptide preparation and purification method with controllable methionine oxidation impurities.
The invention provides a preparation and purification method of high-purity polypeptide, which is used for solving the problems in the prior art. The method is strong in pertinence, simple in process and low in cost, can effectively improve the oxidation efficiency of the linear peptide, and can detect and control methionine oxide of the ularitide to be low, and the purity of the ularitide can reach more than 99.90%.
The invention aims to realize the following steps:
a method for preparing and purifying polypeptide, comprising the following steps:
(1) dissolving and oxidizing a crude polypeptide linear peptide;
(2) concentrating the oxidizing solution;
(3) purifying by high performance liquid chromatography;
(4) transferring salt;
(5) concentrating and freeze-drying the eluent.
The polypeptide is one of ularitide, ziconotide, nesiritide, brain natriuretic peptide, capecitabine and exenatide.
The technical scheme of the invention is explained in detail by taking the ularitide as an example, and specifically comprises the following steps:
(1) dissolving the crude product of the ularitide linear peptide in a stirred buffer salt-water solution, adjusting the pH value, and carrying out air oxidation to obtain an ularitide oxidation solution;
(2) carrying out desalination concentration by using ion exchange resin or nanofiltration according to the buffer salt selected in the step (1) to obtain a concentrated solution;
(3) loading the concentrated solution to a high performance liquid preparative chromatographic column, taking organic solvents and buffer solutions with different volume ratios as A, B two-phase mobile phase gradient elution, and collecting eluent;
(4) and loading the eluent into a high performance liquid chromatography column again, and performing gradient elution by using organic solvents and dilute acid solutions with different volume ratios as A, C two-phase mobile phases to obtain the urotropine eluent.
(5) Concentrating the above Ursolin eluate under reduced pressure to remove organic solvent, and lyophilizing to obtain Ursolin refined product.
Preferably, in the step (1), the buffer salt is sodium salt, potassium salt or ammonium salt, and can be inorganic salt or organic salt thereof; the inorganic salt can be hydrochloride, carbonate, phosphate or sulfate, and the organic salt can be formate, acetate, maleate, oxalate, citrate or lactate. More preferably, the buffer salt is monopotassium phosphate, ammonium dihydrogen phosphate, sodium bicarbonate, ammonium sulfate, ammonium acetate, ammonium chloride, sodium formate, or the like.
Preferably, the concentration of the buffer salt in the step (1) is 0.1-5.0 mol/L; more preferably, the concentration of the buffer salt is 1.0-1.5 mol/L.
In one embodiment, the pH value in the step (1) is 6.0-8.0; in another embodiment, the pH value in step (1) is 7.0-8.0, and when the pH value is 7.0-8.0, the oxidation time is obviously shortened compared with the pH value of 6.0-7.0; in another embodiment, the pH of step (1) is greater than 8.0 and the oxidation process methionine oxidation impurities are significantly increased.
Preferably, in the oxidation process in the step (1), the reaction temperature is 20-30 ℃, the air oxidation time is 5-10 hours, and in the oxidation process, sampling detection is carried out, and the oxidation can be stopped when the oxidation solution does not contain cysteine residues.
Preferably, in the step (2), the desalting and concentrating process can adopt ion exchange resin for desalting and concentrating, and when the buffer salt is monovalent salt, nanofiltration concentration can be selected, and the oxidation solution is concentrated to 1/10-1/8 of the original volume; the ion exchange resin is preferably a cation exchange resin.
Preferably, in step (3), the packing material for HPLC is reversed phase chromatography packing material, optionally C18 bonded packing material, and the pore diameter of the packing material isThe C18 column packing material can be Agela-Venusil-C18--10μm,Kromasil-C18--10μm,Sepax-GP-C18--5μm,Daiso-SP-ODS--5μm,Nano-Unsil-C18--10μm,YMC-ODS--10μm,FUJI-SPS-C18--9μm,Welch-XB-C18--10μm,Daiso-SP-ODS-Any of-10 μm, preferably Sepax-GP-C18--5μm,Welch-XB-C18-10 μm or Daiso-SP-ODS--10μm。
Preferably, the mobile phase in the step (3) is a mixed solution of A, B two solutions; the phase A is an organic solvent, and the phase B is a buffer salt solution; the organic solvent is selected from one of methanol, ethanol and acetonitrile; the buffer salt is potassium salt, sodium salt or ammonium salt, and can be inorganic salt or organic salt. Preferably, one selected from the group consisting of phosphate, sulfate, formate and acetate; more preferably, the buffer salt is sodium dihydrogen phosphate, potassium dihydrogen phosphate, ammonium sulfate, ammonium acetate, sodium formate, etc.; the concentration of the buffer salt is 10-100 mmol/L; the pH value of the buffer solution is 2.0-7.5.
Preferably, the mobile phase, A, B two-phase gradient elution procedure described in step (3) is shown in table 1, and the eluate is collected according to the result of the ultraviolet on-line detection.
TABLE 1 elution gradient during purification
More preferably, the gradient elution schedule of step (3) is as follows:
TABLE 2 elution gradient during purification
And (4) continuously loading the eluent obtained in the step (3) into a preparative chromatographic column for salt conversion, gradient elution and ultraviolet detection, and collecting the glatiramer eluent.
The mobile phase used in the salt conversion step is A, C two-phase mixed solution, wherein phase A is an organic solvent and can be selected from one of methanol, ethanol or acetonitrile, phase C is a dilute acid solution, the acid is one of formic acid, acetic acid, trifluoroacetic acid or hydrochloric acid, preferably, when the acid is formic acid or acetic acid, the volume fraction of formic acid or acetic acid is 0.1-1.0%, when the acid is trifluoroacetic acid, the volume fraction of trifluoroacetic acid is 0.05-0.1%, and when the acid is hydrochloric acid, the volume fraction of hydrochloric acid is 0.01-0.05%. A. The gradient elution procedure for the C two phases is shown in Table 3.
TABLE 3 elution gradient during salt conversion
More preferably, the elution gradient during the salt transfer is shown in table 4 below.
TABLE 4 elution gradient during salt conversion
In the step (5), the freeze-drying condition is that the vacuum degree is less than or equal to 10mbar, the temperature of the clapboard is controlled to be 20 ℃, and the freeze-drying time is 12-36 hours.
After the oxidation step is finished, polypeptide oxidation needs to be inhibited in the subsequent steps of purification, salt conversion, freeze-drying and the like in steps (3) to (5), and preferably, the method comprises one or more of pH adjustment, gas replacement, replacement of purified water by using oxygen-free water, addition of an amino acid protective agent and addition of an antioxidant.
Further preferably, polypeptide oxidation is inhibited by adjusting pH, the pH is 3.0-5.0, and the used acid is one of formic acid, acetic acid, trifluoroacetic acid or hydrochloric acid.
Further preferably, the polypeptide oxidation is suppressed by gas replacement, which is air exchange of the eluent collection device, the reduced-pressure concentration system and the lyophilization system, and the replacement gas used is high-purity nitrogen or carbon dioxide.
Further preferably, the polypeptide oxidation is inhibited by using non-oxygenated water instead of purified water, wherein the non-oxygenated water is relatively oxygen-free and is prepared by heating, refluxing and cooling the purified water, and then blowing off oxygen by using high-purity nitrogen to obtain the non-oxygenated water; can be used as the water for the mobile phase in the step (3) or (4).
Further preferably, polypeptide oxidation is inhibited by adding an amino acid protecting agent which is one of methionine, proline, histidine, glycine and lysine; preferably methionine or lysine. The addition concentration of the amino acid protective agent is 2.0-10.0 mmol/L.
Further preferably, the oxidation of the polypeptide is inhibited by adding an antioxidant, wherein the antioxidant is selected from one of ascorbic acid, citric acid, vitamin E and EDTA, preferably citric acid or EDTA, and the adding concentration of the antioxidant is 0.1-0.3 mmol/L.
In the invention, after the oxidation of the ularitide, the steps of purification, salt transfer, freeze-drying and the like need to select at least one or more methods to carry out oxidation inhibition on the ularitide.
According to verification, the method for inhibiting oxidation of the urotropine is also suitable for other polypeptides containing methionine which is easy to be oxidized, and in one embodiment, the method for preparing and purifying the carpropine can effectively control oxidation impurities of the methionine of the carpropine, so that the HPLC purity of the carpropine is improved to 99.85% from 98.71%. In another embodiment, the purified exenatide prepared by the method of the present invention can effectively control the oxidation impurities of exenatide methionine, so that the HPLC purity of the exenatide is increased from 98.12% to 99.56%.
Through verification, the method for inhibiting the oxidation of the ularitide is also suitable for other polypeptides such as ziconotide, nesiritide, brain natriuretic peptide and the like, achieves the same technical effect, and can effectively control oxidation impurities of the peptides.
Compared with the prior art, the invention achieves the following technical effects:
(1) according to the method, the crude product of the ularitide linear peptide is dissolved by adopting a buffer salt-water solution, the concentration of the crude product is increased to 1.0mg/ml from 0.2mg/ml of water-soluble solution, the pH value is adjusted to 6.0-8.0, the air oxidation reaction time is reduced to 5-10 h from 24 h-48 h, and the oxidation time is greatly shortened.
(2) The invention can effectively separate and remove the polypeptide of the structural analogue by purifying the oxidized urotropine and a two-step salt conversion purification method, and the purity is higher than 99.93 percent.
(3) Methionine oxide impurities of the ularitide are detected and controlled for the first time, a process for inhibiting polypeptide oxidation impurities is adopted, and after freeze-drying, the purity of the refined ularitide reaches 99.90 percent, and the maximum single-hetero polypeptide methionine oxidation impurities is 0.10 percent.
Drawings
FIG. 1 is an HPLC chromatogram of an oxidized solution of crude linear peptide prior to preparation and purification in example 1;
FIG. 2 is an HPLC chromatogram of a lyophilized sample of ularitide after salt conversion in example 1;
FIG. 3 is an HPLC chromatogram of a lyophilized sample of ularitide after salt conversion in example 2;
FIG. 4 is an HPLC chromatogram of a lyophilized sample of ularitide after salt conversion in example 3;
FIG. 5 is an HPLC chromatogram of an oxidized solution of crude linear peptide prior to preparation and purification in comparative example 1;
fig. 6 is an HPLC profile of a lyophilized sample of ularitide obtained in comparative example 2.
Detailed Description
The present invention is further described in detail with reference to the following specific examples, which are provided for illustrative purposes only and are not intended to limit the present invention, and will be apparent to those of ordinary skill in the art to which the present invention pertains.
The crude products of the ularitide linear peptide, the carperitide linear peptide and the exenatide used in the embodiment of the invention are all provided by Shandong New era pharmaceutical industry Co., Ltd, and the crude products in other prior art are also suitable for the method of the invention; other starting materials are commercially available without specific recitation.
The high performance liquid preparative chromatographic column used in the invention is a dynamic axial compression preparative column (DAC50 column) with the size of 50mm multiplied by 450mm, and the prepared chromatographic column packing is Sepax-GP-C18--5μm,Welch-XB-C18-10 μm or Daiso-SP-ODS-10 μm, while maintaining the column temperature with an external water circulation at 30 ℃.
The purification process and sample impurity analysis of the ularitide are detected by HPLC, and the chromatographic conditions are as follows:
a chromatographic column: Welch-Ultimate-LP-C18-5 μm, 4.6X 250 mm;
mobile phase: phase A is 20mmol/L (NH)4)2SO4ACN 9:1, B phase 20mmol/L (NH)4)2SO4ACN 1:1, wherein 20mmol/L (NH)4)2SO4Adjusting pH2.0 with phosphoric acid;
detection wavelength: 210 nm; flow rate: 1.0 ml/min; column temperature: 30 ℃;
the gradient elution procedure runs the table 5 gradient table.
TABLE 5 elution gradient chart for Ursolitin detection
Example 1 preparation and purification of Uralitide
(1) Dissolution and oxidation of crude ularitide linear peptide
Taking 10.0g of crude linear peptide, stirring and dissolving by using 10.0L of ammonium acetate water solution with the pH value of 8.0 and the concentration of 1mol/L, stirring and air oxidizing at the temperature of 25 ℃, detecting that the content of cysteine residue is 0 when oxidizing for 6 hours, and stopping oxidizing; the oxidized liquid was checked by HPLC, and the HPLC purity of ularitide was 51.81%, and the oxidized impurity of methionine (relative retention time ═ 0.61) was 2.24% (fig. 1).
(2) Concentration of the oxidized liquid
And (2) carrying out nanofiltration concentration on 10.0L of the oxidized liquid of the ularitide obtained in the step (1), stopping concentration when the oxidized liquid is concentrated to the volume of 1.0-1.2L, and adjusting the pH value of the concentrated liquid to 6.0 by using acetic acid for later use.
(3) Purification by high performance liquid chromatography
Using DAC50 column, circulating water outside the column, keeping temperature at 30 ℃, and filling Sepax-GP-C18--5 μm. A, B mobile phase is prepared, wherein A is acetonitrile, B is 50mmol/L potassium dihydrogen phosphate anaerobic aqueous solution with pH 6.0.
And (3) loading the concentrated solution with the pH value of 6.0 obtained in the step (2), operating an AB two-phase gradient elution program in the table 1, setting a detection wavelength, and collecting and preparing an eluent. HPLC detection of the eluent shows that the purity of the Ursolitin HPLC is 98.48 percent, and the methionine oxidized impurity is 0.65 percent. The eluate is adjusted to pH3.0 with acetic acid and is ready for use.
(4) Salt conversion
Using DAC50 column, circulating water outside the column, keeping temperature at 30 ℃, and filling Sepax-GP-C18--5 μm. A, C mobile phase is prepared, wherein A is acetonitrile, C is oxygen-free aqueous solution with volume fraction of 1.0% acetic acid.
And (3) loading the Ursolitin eluent with the pH value of 3.0 obtained in the step (3), operating an AC two-phase gradient elution program in the table 2, performing high-purity nitrogen replacement on a receiving bottle, and collecting the eluent. HPLC detection shows that the HPLC purity of the ularitide is 99.93 percent, and the methionine oxidized impurity is 0.07 percent.
(5) Concentration and lyophilization of eluents
Under the condition of high-purity nitrogen replacement, the eluent collected after salt conversion is decompressed and concentrated to remove acetonitrile, and 3.26g of refined product of the ularitide is obtained after the vacuum degree is less than or equal to 10mbar, the temperature of a clapboard is controlled at 10 ℃, and the drying time is 12h, the HPLC purity is 99.90 percent, and the methionine oxidation impurity is 0.10 percent (figure 2).
Example 2 preparation and purification of Uralitide
(1) Dissolution and oxidation of crude ularitide linear peptide
Taking 10.0g of crude linear peptide, stirring and dissolving by using 10.0L of potassium dihydrogen phosphate aqueous solution with pH of 6.0 and 1.5mol/L, stirring and air oxidizing at 20 ℃, detecting that the content of cysteine residue is 0 when oxidizing for 8h, and stopping oxidizing; and (3) detecting the oxidation liquid by HPLC, wherein the HPLC purity of the ularitide is 52.12%, and the methionine oxidation impurity is 2.15%.
(2) Concentration of the oxidized liquid
And (2) loading 10.0L of the oxidized solution of the ularitide obtained in the step (1) to a D152 cation exchange resin column, and desalting and eluting with 0.1N hydrochloric acid aqueous solution, wherein the elution volume is 1.0-1.2L for later use.
(3) Purification by high performance liquid chromatography
DAC50 column is used, the temperature of the column outside is kept at 30 ℃ by water circulation, and the packing of the chromatographic column is Welch-XB-C18--10 μm. A, B mobile phase is prepared, wherein A is methanol, B is 10mmol/L ammonium sulfate aqueous solution with pH2.0.
And (3) loading the concentrated solution with the pH of 2.0 obtained in the step (2), operating an AB two-phase gradient elution program in the table 1, setting a detection wavelength, and collecting and preparing an eluent. HPLC detection of the eluent shows that the HPLC purity of the ularitide is 98.66%, and the oxidation impurity of the methionine is 0.50%. EDTA is added into the eluent to contain EDTA0.2mmol/L for standby.
(4) Salt conversion
DAC50 column is used, the temperature of the column outside is kept at 30 ℃ by water circulation, and the packing of the chromatographic column is Welch-XB-C18--10 μm. A, C mobile phase is prepared, wherein A is methanol and C is 0.1% formic acid in oxygen-free water solution.
And (3) loading the Urapide eluent with the pH value of 2.0 and the EDTA0.2mmol/L obtained in the step (3), operating the AC two-phase gradient elution program in the table 2, and collecting the eluent. EDTA was added to the eluate to give an EDTA0.1mmol/L solution. HPLC detection shows that the HPLC purity of the ularitide is 99.94 percent, and the methionine oxidation impurity is 0.06 percent.
(5) Concentration and lyophilization of eluents
Under the condition of high-purity carbon dioxide replacement, the eluent collected after salt conversion is decompressed and concentrated to remove methanol, and 2.78g of refined product of the ularitide is obtained after the vacuum degree is less than or equal to 10mbar, the temperature of a clapboard is controlled at 10 ℃, and the drying time is 12h, the HPLC purity is 99.91 percent, and the methionine oxidation impurity is 0.09 percent (figure 3).
Example 3 preparation and purification of Uralitide
(1) Dissolution and oxidation of crude ularitide linear peptide
Taking 10.0g of crude linear peptide, stirring and dissolving by using 10.0L of ammonium chloride aqueous solution with pH of 6.5 and 5.0mol/L, stirring and air oxidizing at 30 ℃, detecting that the content of cysteine residue is 0 when oxidizing for 5 hours, and stopping oxidizing; HPLC detection shows that the purity of the oxidized liquid is 51.78 percent and the methionine oxidized impurity is 2.18 percent.
(2) Concentration of the oxidized liquid
And (2) carrying out nanofiltration concentration on 10.0L of the oxidized liquid of the ularitide obtained in the step (1), stopping concentration when the oxidized liquid of the ularitide is concentrated to the volume of 1.0-1.2L, and adjusting the pH value of an elution concentrated liquid to 6.0 for later use by using dilute hydrochloric acid.
(3) Purification by high performance liquid chromatography
Using DAC50 column, circulating water outside the column, keeping temperature at 30 ℃, and filling Daiso-SP-ODS--10 μm. A, B mobile phase is prepared, wherein A is ethanol, and B is 100mmol/L ammonium acetate oxygen-free aqueous solution with pH 6.0.
And (3) loading the concentrated solution with the pH value of 6.0 obtained in the step (2), operating an AB two-phase gradient elution program in the table 1, setting a detection wavelength, and collecting and preparing an eluent. HPLC detection of the eluent shows that the purity of the urapidine HPLC is 98.07 percent, and the methionine oxidized impurity is 0.63 percent. The eluate was added to lysine at a final concentration of 10mmol/L and EDTA at a final concentration of 0.1mmol/L for further use.
(4) Salt conversion
Using DAC50 column, circulating water outside the column, keeping temperature at 30 ℃, and filling Daiso-SP-ODS--10 μm. A, C preparing mobile phase, wherein A is ethanol, and C is 0.01% by volume of hydrochloric acid oxygen-free aqueous solution.
And (3) loading the Urapide eluent containing 10mmol/L lysine obtained in the step (3), operating the AC two-phase gradient elution program in the table 2, collecting the eluent, and adding the lysine with the final concentration of 10mmol/L and the EDTA with the final concentration of 0.1 mmol/L. HPLC detection shows that the HPLC purity of the ularitide is 99.95 percent, and the methionine oxidation impurity is 0.05 percent.
(5) Concentration and lyophilization of eluents
Under the condition of high-purity nitrogen replacement, the eluent collected after salt conversion is decompressed and concentrated to remove ethanol, and 3.05g of refined product of the ularitide is obtained after the vacuum degree is less than or equal to 10mbar, the temperature of a partition plate is controlled at 10 ℃, and the drying time is 12h, the HPLC purity is 99.92%, and the methionine oxidation impurity is 0.08% (figure 4).
Example 4 preparation and purification of Uralitide
(1) Dissolution and oxidation of crude ularitide linear peptide
Taking 10.0g of crude linear peptide, stirring and dissolving by using 10.0L of sodium formate aqueous solution with pH value of 7.5 and 1.5mol/L, stirring and air oxidizing at 30 ℃, detecting that the content of cysteine residue is 0 when oxidizing for 6 hours, and stopping oxidizing; and (3) detecting the oxidation liquid by HPLC, wherein the HPLC purity of the ularitide is 51.86%, and the methionine oxidized impurity is 2.26%.
(2) Concentration of the oxidized liquid
And (2) carrying out nanofiltration concentration on 10.0L of the oxidized liquid of the ularitide obtained in the step (1), stopping concentration when the oxidized liquid is concentrated to the volume of 1.0-1.2L, and adjusting the pH value of an elution concentrated liquid to 4.5 by using dilute hydrochloric acid for later use.
(3) Purification by high performance liquid chromatography
Using DAC50 column, circulating water outside the column, keeping temperature at 30 ℃, and using Agela-Venusil-C18-ion as chromatographic column packing-10 μm. A, B mobile phase is prepared, wherein A is acetonitrile, B is 20mmol/L ammonium dihydrogen phosphate water solution with pH 4.5.
And (3) loading the concentrated solution with the pH value of 4.5 obtained in the step (2), operating an AB two-phase gradient elution program in the table 1, setting a detection wavelength, and collecting and preparing an eluent. HPLC detection of the eluent shows that the HPLC purity of the ularitide is 98.74 percent, and the methionine oxidized impurity is 0.43 percent. The eluate was adjusted to pH3.0 with trifluoroacetic acid and methionine was added to the eluate at 5 mmol/L.
(4) Salt conversion
Using DAC50 column, circulating water outside the column, keeping temperature at 30 ℃, and using Agela-Venusil-C18-ion as chromatographic column packing-10 μm. A, C mobile phase is prepared, wherein A is acetonitrile, C is oxygen-free aqueous solution with 0.05% trifluoroacetic acid by volume fraction.
And (3) loading the Urapide eluent with the pH value of 3.0 and the methionine content of 5mmol/L obtained in the step (3), operating the AC two-phase gradient elution program in the table 2, and collecting the eluent. HPLC detection shows that the HPLC purity of the ularitide is 99.94 percent, and the methionine oxidation impurity is 0.06 percent.
(5) Concentration and lyophilization of eluents
Under the condition of high-purity carbon dioxide replacement, concentrating the eluent collected after salt conversion under reduced pressure to remove acetonitrile, and drying for 12h under the conditions that the vacuum degree is less than or equal to 10mbar, the temperature of a partition plate is controlled at 10 ℃, so as to obtain 2.86g of the refined product of the ularitide, the HPLC purity is 99.90%, and the methionine oxidation impurity is 0.10%.
Example 5 preparation and purification of Uralitide
(1) Dissolution and oxidation of crude ularitide linear peptide
Taking 10.0g of crude linear peptide, stirring and dissolving with 10.0L of 1mol/L sodium bicarbonate water solution with pH of 8.0, stirring and air oxidizing at 30 ℃, detecting that the content of cysteine residue is 0 when oxidizing for 7h, and stopping oxidation; HPLC detection shows that the HPLC purity of the oxidized liquid is 52.03 percent, and the methionine oxidized impurity is 2.11 percent.
(2) Concentration of the oxidized liquid
And (2) loading 10.0L of the oxidized solution of the ularitide obtained in the step (1) to a D152 cation exchange resin column, and desalting and eluting with 0.1N hydrochloric acid aqueous solution, wherein the elution volume is 1.0-1.2L for later use.
(3) Purification by high performance liquid chromatography
DAC50 column is used, the temperature of the column outside is kept at 30 ℃ by water circulation, and the packing of the chromatographic column is Welch-XB-C18--10 μm. A, B mobile phase is prepared, wherein A is acetonitrile, B is 20mmol/L ammonium acetate anaerobic aqueous solution with pH 6.0.
And (3) loading the concentrated solution with the pH value of 6.0 obtained in the step (2), operating an AB two-phase gradient elution program in the table 1, setting a detection wavelength, and collecting and preparing an eluent. HPLC detection of the eluent shows that the purity of the Ursolitin HPLC is 98.22 percent, and the methionine oxidized impurity is 0.39 percent. Adding histidine and citric acid into the eluate until the content of histidine in the eluate is 2mmol/L and the content of citric acid in the eluate is 0.3mmol/L for use.
(4) Salt conversion
DAC50 column is used, the temperature of the column outside is kept at 30 ℃ by water circulation, and the packing of the chromatographic column is Welch-XB-C18--10 μm. A, C mobile phase is prepared, wherein A is acetonitrile, C is 0.5% acetic acid by volume fraction.
And (3) loading the Urariptin eluent containing histidine and citric acid obtained in the step (3), running an AC two-phase gradient elution program in the table 2, collecting the eluent, and adding histidine and citric acid into the eluent until the content of histidine in the eluent is 2mmol/L and the content of citric acid in the eluent is 0.3 mmol/L. HPLC detection shows that the HPLC purity of the ularitide is 99.94 percent, and the methionine oxidation impurity is 0.06 percent.
(5) Concentration and lyophilization of eluents
And (3) under the condition of high-purity nitrogen replacement, concentrating the eluent collected after salt conversion under reduced pressure to remove acetonitrile, and drying for 12 hours at the vacuum degree of less than or equal to 10mbar and the temperature of a partition plate controlled at 10 ℃ to obtain 3.05g of a refined product of the ularitide, wherein the HPLC purity is 99.91 percent and the methionine oxidation impurity is 0.09 percent.
Example 6 preparation and purification of Capecilitide
(1) Dissolution and oxidation of crude carperitide linear peptide
Taking 10.0g of crude linear peptide, stirring and dissolving by using 10.0L of ammonium chloride aqueous solution with pH of 6.5 and 1.5mol/L, stirring and air oxidizing at 30 ℃, detecting that the content of cysteine residue is 0 when oxidizing for 7 hours, and stopping oxidizing; the HPLC detection shows that the HPLC purity of the oxidized liquid is 49.78 percent and the methionine oxidized impurity is 2.73 percent.
(2) Concentration of the oxidized liquid
And (2) carrying out nanofiltration concentration on 10.0L of the capecitabine oxidized liquid obtained in the step (1), stopping concentration when the capecitabine oxidized liquid is concentrated to 1.0-1.2L, and adjusting the pH value of an elution concentrated liquid to 6.0 for later use by using dilute hydrochloric acid.
(3) Purification by high performance liquid chromatography
Using DAC50 column, circulating water outside the column, keeping temperature at 30 ℃, and filling Daiso-SP-ODS--10 μm. A, B mobile phase is prepared, wherein A is ethanol, and B is 100mmol/L ammonium acetate anaerobic aqueous solution with pH 6.0.
And (3) loading the concentrated solution with the pH value of 6.0 obtained in the step (2), operating an AB two-phase gradient elution program in the table 1 which is the same as the ularitide, setting the detection wavelength, and collecting and preparing the eluent. HPLC detection of the eluent shows that the HPLC purity of the carperitide is 98.07 percent, and the methionine oxidized impurity is 0.63 percent. The eluate was adjusted to pH5.0 with dilute hydrochloric acid for further use.
(4) Salt conversion
Using DAC50 column, circulating water outside the column, keeping temperature at 30 ℃, and filling Daiso-SP-ODS--10 μm. A, C mobile phase is prepared, wherein A is ethanol, and C is 0.01% hydrochloric acid in oxygen-free water solution.
And (3) loading the carperitide eluent with the pH value of 5.0 obtained in the step (3), running an AC two-phase gradient elution program in the table 2 which is the same as the uracride, collecting the eluent, and adding lysine with the final concentration of 10mmol/L and EDTA with the final concentration of 0.1 mmol/L. HPLC detection shows that the HPLC purity of the carperitide is 99.88 percent, and the methionine oxidation impurity is 0.12 percent.
(5) Concentration and lyophilization of eluents
And (3) under the condition of high-purity nitrogen replacement, concentrating the eluent collected after salt conversion under reduced pressure to remove ethanol, and drying for 12 hours at the vacuum degree of less than or equal to 10mbar and the temperature of a partition plate controlled at 10 ℃ to obtain 2.95g of a refined product of the carperitide, wherein the HPLC purity is 99.85 percent and the methionine oxidation impurity is 0.15 percent.
Example 7 preparative purification of Exenatide
(1) Dissolution and filtration of crude exenatide
10.0g of crude exenatide was dissolved in 2.0L of 50mmol/L aqueous ammonium acetate solution with stirring. The obtained exenatide sample liquid was subjected to filtration through a 0.45 μm filter. The filtrate was checked by HPLC and the HPLC purity of exenatide was 39.68% and methionine oxidized impurity was 2.35%.
(2) Purification by high performance liquid chromatography
Using DAC50 column, circulating water outside the column, and keeping the temperature at 30 deg.C, wherein the chromatographic column is packed with YMC-ODS--10 μm. A, B mobile phase is prepared, wherein A is acetonitrile, B is 50mmol/L ammonium acetate water solution with pH 6.0.
And (2) loading the sample filtrate obtained in the step (1), operating an AB two-phase gradient elution program in the table 1 which is the same as the ularitide, setting the detection wavelength, and collecting and preparing the eluent. The HPLC detection of the eluent shows that the purity of the exenatide HPLC is 97.87 percent, and the methionine oxidized impurity is 0.53 percent. The eluate was added to a final concentration of 10mmol/L methionine and 0.1mmol/L EDTA for further use.
(3) Salt conversion
Using DAC50 column, circulating water outside the column, and keeping the temperature at 30 deg.C, wherein the chromatographic column is packed with YMC-ODS--10 μm. A, C mobile phase is prepared, wherein A is acetonitrile, C is oxygen-free aqueous solution with volume fraction of 1% acetic acid.
And (3) loading the eluent obtained in the step (2), running an AC two-phase gradient elution program in the table 2 which is the same as the Ursolitin, collecting the eluent, and adding the final concentration of 10mmol/L methionine and 0.1mmol/L EDTA. HPLC detection shows that the HPLC purity of the exenatide is 99.68 percent, and the methionine oxidation impurity is 0.12 percent.
(4) Concentration and lyophilization of eluents
Under the condition of high-purity nitrogen replacement, concentrating the eluate after salt conversion under reduced pressure to remove acetonitrile, and drying at 20 deg.C for 10 hr under vacuum degree of less than or equal to 10mbar and at 20 deg.C of partition plate to obtain refined exenatide product 3.15g, with HPLC purity of 99.56% and methionine oxidation impurity of 0.18%
Comparative example 1 preparation and purification of Uraritide
(1) Dissolution and oxidation of crude ularitide linear peptide
Taking 10.0g of crude linear peptide, stirring and dissolving by using 10.0L of ammonium acetate water solution with pH of 9.0 and 1mol/L, stirring and air oxidizing at 25 ℃, detecting that the content of cysteine residue is 0 when oxidizing for 7 hours, and stopping oxidizing; the oxidized liquid was checked by HPLC, and the HPLC purity of the ularitide was 48.90%, and the methionine oxidation impurity (relative retention time ═ 0.61) was 3.11% (fig. 5).
(2) Concentration of the oxidized liquid
And (2) carrying out nanofiltration concentration on 10.0L of the oxidized liquid of the ularitide obtained in the step (1), stopping concentration when the oxidized liquid is concentrated to the volume of 1.0-1.2L, and adjusting the pH value of the concentrated liquid to 6.0 by using acetic acid for later use.
(4) Purification by high performance liquid chromatography
Using DAC50 column, circulating water outside the column, keeping temperature at 30 ℃, and filling Sepax-GP-C18--5 μm. A, B mobile phase is prepared, wherein A is acetonitrile, B is 50mmol/L potassium dihydrogen phosphate anaerobic aqueous solution with pH 6.0.
And (3) loading the concentrated solution with the pH value of 6.0 obtained in the step (2), operating an AB two-phase gradient elution program in the table 1, setting a detection wavelength, and collecting and preparing an eluent. HPLC detection of the eluent shows that the purity of the Ursolitin HPLC is 98.17 percent, and the methionine oxidized impurity is 0.75 percent. The eluate is adjusted to pH3.0 with acetic acid and is ready for use.
(4) Salt conversion
Using DAC50 column, circulating water outside the column, keeping temperature at 30 ℃, and filling Sepax-GP-C18--5 μm. A, C mobile phase is prepared, wherein A is acetonitrile, C is oxygen-free aqueous solution with volume fraction of 1.0% acetic acid.
And (3) loading the Ursolitin eluent with the pH value of 3.0 obtained in the step (3), operating an AC two-phase gradient elution program in the table 2, performing high-purity nitrogen replacement on a receiving bottle, and collecting the eluent. HPLC detection shows that the HPLC purity of the ularitide is 99.92 percent, and the methionine oxidation impurity is 0.08 percent.
(6) Concentration and lyophilization of eluents
And (3) under the condition of high-purity nitrogen replacement, concentrating the eluent collected after salt conversion under reduced pressure to remove acetonitrile, and drying for 12 hours at the vacuum degree of less than or equal to 10mbar and the temperature of a partition plate controlled at 10 ℃ to obtain 2.92g of the refined product of the ularitide, wherein the HPLC purity is 99.81 percent and the methionine oxidation impurity is 0.19 percent.
Comparative example 2 preparation and purification of ularitide
(1) Dissolution and oxidation of crude ularitide linear peptide
Taking 10.0g of crude linear peptide, stirring and dissolving by using 10.0L of ammonium acetate water solution with the pH value of 8.0 and the concentration of 1mol/L, stirring and air oxidizing at the temperature of 25 ℃, detecting that the content of cysteine residue is 0 when oxidizing for 6 hours, and stopping oxidizing; the oxidized liquid was checked by HPLC, and the HPLC purity of the ularitide was 52.21%, and the oxidation impurity of methionine (relative retention time ═ 0.61) was 2.17% (fig. 6).
(2) Concentration of the oxidized liquid
And (2) carrying out nanofiltration concentration on 10.0L of the oxidized liquid of the ularitide obtained in the step (1), stopping concentration when the oxidized liquid is concentrated to the volume of 1.0-1.2L, and adjusting the pH value of the concentrated liquid to 6.0 by using acetic acid for later use.
(3) Purification by high performance liquid chromatography
Using DAC50 column, circulating water outside the column, keeping temperature at 30 ℃, and filling Sepax-GP-C18--5 μm. A, B mobile phase is prepared, wherein A is acetonitrile, B is 50mmol/L potassium dihydrogen phosphate water solution with pH 6.0.
And (3) loading the concentrated solution with the pH value of 6.0 obtained in the step (2), operating an AB two-phase gradient elution program in the table 1, setting a detection wavelength, and collecting and preparing an eluent. HPLC detection of the eluent shows that the purity of the Ursolitin HPLC is 98.48 percent, and the methionine oxidized impurity is 0.67 percent.
(4) Salt conversion
Using DAC50 column, circulating water outside the column, keeping temperature at 30 ℃, and filling Sepax-GP-C18--5 μm. A, C mobile phase is prepared, wherein A is acetonitrile, C is 1.0% acetic acid solution in volume fraction.
And (3) loading the Ursolitin eluent with the pH value of 6.0 obtained in the step (3), operating an AC two-phase gradient elution program in the table 2, and collecting the eluent. HPLC detection shows that the HPLC purity of the ularitide is 99.59 percent, and the methionine oxidized impurity is 0.41 percent.
(5) Concentration and lyophilization of eluents
Concentrating the eluate after salt conversion under reduced pressure to remove acetonitrile, and drying at vacuum degree of less than or equal to 10mbar and at a temperature controlled by a partition board of 10 deg.C for 12h to obtain refined product of ularitide 3.06g, with HPLC purity of 97.42% and methionine oxidation impurity of 2.58% (FIG. 6).
Comparative example 3 preparation and purification of Uraritide
(1) Dissolution and oxidation of crude ularitide linear peptide
Dissolving 10.0g of crude linear peptide into water, adjusting the pH value to 7-8, placing and stirring the crude linear peptide open at 25 ℃ for air oxidation, adding hydrogen peroxide with the mass concentration of 30% after oxidizing for 48 hours, continuously oxidizing until the content of cysteine residue is detected to be 0, and stopping oxidation, wherein the adding amount of the hydrogen peroxide is 0.05-0.15% of that of the solution of the linear peptide of the ularitide; the HPLC detection shows that the HPLC purity of the oxidized liquid is 41.75 percent, and the methionine oxidized impurity (relative retention time is 0.61) is 8.12 percent.
(2) Concentration of the oxidized liquid
And (2) carrying out nanofiltration concentration on the oxidized liquid of the ularitide obtained in the step (1), stopping concentration when the volume of the oxidized liquid of the ularitide is 1/10 of the original volume, and adjusting the pH value of the concentrated liquid to 6.0 by using acetic acid for later use.
(3) Purification by high performance liquid chromatography
Using DAC50 column, circulating water outside the column, keeping temperature at 30 ℃, and filling Sepax-GP-C18--5 μm. A, B mobile phase is prepared, wherein A is acetonitrile, B is 50mmol/L potassium dihydrogen phosphate water solution with pH 6.0.
And (3) loading the concentrated solution with the pH value of 6.0 obtained in the step (2), operating an AB two-phase gradient elution program in the table 1, setting a detection wavelength, and collecting and preparing an eluent. HPLC detection of the eluent shows that the HPLC purity of the ularitide is 98.10 percent, and the methionine oxidized impurity is 0.98 percent.
(4) Salt conversion
Using DAC50 column, circulating water outside the column, keeping temperature at 30 ℃, and filling Sepax-GP-C18--5μAnd m is selected. A, C mobile phase is prepared, wherein A is acetonitrile, C is 1.0% acetic acid solution in volume fraction.
And (3) loading the Ursolitin eluent with the pH value of 6.0 obtained in the step (3), operating an AC two-phase gradient elution program in the table 2, and collecting the eluent. HPLC detection shows that the HPLC purity of the ularitide is 98.66%, and the methionine oxidized impurity is 0.83%.
(5) Concentration and lyophilization of eluents
Concentrating the eluate after salt conversion under reduced pressure to remove acetonitrile, and drying at vacuum degree of less than or equal to 10mbar and at a temperature controlled by a partition plate of 10 deg.C for 12h to obtain refined product of ularitide 2.16g, with HPLC purity of 96.11% and methionine oxidized impurity of 3.89%.
Claims (10)
1. A method for preparing and purifying polypeptide is characterized by comprising the following steps:
(1) dissolving the crude polypeptide product in a stirred buffer salt-water solution, adjusting the pH, and carrying out air oxidation to obtain a polypeptide oxidation solution;
(2) performing desalination concentration or nanofiltration concentration by using ion exchange resin according to the buffer salt selected in the step (1) to obtain a concentrated solution;
(3) loading the concentrated solution to a high performance liquid preparative chromatographic column, performing gradient elution by using an organic solvent and a buffer solution as mobile phases, and collecting eluent;
(4) loading the eluent into a high performance liquid chromatography column again, and performing gradient elution by using an organic solvent and an acid aqueous solution as mobile phases to obtain a polypeptide eluent;
(5) concentrating the above glatiramer eluate under reduced pressure to remove organic solvent, and lyophilizing to obtain refined polypeptide product.
2. The method for preparing and purifying the polypeptide of claim 1, wherein the polypeptide is one of ularitide, ziconotide, nesiritide, brain natriuretic peptide, capecitabine and exenatide.
3. The method for preparing and purifying the polypeptide according to claims 1-2, wherein the buffer salt in step (1) is selected from one of potassium dihydrogen phosphate, ammonium dihydrogen phosphate, sodium hydrogen carbonate, ammonium sulfate, ammonium acetate, ammonium chloride and sodium formate; the concentration of the buffer salt is 0.1-5.0 mol/L, preferably 1.0-1.5 mol/L, and preferably, the pH value in the oxidation process in the step (1) is 6.0-8.0, the oxidation temperature is 20-30 ℃, and the oxidation time is 5-10 h.
4. The method for preparing and purifying a polypeptide according to claim 1, wherein the mobile phase in step (3) is a mixed solution of two solutions AB, phase a is an organic solvent, and phase B is a buffered salt solution; the organic solvent is methanol, ethanol or acetonitrile, the buffer salt is selected from one of sodium dihydrogen phosphate, potassium dihydrogen phosphate, ammonium sulfate, ammonium acetate and sodium formate, the concentration of the buffer salt is 10-100 mmol/L, and the pH value is 2.0-7.5.
5. The method for preparing and purifying a polypeptide according to claim 1, wherein the mobile phase used in the step (4) of converting the salt is A, C two-phase mixed solution; the phase A is the same as the phase A in the step (3) in organic solvent, and the phase C is diluted acid solution selected from one of formic acid, acetic acid, trifluoroacetic acid or hydrochloric acid; preferably, the volume fraction of formic acid or acetic acid is 0.1 to 1.0% when the acid is formic acid or acetic acid, 0.05 to 0.1% when the acid is trifluoroacetic acid, and 0.01 to 0.05% when the acid is hydrochloric acid.
6. The method for preparing and purifying a polypeptide according to claim 1, wherein the lyophilization conditions in the step (5) are as follows: the vacuum degree is less than or equal to 10mbar, the temperature of the clapboard is controlled to be 20 ℃, and the freeze-drying time is 12-36 h.
7. The method for preparing and purifying a polypeptide according to claim 1, wherein at least one method for inhibiting methionine oxidation is required in steps (3) - (5), and preferably, the method for inhibiting methionine oxidation is to adjust the system pH to 3.0-5.0, replace purified water with oxygen-free water, add an amino acid protectant, add an antioxidant, and use gas substitution.
8. The method of claim 7, wherein the pH-adjusting agent is selected from the group consisting of formic acid, acetic acid, trifluoroacetic acid and hydrochloric acid; preferably, the oxygen-free water is prepared by heating purified water for reflux treatment, cooling and then blowing off oxygen by using high-purity nitrogen.
9. The method of claim 7, wherein the amino acid is selected from the group consisting of methionine, proline, histidine, glycine and lysine, preferably methionine or lysine; the addition concentration of the amino acid protective agent is 2-10 mmol/L; preferably, the antioxidant is selected from one of ascorbic acid, citric acid, vitamin E and EDTA, preferably citric acid or EDTA; the concentration of the antioxidant is 0.1-0.3 mmol/L.
10. The use of the method of claim 1 in the preparation and purification of a polypeptide, wherein the polypeptide is one of ularitide, ziconotide, nesiritide, brain natriuretic peptide, capecitabine, and exenatide.
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