阅读说明：本技术 一种乌拉立肽杂质 (Ursolitin impurity ) 是由 刘�东 任崇飞 于 2018-08-21 设计创作，主要内容包括：本发明属于医药技术领域,具体公开了一种乌拉立肽杂质,通过样品跟踪监测,发现乌拉立肽甲硫氨酸氧化物贯穿整个乌拉立肽纯化工艺,并在乌拉立肽纯化后期,成为主要杂质成分。本发明还公开了该杂质的制备方法和检测方法,本发明制备方法,反应时间短,操作简单,收率高,所得到的乌拉立肽杂质HPLC纯度达到99.5％以上,可以作为标准品用于乌拉立肽制备过程中的杂质研究。(The invention belongs to the technical field of medicines, and particularly discloses a urotropine impurity, which is a main impurity component in the later stage of urotropine purification by finding that urotropine methionine oxide penetrates through the whole urotropine purification process through sample tracking and monitoring. The preparation method has the advantages of short reaction time, simple operation and high yield, and the obtained ularitide impurity HPLC purity reaches more than 99.5 percent and can be used as a standard substance for impurity research in the ularitide preparation process.)

1. An impurity of ularitide, the amino acid sequence of which is connected as follows:

the structural formula is shown in formula I:

2. a preparation method of the ularitide impurity is characterized by comprising the following steps:

(1) oxydol oxidation reaction

Dissolving the pure product of the ularitide in water, adding hydrogen peroxide solution, and stirring and reacting for 10-30 min at room temperature.

(2) Sample purification

Loading the oxidized solution obtained in the step (1) to a high performance liquid chromatography column, performing gradient elution by using an A-phase organic solvent and a B-phase buffer salt solution as mobile phases, and collecting eluent.

(3) Salt conversion

And (3) continuously loading the eluent obtained in the step (2) into a high performance liquid chromatography column, performing gradient elution and salt conversion by taking an A-phase organic solvent and a C-phase acid solution as mobile phases, and collecting an effluent liquid by ultraviolet detection.

(4) Freeze-drying

And (4) concentrating the elution effluent obtained in the step (3) to remove the organic solvent, and freeze-drying for 10-16 h under the conditions that the vacuum degree is less than or equal to 10mbar and the temperature of a partition plate is controlled at 20 ℃ to obtain the pure product of the ularitide impurity.

3. The method for preparing the ularitide impurity according to claim 2, wherein the concentration of the ularitide dissolved in water in the step (1) is 5-20 mg/ml; the mass fraction of hydrogen peroxide in the hydrogen peroxide solution is 2-5%, and the volume ratio of the ularitide sample solution to the hydrogen peroxide solution is 1: 0.8-1.2.

4. The method for preparing the ularitide impurity of formula I as claimed in claim 2, wherein the phase A organic solvent in step (2) and step (3) is selected from one of methanol, ethanol and acetonitrile.

5. The method for preparing the ularitide impurity of formula I as claimed in claim 2, wherein the B-phase buffer salt solution in step (2) is one of sodium dihydrogen phosphate, potassium dihydrogen phosphate, ammonium sulfate, ammonium acetate and sodium formate solution, the concentration of the buffer salt is 20-100 mmol/L, and the pH of the buffer salt solution is 2.0-4.5.

6. The method for preparing the ularitide impurity of formula I as claimed in claim 2, wherein the C-phase acid solution in step (3) is an aqueous solution of formic acid or acetic acid, and the volume fraction of formic acid or acetic acid is 0.1-1%.

7. A detection method of the urapidine impurity is characterized in that a chromatographic column which takes acid-resistant C18 bonding filler as a filling agent is adopted, and mobile phases are a mobile phase and a mobile phase B mobile phase which are prepared from buffer salt solution and organic solvent in different volume ratios, and gradient elution is carried out; wherein the filler is tolerant to a pH of 1-3; the specification of the chromatographic column is 4.6mm multiplied by 250mm, and the grain diameter of the filler is 5 μm or 10 μm; the detection wavelength is 210-220 nm; the flow rate is 0.8-1.0 ml/min, preferably 1.0 ml/min; the column temperature is 25-35 ℃, and preferably 30 ℃.

8. The method of claim 7, wherein the mobile phase is Ursolitin

a. b, mixing solution of two phases, wherein the two phases a and b are both mixed solution of buffer salt solution and organic solvent; preferably, the volume ratio of the buffer salt solution to the organic solvent in the phase a is 9: 1-7: 3, and the volume ratio of the buffer salt solution to the organic solvent in the phase b is 4: 6-6: 4; the organic solvent is selected from one of methanol and 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 10-30 mmol/L; the pH value of the buffer solution is 1.5-3.0.

9. The method of claim 7, wherein the elution gradient of the mobile phase a and b is as follows:

10. an application of the ularitide impurity in the formula I in the research of the ularitide impurity.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a ularitide impurity.

Background

The structure of Ularitide (Ularitide) is polypeptide containing a pair of intramolecular disulfide bonds and consisting of 32 amino acids, and the molecular formula is C₁₄₅H₂₃₄N₅₂O₄₄S₃Molecular weight of about 3506, CAS No.: 118812-69-4, the polypeptide sequence is:

H-Thr-Ala-Pro-Arg-Arg-Ser-Leu-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)。

the ularitide is natriuretic peptide separated from human urine, and is renal natriuretic peptide of Atrial Natriuretic Peptide (ANP) family. 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, natriuretic and diuresis, 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 contractility-enhancing drugs, which, however, have clinical limitations. The second phase clinical research shows that the ularitide can reduce the cardiac filling pressure and improve dyspnea, has no obvious adverse effect on the kidney function of a DHF patient, and shows that the ularitide has wide prospects in treatment of 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 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, the oxidation process of the ularitide is relatively complex, the product yield is not high, and side reactions are more. Impurities generated by these side reactions may cause structural and functional changes of the ularitide. The generation of these impurities has a potential threat to the safety of clinical medication, and in order to better realize the economic and social benefits of the medicine, each impurity must be strictly controlled in the standard, so that the definition of each impurity is an urgent problem to be solved.

Disclosure of Invention

The oxidation step in the preparation process of the ularitide is to oxidize sulfur-containing amino acid residues in cysteine in an ularitide sequence. Meanwhile, the 16 th site of the ularitide is methionine, and belongs to sulfur-containing amino acid residues which are easy to oxidize. Methionine is readily oxidized to methionine-R, S-sulfoxide and can be further irreversibly oxidized to methionine-R, S-sulfone. Methionine is oxidized in the processes of oxidizing and cyclizing polypeptide and purifying polypeptide, impurities are further generated, and the structure and the function of the ularitide are changed.

In the purification process of the ularitide, the higher the purity of the polypeptide, the more easily methionine is oxidized. Through experimental analysis, the methionine oxidation impurity of the polypeptide can not be detected by the general polypeptide detection method. The existing polypeptide medicine preparation literature and patent technology do not have a method for detecting and controlling the polypeptide oxidation process, and the impurities are not listed as key impurities in the quality standard.

Aiming at the defects of the prior art, the invention optimizes the polypeptide analysis method by testing in the development process of the method for detecting the ularitide, and obtains the analysis method capable of effectively detecting the impurities of the ularitide by screening. The sequence analysis proves that the ularitide impurity is polypeptide methionine oxide. 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 HPLC detection spectra of the pure product and the pure product after being placed in the air for 2h and 6h are shown in figure 1, figure 2 and figure 3. Therefore, it is very necessary to study impurities in the process of preparing ularitide.

The invention aims to provide a ularitide impurity, and the amino acid sequence of the polypeptide impurity is connected as follows:

the structural formula is shown as formula I:

the invention also provides a preparation method of the ularitide impurity shown in the formula I, and the ularitide impurity is obtained by oxidizing a pure product of the ularitide. Specifically, the preparation method comprises the following steps:

(1) oxydol oxidation reaction

Dissolving the pure product of the ularitide in water, adding hydrogen peroxide solution, and stirring and reacting for 10-30 min at room temperature.

(2) Sample purification

Loading the oxidized solution obtained in the step (1) to a high performance liquid chromatography column, performing gradient elution by using an A-phase organic solvent and a B-phase buffer salt solution as mobile phases, and collecting eluent.

(3) Salt conversion

And (3) continuously loading the eluent obtained in the step (2) into a high performance liquid chromatography column, performing gradient elution and salt conversion by taking an A-phase organic solvent and a C-phase diluted acid solution as mobile phases, and collecting an effluent liquid through ultraviolet detection.

(4) Freeze-drying

And (4) concentrating the elution effluent obtained in the step (3) to remove the organic solvent, and freeze-drying for 10-16 h under the conditions that the vacuum degree is less than or equal to 10mbar and the temperature of a partition plate is controlled at 20 ℃ to obtain the ularitide impurity shown in the formula I.

Preferably, the concentration of the solution of the urotropine sample in the step (1) is 5-20 mg/ml, the mass fraction of the hydrogen peroxide in the hydrogen peroxide solution is 2-5%, and the volume ratio of the urotropine sample solution to the hydrogen peroxide solution is 1: 0.8-1.2.

The mass concentration of the hydrogen peroxide in the step (1) can be increased, and the increase of the mass concentration of the hydrogen peroxide can improve the oxidation yield to a certain extent, but other impurities can be generated along with the increase of the mass concentration of the hydrogen peroxide, and the generation of the impurities can influence the separation and purification of the urapidine impurity of the formula I.

In the preparation method of the urapidine impurity shown in the formula I, the oxidant hydrogen peroxide can be replaced by other oxidants, such as sodium peroxide, potassium permanganate, potassium chlorate, hypochlorous acid and the like which can provide active oxygen, and the oxidants can also oxidize methionine in the pure product of the urapidine, but other impurities can be generated at the same time, so that the separation and purification of the urapidine impurity shown in the formula I are influenced.

Preferably, the packing used in the high performance liquid preparative chromatographic column in the step (2) is reversed phase chromatographic packing, and preferably, the preparative packing is selected from

One of the above two methods;

preferably, the mobile phase in the step (2) is a mixed solution of an A-phase organic solvent and a B-phase buffer salt solution, wherein the A-phase organic solvent is selected from one of methanol, ethanol and acetonitrile; the B-phase buffer salt solution is selected from one of sodium dihydrogen phosphate, potassium dihydrogen phosphate, ammonium sulfate, ammonium acetate and sodium formate solutions, the concentration of the buffer salt is 20-100 mmol/L, and the pH value of the buffer salt solution is 2.0-4.5.

Preferably, the gradient elution procedure described in step (2) is shown in table 1, and the eluate is collected according to the online ultraviolet detection result.

TABLE 1 elution gradient during purification

More preferably, the gradient elution schedule of step (2) is as follows:

TABLE 2 elution gradient during purification

Preferably, the high performance liquid chromatography column used in step (3) is the same as that in step (2), and the mobile phase A phase is also the same as that in step (2); and (3) the mobile phase C is a dilute acid solution, the acid is formic acid or acetic acid, the volume fraction of the formic acid or the acetic acid is 0.1-1%, and the elution gradient in the step (3) 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

And (3) carrying out amino acid composition analysis on the ularitide impurity sample. The analytical results were as follows:

1. total number of amino acids

The theoretical total number of amino acids is: 32 (a)

2. Molecular weight

The theoretical molecular weight is: 3521.9 Dalton

3. Amino acid composition

The polypeptide amino acid composition is shown in table 5.

TABLE 5 polypeptide amino acid composition information

As shown in Table 5, the ularitide impurity does not contain Met, but contains Met (O), the molecular weight of the pure ularitide is 3505.9 through mass spectrum analysis, and the molecular weight of the impurity in the formula I is 3521.9. The difference in molecular weight between the two is 16, i.e.methionine is oxidized to methionine sulfoxide. The mass spectrum of the pure product of the ularitide is shown in figure 4, and the mass spectrum of the prepared impurity of the formula I is shown in figure 5.

The invention also provides a method for detecting the ularitide impurities. The method is used for solving the defect that the methionine oxidation impurity cannot be detected by the conventional polypeptide analysis method.

The following two main methods are used in conventional polypeptide analysis:

(1) the conventional C18 bonded column, 0.1% TFA/H was used₂O-0.1% TFA/ACN or 0.1% HA/H₂An O-ACN elution system;

(2) a common C18 bonded or ODS chromatographic column and a two-phase flowing phase system with different mixed buffer salts and organic solvents are adopted, and the common pH value is 4.0-7.0.

However, the two analysis methods cannot effectively detect the impurities of the formula I of the urotropine, and the invention provides a method for detecting the oxidized impurities of the methionine, so that the impurities of the formula I of the urotropine are quantitatively detected, and the oxidized impurities of the methionine are controlled in the purification process of the urotropine.

The invention provides a method for detecting impurities in ularitide as shown in formula I, which adopts a chromatographic column taking acid-resistant C18 bonding filler as a filling agent, adopts two mobile phases a and b with different volume ratios of buffer salt solution and organic solvent as mobile phases, and performs gradient elution.

Preferably, the filler is resistant to pH of 1-3; more preferably, the filler is Welch-Ultimate-LP-C18-5 μm orThe specification of the chromatographic column used is 4.6mm multiplied by 250 mm; the detection wavelength is 210-220 nm: the flow rate is 0.8-1.0 ml/min, preferably 1.0 ml/min; the column temperature is 25-35 ℃, and preferably 30 ℃.

Preferably, the mobile phase is a mixed solution of a phase a and a phase b, and the phase a and the phase b are mixed solutions of buffer salt solutions and organic solvents with different volume ratios; more preferably, the volume ratio of the buffer salt solution to the organic solvent in the phase a is 9: 1-7: 3, and the volume ratio of the buffer salt solution to the organic solvent in the phase b is 4: 6-6: 4; the organic solvent is selected from one of methanol and 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 10-30 mmol/L; the pH value of the buffer solution is 1.5-3.0, and the pH regulator is an acid corresponding to buffer salt in the mobile phase.

Preferably, the gradient elution procedure is shown in table 6.

TABLE 6 Uralitide impurity HPLC detection elution gradient chart

More preferably, HPLC detection chromatographic conditions of the oxidized impurities of the ularitide are as follows:

a chromatographic column: Welch-Ultimate-LP-C18-5 μm, 4.6X 250 mm;

mobile phase: the volume ratio of the ammonium sulfate solution of 20mmol/L to the acetonitrile of the phase a is 9: 1, the volume ratio of the ammonium sulfate solution of 20mmol/L to the acetonitrile of the phase b is 1: 1, wherein the pH value of the ammonium sulfate solution of 20mmol/L is adjusted to 2.0 by using phosphoric acid;

detection wavelength: 210 nm; flow rate: 1.0 ml/min; column temperature: 30 ℃;

the gradient elution is shown in Table 7.

TABLE 7 elution gradient chart for HPLC detection of Ursolitin impurities

The HPLC (high performance liquid chromatography) chromatogram of the impurities of the ularitide shown in the formula I prepared by the invention is shown in a figure 6, the analysis method adopts an acid-resistant filler chromatographic column, and the chromatographic column can tolerate pH 1-3 which is lower than that of a common C18 reverse phase column, namely, the pH 2-8; the mobile phase adopts two mobile phases a and b with different volume ratios of buffer salt solution and organic solvent to carry out gradient elution; the analysis method can effectively detect the impurities of the ularitide and the ularitide shown in the formula I, and can realize effective detection and control of the impurities of the ularitide shown in the formula I in the preparation and purification process of the ularitide.

Compared with the prior art, the invention achieves the following technical effects:

(1) the preparation method has the advantages of short reaction time, simple operation and high yield, and the obtained urapidine impurity HPLC purity reaches more than 99.5 percent and can be used as a standard substance for impurity research in the preparation process of the urapidine;

(2) the analysis method can complete high-efficiency analysis of the ularitide sample, can be applied to detection and control of the ularitide and methionine oxide impurities in the purification process of the ularitide, and the conventional polypeptide detection method cannot effectively detect the polypeptide amino acid oxidation impurities and cannot realize effective detection of the ularitide.

(3) The method has strong pertinence and is simple, and the method has reference significance for establishing other analysis methods of the sulfur-containing amino acid polypeptide.

Drawings

FIG. 1: HPLC detection spectrum of pure product of ularitide;

FIG. 2: placing the pure product of the ularitide for 2h, and detecting a map by HPLC;

FIG. 3: placing the pure product of the ularitide for 6 hours, and detecting a map by HPLC;

FIG. 4: a pure wuralitide mass spectrogram;

FIG. 5: an urapidide impurity mass spectrum of formula I;

FIG. 6: HPLC detection profile of the urapidine impurity of formula I;

FIG. 7: example 11 HPLC detection profile of ularitide impurities;

FIG. 8: example 12 HPLC detection profile of ularitide impurities;

FIG. 9: example 13 HPLC detection profile of pure ularitide;

FIG. 10: example 14 HPLC detection profile of pure ularitide.

Detailed Description

The present invention is fully illustrated by the following specific examples, which are intended to represent only a part of the present invention, but not all examples, and which should not be construed as limiting the scope of the present invention, which will be obvious to those skilled in the art to which the present invention pertains.

The pure product of the ularitide is provided by Shandong New times pharmaceutical industry Co., Ltd, and other used raw materials/fillers and the like can be obtained by commercial means without special description. The high performance liquid preparative chromatographic column adopted in the preparation process of the urapidine impurity adopts a dynamic axial compression preparative column (DAC50 column) with the thickness of 50mm multiplied by 450mm, and the HPLC instrument in the analysis and detection process is a Thermo Ultimate3000 high performance liquid chromatograph (American Saimer Feishell science and technology).