阅读说明：本技术 一种手性多肽类药物非对映异构体杂质的检测方法 (Method for detecting chiral polypeptide drug diastereoisomer impurities ) 是由 黄秀坤 胡沙 袁瑜 刘宏 付晓平 于 2021-08-23 设计创作，主要内容包括：本发明提供了一种手性多肽类药物非对映异构体杂质的检测方法,包括以下步骤：S1)多肽药物样品溶解后过滤制得检测样品溶液；S2)采用反相色谱法,以十八烷基硅烷键合硅胶为固定相,无机盐水溶液为A相,甲醇或乙腈为B相,对检测样品溶液进行梯度洗脱,实现非对映异构体杂质与主成分的分离。该反相色谱分析方法主要解决了常规分析方法无法有效检测出非对映异构体杂质,无法准确进行该药物的定性定量检测,为有效控制产品有关物质,监测产品质量水平提供了检测手段保障。(The invention provides a method for detecting chiral polypeptide drug diastereoisomer impurities, which comprises the following steps: s1) dissolving the polypeptide drug sample and filtering to prepare a detection sample solution; s2) adopting a reversed phase chromatography, taking octadecylsilane chemically bonded silica as a stationary phase, taking an inorganic salt water solution as an A phase and taking methanol or acetonitrile as a B phase, and carrying out gradient elution on the detection sample solution to realize the separation of diastereoisomer impurities and main components. The reversed phase chromatographic analysis method mainly solves the problems that the conventional analysis method cannot effectively detect the diastereoisomer impurities and accurately perform qualitative and quantitative detection on the medicine, and provides a detection means guarantee for effectively controlling related substances of the product and monitoring the quality level of the product.)

1. A method for detecting chiral polypeptide drug diastereoisomer impurities comprises the following steps:

s1) dissolving the polypeptide drug sample and filtering to prepare a detection sample solution;

s2) adopting a reversed phase chromatography, taking octadecylsilane chemically bonded silica as a stationary phase, taking an inorganic salt water solution as an A phase and taking methanol or acetonitrile as a B phase, and carrying out gradient elution on the detection sample solution to realize the separation of diastereoisomer impurities and main components.

2. The assay of claim 1, wherein said polypeptide drug is a crude [ (S) -1- (D-phenylalanyl-D-leucyl-D-lysyl) pyrrolidin-3-yl ] borate salt comprising an impurity of [ (R) -1- (D-phenylalanyl-D-leucyl-D-lysyl) pyrrolidin-3-yl ] borate.

3. The method of claim 1, wherein the solvent in which the polypeptide drug sample is dissolved is a polar hydrophilic solvent.

4. The detection method according to claim 3, wherein the solvent in which the polypeptide drug sample is dissolved is selected from one or more of water, methanol, ethanol, and acetonitrile.

5. The detection method according to claim 3, wherein the concentration of the detection sample solution is 5mg/ml or less.

6. The detection method according to claim 1, wherein the phase A is an aqueous ammonium sulfate solution.

7. The detection method according to claim 6, wherein the concentration of the aqueous ammonium sulfate solution is 5mM to 20 mM; the pH value is 1.5-2.5.

8. The detection method according to claim 1, wherein the conditions of the reverse phase chromatography are:

the flow rate is 0.8-1.2 ml/min; the column temperature is 30-45 ℃; the detection wavelength is 220 nm; the sample volume is 10-100 μ l.

9. The detection method according to claim 1, wherein the gradient elution is in particular:

maintaining 75% of phase A in 0-10min, reducing phase A from 75% to 70% in 10.01-25 min, reducing phase A from 70% to 68% in 25.01-35 min, reducing phase A from 68% to 50% in 35.01-55 min, maintaining phase A to 65min, and recovering 75% of mobile phase A from 65.01 min-75 min to perform chromatographic column balance.

Technical Field

The invention relates to the technical field of detection, in particular to a method for detecting chiral polypeptide drug diastereoisomer impurities.

Background

Opioid receptors are a major class of G protein-coupled receptors, are endogenous opioid peptides and opioid drug binding targets, and are widely found in the central and peripheral nervous systems. Opioid receptors, which are activated to regulate the immune and endocrine systems of the nervous system, are the most powerful and commonly used central analgesics. Endogenous opioid peptides are naturally occurring opioid active substances in mammals, and currently known endogenous opioid peptides are broadly classified into several classes, namely enkephalins, endorphins, dynorphins and neorphins. Its corresponding opioid receptors, i.e., μ, δ and κ receptors, are present in the central nervous system. Mu receptor has the strongest analgesic activity and the strongest addiction, and is the main reason for generating side effects. The delta receptor has small addiction and also has unobvious analgesic effect. Kappa receptor (KOR) analgesic activity is intermediate between the first two. The polypeptide KOR agonist can exert analgesic effect in periphery without entering into center, and has no adverse side effects such as respiratory depression and constipation, and lower addiction, thus having potential of drug addiction treatment.

Patent CN111233974B reports a series of novel KOR agonists with excellent agonistic activity, including a compound represented by the structural formula I-1 (chemical name: [ (S) -1- (D-phenylalanyl-D-leucyl-D-lysyl) pyrrolidin-3-yl ] boronic acid hydrochloride). The crude product of the compound I-1 contains various process impurities, degradation impurities and chiral isomer impurities (formula I-2, chemical name: [ (R) -1- (D-phenylalanyl-D-leucyl-D-lysyl) pyrrolidine-3-yl ] boric acid hydrochloride) which are difficult to separate, the polarity difference of the isomer impurities and the main component is very small, the physicochemical properties are extremely similar, the isomer impurities are extremely difficult to be effectively separated in HPLC detection, great challenges are brought to detection and analysis, and samples meeting clinical requirements cannot be effectively monitored and guided to be produced. Therefore, developing a simple, convenient, reproducible, stable and reliable analysis method to monitor the impurities, especially chiral diastereomer impurities, to ensure the quality controllability in the process of drug development is a problem to be solved at present.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a method for detecting diastereomer impurities of chiral polypeptide drugs, which can effectively detect diastereomer impurities that cannot be detected under conventional detection conditions by reasonably optimizing chromatographic conditions, thereby providing an effective monitoring means for ensuring the quality level of the polypeptide drugs.

In order to achieve the above purpose, the invention provides a method for detecting chiral polypeptide drug diastereoisomer impurities, which comprises the following steps:

s1) dissolving the polypeptide drug sample and filtering to prepare a detection sample solution;

s2) adopting a reversed phase chromatography, taking octadecylsilane chemically bonded silica as a stationary phase, taking an inorganic salt water solution as an A phase and taking methanol or acetonitrile as a B phase, and carrying out gradient elution on the detection sample solution to realize the separation of diastereoisomer impurities and main components.

The invention adopts HPLC reversed phase chromatography to detect the diastereoisomer impurities, provides an effective monitoring means, can accurately perform qualitative and quantitative analysis on the medicament, and effectively ensures the quality of the chiral polypeptide medicament.

In the invention, the polypeptide drug is a crude [ (S) -1- (D-phenylalanyl-D-leucyl-D-lysyl) pyrrolidine-3-yl ] borate (shown as the formula I-1), wherein the crude [ (R) -1- (D-phenylalanyl-D-leucyl-D-lysyl) pyrrolidine-3-yl ] borate impurity (shown as the formula I-2) is contained.

The polypeptide drug is a polypeptide compound obtained by a solid phase or liquid phase synthesis method.

In a preferred embodiment of the present invention, the solvent in which the polypeptide drug sample is dissolved is a polar hydrophilic solvent. Further preferably one or more of water, methanol, ethanol, and acetonitrile. More preferably methanol, water or a mixture of both.

The solubility of the polypeptide drug sample in the polar hydrophilic solvent is from soluble to very soluble.

Preferably, the concentration of the detection sample solution is less than or equal to 5 mg/ml. More preferably 0.5 to 1 mg/ml.

The method adopts the reversed phase chromatography to carry out gradient elution on the detection sample solution, thereby realizing the separation of the diastereoisomer impurities and the main component.

The reverse phase chromatographic column is preferably a reverse phase C18 liquid chromatographic column or other column equivalent chromatographic column. More preferably a YMC-Pack ODS-AQ or Aeris TM PEPTIDE XB-C18 column. Further preferred is YMC-Pack ODS-AQ, 4.6 x 150mm, 3 μm or Aeris TM PEPTIDE XB-C18 liquid chromatography column (4.6X 250mm, 3.6 μm).

Preferably, the phase A is an aqueous solution of ammonium sulfate.

The concentration of the ammonium sulfate aqueous solution is preferably 5 mM-20 mM; more preferably 5 mM.

The pH value of the ammonium sulfate aqueous solution is preferably 1.5-2.5, and more preferably 2.0-2.5.

The pH value of the ammonium sulfate aqueous solution is preferably adjusted by using sulfuric acid.

In the present invention, the phase B is preferably methanol or acetonitrile, and more preferably methanol.

In a preferred embodiment of the present invention, the conditions of the reverse phase chromatography are:

the flow rate is 0.8-1.2 ml/min; the column temperature is 30-45 ℃; the detection wavelength is 220 nm; the sample volume is 10-100 μ l.

The flow rate is further preferably 0.8, 1.0 or 1.2 ml/min; most preferably 1.0 ml/min.

The column temperature is further preferably 35 to 45 ℃, and most preferably 45 ℃.

The detection wavelength is preferably 220 nm.

The sample injection amount is further preferably 10 to 20 mu l.

In some embodiments of the invention, the chromatographic conditions are as shown in table 1:

table 1 chromatographic conditions of the invention

Preferably, the gradient elution is specifically as follows:

maintaining 75% of phase A in 0-10min, reducing phase A from 75% to 70% in 10.01-25 min, reducing phase A from 70% to 68% in 25.01-35 min, reducing phase A from 68% to 50% in 35.01-55 min, maintaining phase A to 65min, and recovering 75% of mobile phase A from 65.01 min-75 min to perform chromatographic column balance.

The invention develops a detection method of diastereoisomers which only belongs to chiral polypeptide drugs through a large amount of experimental investigation. The experimental result shows that in the detection map obtained by the detection method, the retention time (RRT) of the diastereoisomer impurities in front of the main component medicament relative to the chiral polypeptide medicament is about 0.96.

Compared with the prior art, the invention provides a method for detecting chiral polypeptide drug diastereoisomer impurities, which comprises the following steps: s1) dissolving the polypeptide drug sample and filtering to prepare a detection sample solution; s2) adopting a reversed phase chromatography, taking octadecylsilane chemically bonded silica as a stationary phase, taking an inorganic salt water solution as an A phase and taking methanol or acetonitrile as a B phase, and carrying out gradient elution on the detection sample solution to realize the separation of diastereoisomer impurities and main components. The reversed phase chromatographic analysis method mainly solves the problems that the conventional analysis method cannot effectively detect the diastereoisomer impurities and accurately perform qualitative and quantitative detection on the medicine, and provides a detection means guarantee for effectively controlling related substances of the product and monitoring the quality level of the product.

Drawings

FIG. 1 is a conventional HPLC profile of crude polypeptide drug;

FIG. 2 is a HPLC analysis detection profile of example 1;

FIG. 3 is a HPLC analysis detection profile of example 2;

FIG. 4 is a HPLC analysis detection map of comparative example 1;

FIG. 5 is a HPLC analysis detection map of comparative example 2.

Detailed Description

In order to further illustrate the present invention, the following examples are provided to describe the detection method of the chiral polypeptide drug diastereomer impurity.

The following examples are prepared by the method disclosed in patent CN111233974B example 2.

Example 1

A method for detecting diastereoisomers of a chiral polypeptide drug, comprising:

(1) test solution: taking 50mg of the crude chiral polypeptide drug, adding water to dissolve and dilute the crude chiral polypeptide drug to prepare a solution containing about 0.5mg of the crude chiral polypeptide drug in each 1ml, shaking up the solution, and filtering the solution by using a 0.22um organic filter membrane to obtain a crude filtrate for later use.

(2) Chromatographic conditions are as follows: using YMC-Pack ODS-AQ, 4.6 x 150mm, 3 μm column; taking ammonium sulfate buffer solution (5mmol/L ammonium sulfate, pH adjusted to 2.5 with sulfuric acid) as mobile phase A and methanol as mobile phase B, performing gradient elution according to the following table, with flow rate of 1.0 ml/min; the column temperature is 45 ℃; the detection wavelength is 220 nm; the amount of the sample was 20. mu.l.

(3) The assay was run at the following gradient 2:

table 2 example 1 gradient elution conditions

FIG. 1 is a conventional HPLC chromatogram of a crude polypeptide drug, and it can be seen that chiral diastereomers and the main peaks overlap.

The HPLC analysis detection spectrum of the embodiment is shown in figure 2, the retention time of the main component is 104.273min, and the retention time of the chiral diastereoisomer impurity is 98.000 min. Through calculation: the diastereomeric impurities of this example were able to be separated efficiently from the main component with a degree of separation of 1.46.

Example 2

A method for detecting diastereoisomers of a chiral polypeptide drug, comprising:

(1) test solution: taking 50mg of the crude chiral polypeptide drug, adding water to dissolve and dilute the crude chiral polypeptide drug to prepare a solution containing about 0.5mg of the crude chiral polypeptide drug in each 1ml, shaking up the solution, and filtering the solution by using a 0.22um organic filter membrane to obtain a crude filtrate for later use.

(2) Chromatographic conditions are as follows: an Aeris PEPTIDE XB-C18 liquid chromatography column (4.6X 250mm, 3.6 μm) was used; taking ammonium sulfate buffer solution (5mmol/L ammonium sulfate, pH adjusted to 2.2 with sulfuric acid) as mobile phase A and methanol as mobile phase B, performing gradient elution according to the following table, with flow rate of 1.0 ml/min; the column temperature is 45 ℃; the detection wavelength is 220 nm; the amount of the sample was 20. mu.l.

(3) The assay was run at the following gradient 3:

table 3 example 2 gradient elution conditions

Through calculation: the diastereomeric impurities of this example were able to be separated completely and effectively from the main component with a degree of separation of 1.45.

The HPLC analysis detection spectrum is shown in figure 3.

Comparative example 1

A method for detecting diastereoisomers of a chiral polypeptide drug, comprising:

(1) test solution: taking 50mg of the crude chiral polypeptide drug, adding water to dissolve and dilute the crude chiral polypeptide drug to prepare a solution containing about 0.5mg of the crude chiral polypeptide drug in each 1ml, shaking up the solution, and filtering the solution by using a 0.22um organic filter membrane to obtain a crude filtrate for later use.

(2) Chromatographic conditions are as follows: using an AQ-C18 liquid chromatography column (4.6X 250mm, 5 μm); taking ammonium chloride buffer solution (10mmol/L ammonium sulfate, pH adjusted to 2.5 with hydrochloric acid) as mobile phase A and methanol as mobile phase B, performing gradient elution according to the following table, with flow rate of 1.0 ml/min; the column temperature is 40 ℃; the detection wavelength is 220 nm; the amount of the sample was 10. mu.l.

(3) Detection, run at the following gradient 4:

TABLE 4 comparative example 1 gradient elution conditions

Through calculation: the detection method of the comparative example can detect diastereoisomer impurities, and obviously under the conditions that the elution gradient is not changed, and other chromatographic conditions such as only a mobile phase, a chromatographic column and the like are changed, the diastereoisomer impurities cannot be completely and effectively separated from the main component, the separation degree is only 1.0, and the accurate integration and quantification requirements cannot be realized.

The HPLC analysis detection spectrum is shown in figure 4.

Comparative example 2

A method for detecting diastereoisomers of a chiral polypeptide drug, comprising:

(1) test solution: taking 50mg of the crude chiral polypeptide drug, adding water to dissolve and dilute the crude chiral polypeptide drug to prepare a solution containing about 0.5mg of the crude chiral polypeptide drug in each 1ml, shaking up the solution, and filtering the solution by using a 0.22um organic filter membrane to obtain a crude filtrate for later use.

(2) Chromatographic conditions are as follows: a liquid chromatography column (4.6X 250mm, 5 μm) using DAISOPAK SP-ODS-P (C18); taking ammonium sulfate buffer solution (5mmol/L ammonium sulfate, pH adjusted to 2.5 with sulfuric acid) as mobile phase A and methanol as mobile phase B, performing gradient elution according to the following table, with flow rate of 1.2 ml/min; the column temperature is 30 ℃; the detection wavelength is 220 nm; the amount of the sample was 20. mu.l.

(3) Detection, run at gradient as in Table 5 below

TABLE 5 comparative example 2 gradient elution conditions

Through calculation: the detection method of the embodiment can detect diastereoisomer impurities, and obviously, under the conditions that the elution gradient and the mobile phase are not changed, and only other chromatographic conditions such as a chromatographic column, flow rate, column temperature and the like are changed, the diastereoisomer impurities cannot be completely and effectively separated from main components, the separation degree is only 0.7, and the requirements of accurate integration and quantification cannot be met.

The HPLC analysis detection spectrum is shown in figure 5.

The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core idea. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.