CN113702561B - Method for detecting diastereoisomeric impurities of chiral polypeptide drugs - Google Patents
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/89—Inverse chromatography
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- Physics & Mathematics (AREA)
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- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention provides a method for detecting diastereoisomeric impurities of chiral polypeptide drugs, which comprises the following steps: s1) dissolving a polypeptide drug sample, and filtering to prepare a detection sample solution; s2) adopting a reversed phase chromatography method, taking octadecylsilane chemically bonded silica as a stationary phase, taking inorganic salt aqueous solution as a phase A and methanol or acetonitrile as a phase B, and carrying out gradient elution on a detection sample solution to realize separation of diastereoisomeric impurities and main components. The reverse phase chromatographic analysis method mainly solves the problems that diastereoisomeric impurities cannot be effectively detected by the conventional analysis method, and the qualitative and quantitative detection of the medicine cannot be accurately carried out, and provides detection means guarantee for effectively controlling related substances of products and monitoring the quality level of the products.
Description
Technical Field
The invention relates to the technical field of detection, in particular to a method for detecting diastereoisomeric impurities of chiral polypeptide drugs.
Background
Opioid receptors are a major class of G protein-coupled receptors, which are endogenous opioid peptides and opioid drug-binding targets, and are widely found in the central and peripheral nervous systems. The opioid receptor has an effect of regulating the immune and endocrine systems of the nervous system after activation, and is the strongest and commonly used central analgesic at present. Endogenous opioid peptides are naturally occurring opioid actives in mammals, and currently known endogenous opioid peptides are broadly classified into enkephalins, endorphins, dynorphins, and neorphins. There are their corresponding opioid receptors in the central nervous system, namely the μ, δ and κ receptors. Mu receptor has the strongest analgesic activity and the strongest addiction and is the main cause of side effects. The delta receptor has small addiction and insignificant analgesic effect. Kappa receptor (KOR) analgesic activity is intermediate between the former two. The polypeptide KOR agonist can play an analgesic role in the periphery without entering the center, cannot cause toxic and side effects such as respiratory depression, constipation and the like, and has lower addiction, so that the polypeptide KOR agonist has potential for treating drug addiction.
Patent CN111233974B reports a series of novel KOR agonists with excellent agonistic activity, including compounds of the formula I-1 (chemical name: [ (S) -1- (D-phenylalanyl-D-leucyl-D-lysyl) pyrrolidin-3-yl ] borate). 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) pyrrolidin-3-yl ] borate) which are most difficult to separate, the polarity difference between the isomer impurities and the main component is extremely small, the physical and chemical properties are extremely similar, the chiral isomer impurities are extremely difficult to separate effectively in HPLC detection, great challenges are brought to detection and analysis, and samples which are required by clinic cannot be effectively monitored and guided to be produced. Therefore, the problem of monitoring these impurities, particularly chiral diastereoisomeric impurities, to ensure 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 invention is to provide a method for detecting diastereoisomeric impurities of chiral polypeptide drugs, which can effectively detect diastereoisomeric impurities which cannot be detected under conventional detection conditions through reasonable optimization of chromatographic conditions, thereby providing an effective monitoring means for guaranteeing the quality level of the polypeptide drugs.
In order to achieve the above purpose, the invention provides a method for detecting diastereoisomeric impurities of chiral polypeptides drugs, which comprises the following steps:
s1) dissolving a polypeptide drug sample, and filtering to prepare a detection sample solution;
s2) adopting a reversed phase chromatography method, taking octadecylsilane chemically bonded silica as a stationary phase, taking inorganic salt aqueous solution as a phase A and methanol or acetonitrile as a phase B, and carrying out gradient elution on a detection sample solution to realize separation of diastereoisomeric impurities and main components.
The invention adopts HPLC reversed phase chromatography to detect the diastereoisomeric impurity, provides an effective monitoring means, can accurately carry out qualitative and quantitative analysis of the medicine, and effectively ensures the quality of the chiral polypeptide medicine.
In the invention, the polypeptide medicine is [ (S) -1- (D-phenylalanyl-D-leucyl-D-lysyl) pyrrolidin-3-yl ] borate hydrochloride crude product (shown in the formula I-1 above), and contains [ (R) -1- (D-phenylalanyl-D-leucyl-D-lysyl) pyrrolidin-3-yl ] borate hydrochloride impurity (shown in the formula I-2 above).
The polypeptide medicine is a polypeptide compound obtained through a solid-phase or liquid-phase synthesis method.
Preferably, the solvent for dissolving the polypeptide drug sample is a polar hydrophilic solvent. Further preferred is one or more of water, methanol, ethanol, acetonitrile. More preferably methanol, water or a mixture of both.
The solubility of the polypeptide drug sample in the polar hydrophilic solvent is from dissolution to very dissolution.
In the invention, the concentration of the detection sample solution is preferably less than or equal to 5mg/ml. More preferably 0.5 to 1mg/ml.
The invention adopts a reversed phase chromatography to carry out gradient elution on the detection sample solution, thereby realizing the separation of diastereoisomeric impurities and main components.
The reversed phase chromatographic column of the reversed phase chromatography is preferably a reversed phase C18 liquid chromatographic column or other column with equivalent column efficiency. More preferably YMC-Pack ODS-AQ or AerisTM PEPTIDE XB-C18 column. Further preferred are YMC-Pack ODS-AQ, 4.6X105 mm,3 μm or AerisTM PEPTIDE XB-C18 liquid chromatography columns (4.6X105 mm,3.6 μm).
In the present invention, the phase a is preferably an aqueous solution of ammonium sulfate.
The concentration of the ammonium sulfate aqueous solution is preferably 5 mM-20 mM; further preferably 5mM.
The pH of the aqueous ammonium sulfate solution is preferably 1.5 to 2.5, and more preferably 2.0 to 2.5.
The invention preferably uses sulfuric acid to adjust the pH of the aqueous solution of ammonium sulfate.
In the present invention, the phase B is preferably methanol or acetonitrile, more preferably methanol.
Preferably, the conditions of the reverse phase chromatography are as follows:
the flow rate is 0.8-1.2 ml/min; the column temperature is 30-45 ℃; the detection wavelength is 220nm; the sample injection amount is 10-100 mu l.
The flow rate is further preferably 0.8, 1.0 or 1.2ml/min; most preferably 1.0ml/min.
The column temperature is further preferably 35 to 45℃and most preferably 45 ℃.
The detection wavelength is preferably 220nm.
The sample amount is more preferably 10 to 20. Mu.l.
In some embodiments of the invention, the chromatographic conditions are as shown in table 1:
table 1 chromatographic condition examples of the present invention
Preferably, the gradient elution specifically comprises:
maintaining the phase A for 0-10min to 75%, reducing the phase A from 75% to 70% between 10.01-25 min, reducing the phase A from 70% to 68% between 25.01 min-35 min, reducing the phase A from 68% to 50% between 35.01 min-55 min, maintaining the phase A to 65min, and recovering the mobile phase A from 75% between 65.01 min-75 min for chromatographic column balance.
The invention develops a method for detecting diastereoisomers which are exclusively chiral polypeptides through a large number of experiments. Experimental results show that the diastereoisomeric impurity is positioned before the main component medicament in the detection map obtained according to the detection method, and the retention time (RRT) of the diastereoisomeric impurity relative to the chiral polypeptide medicament is about 0.96.
Compared with the prior art, the invention provides a method for detecting diastereoisomeric impurities of chiral polypeptide drugs, which comprises the following steps: s1) dissolving a polypeptide drug sample, and filtering to prepare a detection sample solution; s2) adopting a reversed phase chromatography method, taking octadecylsilane chemically bonded silica as a stationary phase, taking inorganic salt aqueous solution as a phase A and methanol or acetonitrile as a phase B, and carrying out gradient elution on a detection sample solution to realize separation of diastereoisomeric impurities and main components. The reverse phase chromatographic analysis method mainly solves the problems that diastereoisomeric impurities cannot be effectively detected by the conventional analysis method, and the qualitative and quantitative detection of the medicine cannot be accurately carried out, and provides detection means guarantee for effectively controlling related substances of products and monitoring the quality level of the products.
Drawings
FIG. 1 is a conventional HPLC profile of crude polypeptide drug;
FIG. 2 is an HPLC analysis detection chart of example 1;
FIG. 3 is an HPLC analysis detection chart of example 2;
FIG. 4 is an HPLC analysis detection chart of comparative example 1;
FIG. 5 is an HPLC analysis detection chart of comparative example 2.
Detailed Description
In order to further illustrate the present invention, the following describes in detail the method for detecting diastereoisomeric impurities of chiral polypeptides provided by the present invention with reference to examples.
The crude chiral polypeptide drug in the following examples was prepared according to the method disclosed in example 2 of patent CN 111233974B.
Example 1
A method for detecting diastereomers of chiral polypeptides, comprising:
(1) Test solution: taking 50mg of chiral polypeptide medicine crude product, adding water to dissolve and dilute the crude product to prepare a solution containing about 0.5mg of chiral polypeptide medicine per 1ml, shaking the solution uniformly, and filtering the solution by using an organic filter membrane with 0.22um to obtain crude filtrate for later use.
(2) Chromatographic conditions: using YMC-Pack ODS-AQ, 4.6X105 mm,3 μm column; ammonium sulfate buffer solution (5 mmol/L ammonium sulfate, pH is regulated to 2.5 by sulfuric acid) is used as a mobile phase A, methanol is used as a mobile phase B, gradient elution is carried out according to the following table, and the flow rate is 1.0ml/min; column temperature 45 ℃; the detection wavelength is 220nm; the sample was introduced in an amount of 20. Mu.l.
(3) The test was run gradient-wise as in table 2 below:
TABLE 2 example 1 gradient elution conditions
FIG. 1 is a conventional HPLC profile of crude polypeptide drug, and it can be seen that chiral diastereomers and main peaks overlap.
The HPLC analysis and detection chart of this example is shown in FIG. 2, the main component retention time is 104.273min, and the chiral diastereomeric impurity retention time is 98.000min. The method comprises the following steps of: the diastereomeric impurity of this example was effectively separated from the main component with a degree of separation of 1.46.
Example 2
A method for detecting diastereomers of chiral polypeptides, comprising:
(1) Test solution: taking 50mg of chiral polypeptide medicine crude product, adding water to dissolve and dilute the crude product to prepare a solution containing about 0.5mg of chiral polypeptide medicine per 1ml, shaking the solution uniformly, and filtering the solution by using an organic filter membrane with 0.22um to obtain crude filtrate for later use.
(2) Chromatographic conditions: using a AerisTM PEPTIDE XB-C18 liquid chromatography column (4.6X105 mm,3.6 μm); ammonium sulfate buffer solution (5 mmol/L ammonium sulfate, pH is regulated to 2.2 by sulfuric acid) is used as a mobile phase A, methanol is used as a mobile phase B, gradient elution is carried out according to the following table, and the flow rate is 1.0ml/min; column temperature 45 ℃; the detection wavelength is 220nm; the sample was introduced in an amount of 20. Mu.l.
(3) The test was run gradient-wise as shown in table 3 below:
TABLE 3 gradient elution conditions example 2
The method comprises the following steps of: the diastereomeric impurities of this example were completely and effectively separated from the main component with a degree of separation of 1.45.
HPLC analysis detection patterns are shown in figure 3.
Comparative example 1
A method for detecting diastereomers of chiral polypeptides, comprising:
(1) Test solution: taking 50mg of chiral polypeptide medicine crude product, adding water to dissolve and dilute the crude product to prepare a solution containing about 0.5mg of chiral polypeptide medicine per 1ml, shaking the solution uniformly, and filtering the solution by using an organic filter membrane with 0.22um to obtain crude filtrate for later use.
(2) Chromatographic conditions: AQ-C18 liquid chromatography column (4.6X105 mm,5 μm) was used; ammonium chloride buffer solution (10 mmol/L ammonium sulfate, pH is regulated to 2.5 by hydrochloric acid) is used as a mobile phase A, methanol is used as a mobile phase B, gradient elution is carried out according to the following table, and the flow rate is 1.0ml/min; column temperature 40 ℃; the detection wavelength is 220nm; the sample was introduced in an amount of 10. Mu.l.
(3) The test was run gradient according to table 4 below:
table 4 comparative example 1 gradient elution conditions
The method comprises the following steps of: the detection method of the comparative example can detect diastereoisomeric impurities, and obviously, under the condition that the elution gradient is not changed and only the mobile phase, the chromatographic column and other chromatographic conditions are replaced, the diastereoisomeric impurities and the main components cannot be completely and effectively separated, the separation degree is only 1.0, and the requirements of accurate integration and quantification cannot be met.
HPLC analysis detection patterns are shown in figure 4.
Comparative example 2
A method for detecting diastereomers of chiral polypeptides, comprising:
(1) Test solution: taking 50mg of chiral polypeptide medicine crude product, adding water to dissolve and dilute the crude product to prepare a solution containing about 0.5mg of chiral polypeptide medicine per 1ml, shaking the solution uniformly, and filtering the solution by using an organic filter membrane with 0.22um to obtain crude filtrate for later use.
(2) Chromatographic conditions: a DAISOPAK SP-ODS-P (C18) liquid chromatography column (4.6X105 mm,5 μm) was used; ammonium sulfate buffer solution (5 mmol/L ammonium sulfate, pH is regulated to 2.5 by sulfuric acid) is used as a mobile phase A, methanol is used as a mobile phase B, gradient elution is carried out according to the following table, and the flow rate is 1.2ml/min; column temperature is 30 ℃; the detection wavelength is 220nm; the sample was introduced in an amount of 20. Mu.l.
(3) The test was run gradient in Table 5 below
TABLE 5 comparative example 2 gradient elution conditions
The method comprises the following steps of: the detection method of the embodiment can detect diastereoisomeric impurities, and obviously can not completely and effectively separate the diastereoisomeric impurities from the main component under the condition that the elution gradient and the mobile phase are not changed and other chromatographic conditions such as chromatographic columns, flow rates, column temperatures and the like are only replaced, and the separation degree is only 0.7, so that the requirements of accurate integration and quantification can not be met.
HPLC analysis detection patterns are shown in figure 5.
The above description of the embodiments is only for aiding in the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that various modifications and adaptations of the invention can be made without departing from the principles of the invention and these modifications and adaptations are intended to be within the scope of the invention as defined in the following claims.
Claims (5)
1. A method for detecting diastereoisomeric impurities of chiral polypeptides comprises the following steps:
s1) dissolving a 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 ammonium sulfate aqueous solution as a phase A and methanol or acetonitrile as a phase B, and carrying out gradient elution on a detection sample solution to realize separation of diastereoisomeric impurities and main components;
the polypeptide medicine is [ (S) -1- (D-phenylalanyl-D-leucyl-D-lysyl) pyrrolidin-3-yl ] borate crude product, which contains [ (R) -1- (D-phenylalanyl-D-leucyl) pyrrolidin-3-yl ] borate impurity;
the reversed phase chromatographic column of the reversed phase chromatography is YMC-Pack ODS-AQ or AerisTM PEPTIDE XB-C18 chromatographic column;
the conditions of the reverse phase chromatography are:
the flow rate is 1.0ml/min; column temperature is 35-45 ℃; the detection wavelength is 220nm; the sample injection amount is 10-20 mu l;
the gradient elution specifically comprises the following steps:
maintaining the phase A for 0-10min to 75%, reducing the phase A from 75% to 70% between 10.01-25 min, reducing the phase A from 70% to 68% between 25.01 min-35 min, reducing the phase A from 68% to 50% between 35.01 min-55 min, maintaining the phase A to 65min, and recovering the mobile phase A from 75% between 65.01 min-75 min for chromatographic column balance.
2. The method of claim 1, wherein the solvent in which the polypeptide drug sample is dissolved is a polar hydrophilic solvent.
3. The method of claim 2, wherein the solvent in which the polypeptide drug sample is dissolved is selected from one or more of water, methanol, ethanol, acetonitrile.
4. The method according to claim 1, wherein the concentration of the detection sample solution is 5mg/ml or less.
5. The method according to claim 1, wherein the concentration of the aqueous ammonium sulfate solution is 5 mM-20 mM; the pH value is 1.5-2.5.
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| EP1664108A1 (en) * | 2003-08-21 | 2006-06-07 | Novo Nordisk A/S | Separation of polypeptides comprising a racemized amino acid |
| CN106290601A (en) * | 2015-06-29 | 2017-01-04 | 深圳翰宇药业股份有限公司 | Aminoacid racemization and the detection method of diastereomer impurity in polypeptide drugs |
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