CN116183752A - Liquid chromatography detection method for polypeptide impurities - Google Patents
Liquid chromatography detection method for polypeptide impurities Download PDFInfo
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- CN116183752A CN116183752A CN202211710398.9A CN202211710398A CN116183752A CN 116183752 A CN116183752 A CN 116183752A CN 202211710398 A CN202211710398 A CN 202211710398A CN 116183752 A CN116183752 A CN 116183752A
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- liquid chromatography
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- polypeptide impurities
- liraglutide
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- 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
- G01N30/02—Column chromatography
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- 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
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
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- G—PHYSICS
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- 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
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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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
- G01N30/02—Column chromatography
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Abstract
The invention relates to a liquid chromatography detection method of polypeptide impurities, belonging to the technical field of pharmaceutical chemistry. The invention provides a liquid chromatography detection method of polypeptide impurities, which comprises the following steps: the method comprises the steps of taking phosphate buffer solution as a mobile phase A, taking a mixed solution of an organic reagent and water as a mobile phase B, taking a C18 chromatographic column as a stationary phase, taking a detector as an ultraviolet detector, and adopting a high performance liquid chromatography gradient to elute polypeptide impurities. The method can effectively detect the impurities which are difficult to separate in the polypeptide impurities, and can be used as an orthogonal method for detecting related substances of the polypeptide.
Description
Technical Field
The invention belongs to the technical field of pharmaceutical chemistry, and particularly relates to a liquid chromatography detection method of polypeptide impurities and application thereof.
Background
The synthetic polypeptide drugs have a complex impurity profile, including impurities introduced from the starting materials, process-related impurities, and impurities resulting from degradation, among others. In order to achieve effective detection of each potential impurity, detection methods employing a variety of different principles are generally required, such as reverse phase HPLC, ion exchange HPLC, size exclusion HPLC, and the like. The polypeptide impurities mainly comprise diastereoisomers, missing peptides, inserted peptides, broken peptides, bad peptides, substituted peptides, side chain groups modified, disulfide bond modified, deamidated impurities, amino acetylation and other impurities, and due to the complexity of the polypeptide impurities, a new impurity is generated when a certain amino acid is changed, theoretical impurities are many, and the difference between the impurity and a main component is small. For polypeptides with a high number of amino acids, it is difficult to control all impurities by one method, and certain special impurities need to be controlled by a special method.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provide a liquid chromatography detection method of polypeptide impurities and application thereof. The method provides a special impurity separation method aiming at the structural characteristics of polypeptide impurities, and can be used for separating impurities co-eluted with main components under a common acidic system.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a liquid chromatography detection method of polypeptide impurities is characterized in that: the method comprises the steps of taking phosphate buffer solution as a mobile phase A, taking a mixed solution of an organic reagent and water as a mobile phase B, taking a C18 chromatographic column as a stationary phase, taking a detector as an ultraviolet detector, and adopting a high performance liquid chromatography gradient to elute polypeptide impurities.
The invention relates to a liquid chromatography detection method of polypeptide impurities, which further adopts the preferable technical scheme that: the chromatography column comprises a C18 reverse phase chromatography column or a C8 reverse phase chromatography column, preferably a C18 reverse phase chromatography column.
The invention relates to a liquid chromatography detection method of polypeptide impurities, which further adopts the preferable technical scheme that: the packed particle size of the chromatographic column is 1.7-10 um, and the preferred packed particle size is 3um.
The invention relates to a liquid chromatography detection method of polypeptide impurities, which further adopts the preferable technical scheme that: the phosphate buffer solution comprises at least one of sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate and dipotassium hydrogen phosphate, preferably the phosphate buffer solution is sodium dihydrogen phosphate aqueous solution, the pH range is 7.0-10.0, preferably the pH range is 9.0, and the organic reagent is at least one of acetonitrile, methanol, ethanol and isopropanol, preferably acetonitrile.
The invention relates to a liquid chromatography detection method of polypeptide impurities, which further adopts the preferable technical scheme that: the volume ratio of the organic reagent to the water in the mixed solution of the organic reagent and the water is 3: 7-9: 1, the volume ratio of the organic reagent to water is preferably 7:3.
the invention relates to a liquid chromatography detection method of polypeptide impurities, which further adopts the preferable technical scheme that: the gradient range of the mobile phase B is 20-90%.
The invention relates to a liquid chromatography detection method of polypeptide impurities, which further adopts the preferable technical scheme that: the column temperature is 25-45 ℃ in the gradient elution process, preferably 30 ℃, the flow rate is 0.4-1.5mL/min, and the flow rate is preferably 1.0mL/min.
The invention relates to a liquid chromatography detection method of polypeptide impurities, which further adopts the preferable technical scheme that: the wavelength of the ultraviolet detector is 210nm.
The liquid chromatography detection method of the polypeptide impurities is applied to detection and identification of the polypeptide impurities generated in the process of drug production or storage. In said application, said impurity is selected from D- [ Phe ]] 22 Liraglutide, D- [ Gln ]] 17 Liraglutide, des- [ Thr] 5 Liraglutide, des- [ Ala ]] 24 Liraglutide and Des- [ Gly ]] 29 Liraglutide.
Compared with the prior art, the invention has the following beneficial effects:
the method can effectively detect the impurities which are difficult to separate in the polypeptide impurities, and can be used as an orthogonal method for detecting related substances of the polypeptide. The method for separating the impurities which are not easy to separate in the alkaline system can be used as a complement of the method of an acidic system and can also be used as an orthogonal method of a main method, and has good application prospect.
Drawings
FIG. 1 is a schematic illustration of example 1 using the present method to detect a polypeptide comprising D- [ Phe] 22 Liraglutide, D- [ Gln ]] 17 Liraglutide, des- [ Thr] 5 Liraglutide, des- [ Ala ]] 24 Liraglutide and Des- [ Gly ]] 29 Resolution chromatograms of 5 impurities of liraglutide.
Detailed Description
The following detailed description of the present invention will be made in order to make the above-mentioned objects, features and advantages of the present invention more obvious, but the present invention is not limited thereto.
Example 1, a liquid chromatography detection method for polypeptide impurities,
the method uses phosphoric acid buffer solution as a mobile phase A, uses a mixed solution of an organic reagent and water as a mobile phase B, uses a C18 chromatographic column as a stationary phase, uses an ultraviolet detector as a detector, and adopts a high performance liquid chromatography gradient to elute polypeptide impurities.
The chromatographic column is a C18 reversed phase chromatographic column;
the packed particle size of the chromatographic column is 1.7um;
the phosphate buffer solution is sodium dihydrogen phosphate or disodium hydrogen phosphate, the pH value is 7.0, and the organic reagent is acetonitrile or methanol;
the volume ratio of the organic reagent to the water in the mixed solution of the organic reagent and the water is 3:7, preparing a base material;
the gradient range of the mobile phase B is 20-90%;
the column temperature is 25 ℃ and the flow rate is 0.4mL/min in the gradient elution process;
the wavelength of the ultraviolet detector is 210nm.
The liquid chromatography detection method of the polypeptide impurities is applied to detection and identification of the polypeptide impurities generated in the process of drug production or storage. The impurity is selected from D- [ Phe] 22 Liraglutide, D- [ Gln ]] 17 Liraglutide, des- [ Thr] 5 Liraglutide, des- [ Ala ]] 24 Liraglutide and Des- [ Gly ]] 29 Liraglutide.
Example 2, a liquid chromatography detection method for polypeptide impurities,
the method uses phosphoric acid buffer solution as a mobile phase A, uses a mixed solution of an organic reagent and water as a mobile phase B, uses a C18 chromatographic column as a stationary phase, uses an ultraviolet detector as a detector, and adopts a high performance liquid chromatography gradient to elute polypeptide impurities.
The chromatographic column is a C8 reversed phase chromatographic column;
the packed particle size of the chromatographic column is 10um;
the phosphate buffer solution is monopotassium phosphate or dipotassium phosphate, the pH value is 10.0, and the organic reagent is ethanol or isopropanol;
the volume ratio of the organic reagent to the water in the mixed solution of the organic reagent and the water is 9:1, a step of;
the gradient range of the mobile phase B is 20-90%;
the column temperature is 45 ℃ and the flow rate is 1.5mL/min in the gradient elution process;
the wavelength of the ultraviolet detector is 210nm.
The liquid chromatography detection method of the polypeptide impurities is applied to detection and identification of the polypeptide impurities generated in the process of drug production or storage. The impurity is selected from D- [ Phe] 22 Liraglutide, D- [ Gln ]] 17 Liraglutide, des- [ Thr] 5 Liraglutide, des- [ Ala ]] 24 Liraglutide and Des- [ Gly ]] 29 Liraglutide.
Example 3, detection experiment:
instrument: siemens flying U3000 high performance liquid chromatograph
Chromatographic column: a C18 chromatographic column;
detection wavelength: 210nm;
flow rate: 1.0ml/min;
column temperature: 30 ℃;
sample: liraglutide split solution (containing liraglutide: 0.5mg/ml, Z28:5 [ mu ] g/ml, Z2:5 [ mu ] g/ml, Z57:5 [ mu ] g/ml, Z48:5 [ mu ] g/ml, Z73:5 [ mu ] g/ml)
Sample injection amount: 40 μl;
mobile phase a:50mmol of sodium dihydrogen phosphate to 1L of water, and adjusting the pH to 9.0 by using a NaOH solution;
mobile phase B: acetonitrile: water=70:30%v/v)；
Acquisition time: 60min
Elution conditions: table 1 below
TABLE 1 gradient elution
The separation effect is that as shown in figure 1, the separation degree of 5 impurities and the main component is more than 1.5, and the separation effect is good; the detection limit of the three impurities is 0.002%, and the sensitivity is good.
TABLE 2 high performance liquid chromatography detection results
Claims (10)
1. A liquid chromatography detection method of polypeptide impurities is characterized in that: the method uses phosphoric acid buffer solution as a mobile phase A, uses a mixed solution of an organic reagent and water as a mobile phase B, uses a C18 chromatographic column as a stationary phase, uses an ultraviolet detector as a detector, and adopts a high performance liquid chromatography gradient to elute polypeptide impurities.
2. The method for liquid chromatography detection of polypeptide impurities according to claim 1, wherein: the chromatography column comprises a C18 reverse phase chromatography column or a C8 reverse phase chromatography column, preferably a C18 reverse phase chromatography column.
3. The method for liquid chromatography detection of polypeptide impurities according to claim 2, wherein: the packed particle size of the chromatographic column is 1.7-10 um, and the preferred packed particle size is 3um.
4. The method for liquid chromatography detection of polypeptide impurities according to claim 1, wherein: the phosphate buffer solution comprises at least one of sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate and dipotassium hydrogen phosphate, preferably the phosphate buffer solution is sodium dihydrogen phosphate aqueous solution, the pH range is 7.0-10.0, preferably the pH range is 9.0, and the organic reagent is at least one of acetonitrile, methanol, ethanol and isopropanol, preferably acetonitrile.
5. The method for liquid chromatography detection of polypeptide impurities according to claim 1, wherein: the volume ratio of the organic reagent to the water in the mixed solution of the organic reagent and the water is 3: 7-9: 1, the volume ratio of the organic reagent to water is preferably 7:3.
6. the method for liquid chromatography detection of polypeptide impurities according to claim 1, wherein: the gradient range of the mobile phase B is 20-90%.
7. The method for liquid chromatography detection of polypeptide impurities according to claim 1, wherein: the column temperature is 25-45 ℃ in the gradient elution process, preferably 30 ℃, the flow rate is 0.4-1.5mL/min, and the flow rate is preferably 1.0mL/min.
8. The method for liquid chromatography detection of polypeptide impurities according to claim 1, wherein: the wavelength of the ultraviolet detector is 210nm.
9. Use of a liquid chromatography detection method for polypeptide impurities according to any one of claims 1-8 for detecting and identifying polypeptide impurities generated during the manufacture or storage of a pharmaceutical.
10. The use according to claim 9, wherein: the impurity is selected from D- [ Phe] 22 Liraglutide, D- [ Gln ]] 17 Liraglutide, des- [ Thr] 5 Liraglutide, des- [ Ala ]] 24 Liraglutide and Des- [ Gly ]] 29 Liraglutide.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA3018627A1 (en) * | 2016-03-23 | 2017-09-28 | Bachem Holding Ag | Purification of glucagon-like peptide 1 analogs |
| CN107290438A (en) * | 2016-03-31 | 2017-10-24 | 深圳翰宇药业股份有限公司 | A kind of HPLC analytical method of polypeptide about material |
| CN108794618A (en) * | 2018-06-25 | 2018-11-13 | 杭州诺泰澳赛诺医药技术开发有限公司 | A method of purifying Liraglutide |
| CN111208242A (en) * | 2020-03-24 | 2020-05-29 | 东莞市东阳光生物药研发有限公司 | Method for detecting GLP-1 or analog thereof by using high performance liquid chromatography |
| WO2022178737A1 (en) * | 2021-02-25 | 2022-09-01 | 杭州九源基因工程有限公司 | Treatment method for stable liraglutide pharmaceutical preparation |
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- 2022-12-29 CN CN202211710398.9A patent/CN116183752B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA3018627A1 (en) * | 2016-03-23 | 2017-09-28 | Bachem Holding Ag | Purification of glucagon-like peptide 1 analogs |
| CN107290438A (en) * | 2016-03-31 | 2017-10-24 | 深圳翰宇药业股份有限公司 | A kind of HPLC analytical method of polypeptide about material |
| CN108794618A (en) * | 2018-06-25 | 2018-11-13 | 杭州诺泰澳赛诺医药技术开发有限公司 | A method of purifying Liraglutide |
| CN111208242A (en) * | 2020-03-24 | 2020-05-29 | 东莞市东阳光生物药研发有限公司 | Method for detecting GLP-1 or analog thereof by using high performance liquid chromatography |
| WO2022178737A1 (en) * | 2021-02-25 | 2022-09-01 | 杭州九源基因工程有限公司 | Treatment method for stable liraglutide pharmaceutical preparation |
Non-Patent Citations (2)
| Title |
|---|
| JIA MENGXUAN等: "Multi-dimensional plug-and-play liquid chromatography-native ion mobility mass spectrometry method for the analysis of biotherapeutics", INTERNATIONAL JOURNAL OF MASS SPECTROMETRY., vol. 471, pages 1 - 10 * |
| 胡馨月等: "利拉鲁肽中长链脂肪酸——棕榈酰谷氨酸残留量的检测方法研究", 中国医药生物技术, vol. 17, no. 5, pages 405 - 411 * |
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