Analysis method for determining impurity content of liraglutide

Abstract

The invention relates to the technical field of pharmaceutical analysis, in particular to an analysis method for determining the impurity content of liraglutide. According to the method, the liraglutide and the impurities thereof are subjected to enzyme digestion by adopting V8 protease, and fragments subjected to enzyme digestion are separated by adopting a high performance liquid chromatography method, so that the content of the impurity [D-Glu3]-liraglutide in the liraglutide is indirectly measured. The method disclosed by the invention is high in specificity, and the content of the impurity [D-Glu3]-liraglutide can be rapidly measured, so that an effective guarantee is provided for quality control of a liraglutide product.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis, and in particular to an analytical method for determining the impurity content of liraglutide. Background Art

[0002] Liraglutide, also known as Liraglutide in English, is a new long-acting glucagon-like peptide-1 (GLP-1) analog developed and produced by Novo Nordisk of Denmark for the treatment of type II diabetes. Liraglutide has the effects of lowering blood sugar, losing weight, and reducing the risk of major adverse cardiovascular events in diabetic patients. It was first approved for marketing in the European Union in September 2007.

[0003] Liraglutide replaces Lys34 of human GLP-1 (7-36) with Arg, and connects a 16-carbon palmitic fatty acid derivative to the side chain of Lys26. Chemically synthesized liraglutide is a 31-mer peptide with a molecular formula of C 172 H 265 N 43 O 51 , molecular weight is 3751.20g / mol. Appearance is white to off-white powder or loose block. The peptide sequence is:

[0004] H-His1-Ala2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-Ala18- Ala19-Lys20(N-ε-(γ-Glu(N-α-Pal))-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-OH.

[0005] Peptides are mainly composed of amino acids (including natural amino acids and unnatural amino acids), which makes peptide drugs different from general drugs in terms of quality research. Due to the uniqueness of the synthesis process and structure, the sources of process impurities in synthetic peptide APIs are different from those of general chemical drugs. The possible process impurities include: missing peptides, misconnected peptides, broken peptides, deamidated peptides, by-products formed by incomplete deprotection of amino acid side chains, oxidized peptides, disulfide bond exchange products, diastereoisomers of peptides, etc. The currently disclosed methods for detecting liraglutide-related substances have poor separation capabilities and are unable to accurately separate and quantify specific impurities, especially diastereoisomers of impurities [D-Glu 3]-Liraglutide, H-His1-Ala2-D-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Le u14-Glu15-Gly16-Gln17-Ala18-Ala19-Lys20(N-ε-(γ-Glu(N-α-Pal))-Glu21-Phe22-Ile23-Ala 24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-OH. Because of its similar structure to liraglutide and almost no difference in physical and chemical properties, it is impossible to separate and accurately quantify it using conventional liquid chromatography methods. At present, there is no report on the analysis and detection method of specific impurities in liraglutide. In order to effectively analyze and detect the quality of drugs and ensure the safety of drug use, it is necessary to develop a convenient and effective method for detecting impurities [D-Glu 3 In order to solve the shortcomings of the prior art, the present invention provides a method for effectively separating and accurately analyzing the impurity [D-Glu 3 ]-Analytical methods for liraglutide. Summary of the invention

[0006] The purpose of the present invention is to provide an analytical method for determining the impurity content of liraglutide, aiming to solve the problem of liraglutide and its specific impurity [D-Glu 3 ]-The separation and quantitative determination of liraglutide can effectively control the quality of liraglutide products.

[0007] In order to achieve the purpose of the present invention, the present invention provides an analytical method for determining the impurity content of liraglutide, comprising the following steps: using V8 protease to enzymatically hydrolyze liraglutide and its impurity specific sites, and using high performance liquid chromatography to separate the specific enzymatic peptide segments produced by hydrolysis, and using an external standard method to accurately quantify. The V8 protease used in the present invention, also known as glutaminase, can specifically act on liraglutide, [D-Glu 3 ]-The carboxyl-terminal peptide bonds of glutamic acid at positions 3, 15, and 21 and asparagine at position 9 in the liraglutide sequence can be enzymatically hydrolyzed into the following peptide fragments.

[0008] Fragment 1: His-Ala-Glu, His-Ala-D-Glu;

[0009] Fragment 2: Gly-Thr-Phe-Thr-Ser-Asp;

[0010] Fragment 3: Val-Ser-Ser-Tyr-Leu-Glu;

[0011] Fragment 4: Gly-Gln-Ala-Ala-Lys(Pal-Gama-Glu)-Glu;

[0012] Fragment 5: Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH;

[0013] Taking advantage of the difference between the enzymatic peptide fragments (His-Ala-Glu) and the impurity peptide fragments (His-Ala-D-Glu) of liraglutide, the peptide fragments were separated by high performance liquid chromatography, thereby achieving the purpose of quantitative detection of specific impurities in liraglutide.

[0014] The method comprises the following steps of enzymatic hydrolysis: taking liraglutide raw material, adding buffer to dissolve and prepare a test sample mother solution; taking an appropriate amount of the mother solution and V8 protease solution to mix, placing in a 37°C oven for enzymatic hydrolysis, and obtaining a test sample enzymatic hydrolyzate; placing the enzymatic hydrolyzate at 37°C and drying by rotary evaporation, and then adding 0.1% TFA aqueous solution to dissolve, and obtaining the test sample.

[0015] In some embodiments of the present invention, the buffer is one of ammonium bicarbonate and phosphate, and the pH is between 7.8 and 8.0.

[0016] In some embodiments of the present invention, the ratio of the test sample to the V8 protease concentration during enzymatic hydrolysis is 12.5:1 to 25:1.

[0017] In some embodiments of the present invention, the enzymatic hydrolysis time in a 37° C. oven is 5 to 24 hours.

[0018] Whether the test sample can be completely hydrolyzed is closely related to the type and pH of the buffer, the concentration of the test sample and V8 protease, and the hydrolysis time. When the buffer is ammonium bicarbonate or phosphate, the pH is between 7.8 and 8.0; the ratio of the test sample to V8 protease concentration during hydrolysis is 12.5:1 to 25:1; and the hydrolysis time is 5 to 24 hours, the test sample can be completely hydrolyzed.

[0019] In the method, the chromatographic column used in the high performance liquid chromatography is C18, TFA aqueous solution is used as mobile phase A, TFA acetonitrile solution is used as mobile phase B, and the elution method is gradient elution.

[0020] Preferably, the particle size of the chromatographic column filler used is 4 μm, the length of the chromatographic column is 250 mm, and the inner diameter of the chromatographic column is 4.6 mm.

[0021] Preferably, the volume ratio of TFA to water in the TFA aqueous solution is 1:1000, and the volume ratio of TFA to acetonitrile in the TFA acetonitrile solution is 1:1000.

[0022] Preferably, the gradient elution program is: mobile phase B: 0%→4% (0-20 min)→4% (20-25 min)→70% (25-35 min).

[0023] In some embodiments of the present invention, the flow rate is 0.8-1.2 ml / min, the column temperature is 30-40° C., the detection wavelength is 214 nm, and the injection volume is 50 μl.

[0024] The positive and progressive effects of the present invention are:

[0025] The present invention uses an enzymatic method to hydrolyze liraglutide and its impurities into different peptide segments, and uses high performance liquid chromatography to determine the different enzymatic peptide segments, thereby indirectly achieving the determination of the impurity [D-Glu 3 ] The determination of liraglutide solves the problem of [D-Glu 3 ] Technical issues of the liraglutide method. This method has the characteristics of strong specificity and fast analysis speed, which provides effective guarantee for the quality control of liraglutide products. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the HPLC spectrum of the H-His-Ala-Glu reference substance solution in Example 1;

[0027] Figure 2 is the HPLC spectrum of the H-His-Ala-D-Glu reference substance solution in Example 1;

[0028] Figure 3 HPLC spectrum of the test solution in Example 1; DETAILED DESCRIPTION

[0029] The analytical method of the present invention can be implemented as follows. The described embodiments are only for the purpose of illustrating and explaining the present invention and do not constitute the sole limitation of the present invention:

[0030] The sample preparation method, chromatographic conditions, and impurity calculation method in the embodiments of the present invention are as follows:

[0031] 1) Sample preparation:

[0032] V8 protease solution: Take 1 tube of V8 protease (1 mg), dissolve it in water and dilute it to make a 0.5-1 mg / ml solution.

[0033] Buffer solution: Weigh 3.987 g of ammonium bicarbonate and add 1 L of water to dissolve; or take 1.15 g of anhydrous disodium hydrogen phosphate and 0.34 g of sodium dihydrogen phosphate, place in a beaker, add 500 ml of water to dissolve, and adjust the pH to 7.8-8.0 with sodium hydroxide solution.

[0034] Test solution: Take liraglutide raw material, add buffer to dissolve to make 15 mg / ml solution as test sample mother solution; measure 170 μl of mother solution, add 200 μl of V8 protease solution and mix well, place in a 37°C oven for enzymolysis for 5 to 24 hours to obtain test sample hydrolyzate; place the hydrolyzate at 37°C and dry by rotary evaporation, then add 1.5 ml of 0.1% TFA aqueous solution to dissolve, and obtain.

[0035] H-His-Ala-Glu reference solution: Dissolve the H-His-Ala-Glu reference in 0.1% TFA aqueous solution to make a 0.5 mg / ml solution;

[0036] H-His-Ala-D-Glu reference solution: Dissolve the H-His-Ala-D-Glu reference in 0.1% TFA aqueous solution to make a 0.025 mg / ml solution;

[0037] 2) Chromatographic conditions

[0038] Instrument: High performance liquid chromatography-ultraviolet detector

[0039] Chromatographic column: Phenomenex Synergi Hydro C18; column particle size: 4 μm; column length: 250 mm; column inner diameter: 4.6 mm;

[0040] Mobile phase A: Mobile phase A: 0.1% TFA in water;

[0041] Mobile phase B: 0.1% TFA acetonitrile solution;

[0042] Flow rate: 0.8~1.2ml / min;

[0043] Column temperature: 30℃~40℃;

[0044] Detection wavelength: 214nm;

[0045] Injection volume: 40 μl

[0046] Elution gradient:

[0047] Time (min) Mobile phase A (%) Mobile phase B (%) 0 100 0 20 96 4 25 96 4 25.01 30 70 35 30 70 35.01 100 0 45 100 0

[0048] 3) Calculation of impurities

[0049] According to the external standard method, the calculation formula is as follows:

[0050] Impurity % = (Sample A / Sample C) / (Pair A / Pair C)*100%

[0051] A pair: peak area of ​​H-His-Ala-D-Glu in H-His-Ala-D-Glu reference solution;

[0052] Sample A: Peak area of ​​H-His-Ala-D-Glu in the test solution;

[0053] C pair: concentration of H-His-Ala-D-Glu reference solution;

[0054] Sample C: test solution concentration*H-His-Ala-D-Glu molecular weight / liraglutide molecular weight;

[0055] Example 1

[0056] 1) Sample preparation:

[0057] V8 protease solution concentration: 1 mg / ml;

[0058] Buffer solution: weigh 3.987 g of ammonium bicarbonate and dissolve in 1 L of water;

[0059] The enzymatic hydrolysis time of the test solution is 24h.

[0060] 2) Chromatographic conditions

[0061] Flow rate: 1.0ml / min;

[0062] Column temperature: 30°C;

[0063] Take 50 μl of H-His-Ala-Glu reference solution, H-His-Ala-D-Glu reference solution and test solution, inject into liquid chromatograph and record chromatogram. The HPLC spectra of H-His-Ala-Glu reference solution, H-His-Ala-D-Glu reference solution and test solution are as follows: Figure 1 , 2 , as shown in Figure 3.

[0064] Table 1 Example 1 Results Statistics

[0065]

[0066] Experimental conclusion: The above results show that H-His-Ala-Glu and H-His-Ala-D-Glu can be effectively separated. 3 ]-The liraglutide content is 0.35%.

[0067] Example 2

[0068] 1) Sample preparation:

[0069] V8 protease solution concentration: 1 mg / ml;

[0070] Buffer solution: Take 1.15g of anhydrous disodium hydrogen phosphate and 0.34g of sodium dihydrogen phosphate, place in a beaker, add 500ml of water to dissolve, and adjust the pH to 7.8 with sodium hydroxide solution;

[0071] Enzymatic hydrolysis time of test solution: 5h.

[0072] 2) Chromatographic conditions

[0073] Flow rate: 0.8ml / min;

[0074] Column temperature: 40°C;

[0075] Take 50 μl of H-His-Ala-Glu reference solution, H-His-Ala-D-Glu reference solution and test solution, inject into liquid chromatograph and record the chromatogram. 3 ]-The liraglutide content is 0.32%.

[0076] Example 3

[0077] 1) Sample preparation:

[0078] V8 protease solution concentration: 0.5 mg / ml;

[0079] Buffer solution: Take 1.15g of anhydrous disodium hydrogen phosphate and 0.34g of sodium dihydrogen phosphate, place in a beaker, add 500ml of water to dissolve, and adjust the pH to 8.0 with sodium hydroxide solution;

[0080] Enzymatic hydrolysis time of test solution: 24h.

[0081] 2) Chromatographic conditions

[0082] Flow rate: 1.2 ml / min;

[0083] Column temperature: 30°C;

[0084] Take 50 μl of H-His-Ala-Glu reference solution, H-His-Ala-D-Glu reference solution and test solution, inject into liquid chromatograph and record the chromatogram. 3 ]-The liraglutide content is 0.35%.

[0085] Example 4

[0086] 1) Sample preparation:

[0087] V8 protease solution concentration: 0.5 mg / ml;

[0088] Buffer solution: weigh 3.987 g of ammonium bicarbonate and dissolve in 1 L of water;

[0089] Enzymatic hydrolysis time of test solution: 5h.

[0090] 2) Chromatographic conditions

[0091] Flow rate: 1.0ml / min;

[0092] Column temperature: 40°C;

[0093] Take 50 μl of H-His-Ala-Glu reference solution, H-His-Ala-D-Glu reference solution and test solution, inject into liquid chromatograph and record the chromatogram. 3 ]The liraglutide content is 0.34%.

Claims

1. An analytical method for determining the impurity content of liraglutide, characterized in that: V8 protease was used to treat liraglutide and its impurities [D-Glu 3 ]-The specific sites of liraglutide were enzymatically hydrolyzed, and the specific enzymatic peptide fragments produced by hydrolysis were separated by high performance liquid chromatography, and the impurity content was determined by the external standard method.

2. The method according to claim 1, characterized in that The enzymatic hydrolysis steps are as follows: taking liraglutide raw material, adding buffer to dissolve to prepare a test sample mother solution; taking an appropriate amount of the mother solution and V8 protease solution to mix, placing in a 37°C oven for enzymatic hydrolysis to obtain a test sample enzymatic hydrolyzate; placing the enzymatic hydrolyzate at 37°C and drying by rotary evaporation, and then adding 0.1% TFA aqueous solution to dissolve, thereby obtaining the test sample.

3. The method according to claim 2, characterized in that The buffer is one of ammonium bicarbonate and phosphate.

4. The method according to claim 2, characterized in that: The pH of the buffer solution is between 7.8 and 8.

0.

5. The method according to claim 2, characterized in that: During enzymatic hydrolysis, the ratio of the test sample to the V8 protease concentration was 12.5:1 to 25:

1.

6. The method according to claim 2, characterized in that The enzymatic hydrolysis time in a 37°C oven is 5 to 24 hours.

7. The method according to claim 1, characterized in that: In the high performance liquid chromatography method, the chromatographic column used was C18, TFA aqueous solution was used as mobile phase A, TFA acetonitrile solution was used as mobile phase B, and the elution method was gradient elution.

8. The method according to claim 7, characterized in that: The particle size of the chromatographic column filler used is 4 μm, the length of the chromatographic column is 250 mm, and the inner diameter of the chromatographic column is 4.6 mm.

9. The method according to claim 7, characterized in that: The volume ratio of TFA to water in the TFA aqueous solution is 1:1000, and the volume ratio of TFA to acetonitrile in the TFA acetonitrile solution is 1:1000.

10. The method according to claim 7, characterized in that The gradient elution program was: mobile phase B: 0% → 4% (0-20 min) → 4% (20-25 min) → 70% (25-35 min).

11. The method according to claim 1 or 7, characterized in that: In the high performance liquid chromatography method, the flow rate is 0.8-1.2 ml / min, the column temperature is 30-40° C., the detection wavelength is 214 nm, and the injection volume is 50 μl.

12. Use of the method according to any one of claims 1 to 11 in the detection of liraglutide impurities.

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