A chromatographic method for simultaneously analyzing liraglutide and its Boc-liraglutide backbone

Abstract

The present invention discloses a chromatographic method for simultaneously analyzing liraglutide and its Boc-liraglutide backbone. Specifically, a sample solution containing liraglutide and its Boc-liraglutide backbone is injected into a high-performance liquid chromatograph, and the following high-performance liquid chromatography conditions are used for detection: a bonded silica gel chromatographic column; mobile phase A is an aqueous solution of ammonium dihydrogen phosphate at 0.05 mol / mL - 0.15 mol / mL, and mobile phase B is acetonitrile; among them, the volume ratio of mobile phase B is 15% → 95% for gradient elution. This analysis method has the characteristics of fast analysis speed, accuracy, simplicity, and rapidity, and can play a good role in quality control and analysis and purification during the production process of liraglutide, and has good specificity, high sensitivity, and low cost, etc.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and pertains to the establishment of quality standards and the development of detection methods, and relates to a chromatographic method for simultaneously analyzing liraglutide and its Boc-liraglutide backbone. Background Art

[0002] Diabetes is a highly prevalent disease, and the global incidence is continuously increasing. According to the data released by the World Health Organization: the number of patients in 2000 was 175 million, in 2010 it was 239 million, it is expected to exceed 300 million in 2025, and the number of patients will be close to 600 million in 2035. The global diabetes incidence is growing rapidly. The number of diabetic patients in China accounts for 1 / 4 of the world, and the prevalence rate has reached as high as 11.6%, ranking first in the world. Diabetes involves all life systems of the human body, seriously affecting people's labor and life, and threatening human life safety.

[0003] Liraglutide is an analogue of the incretin hormone peptide secreted by intestinal cells, with high homology, and thus almost has all the physiological functions of the endogenous substance. It can be used to treat diabetes and its cardiovascular complications. It is a long-acting glucagon-like peptide-1 analogue developed by Novo Nordisk A / S in Denmark. As a new generation of hypoglycemic drug based on incretin, it can safely and effectively lower blood sugar and may protect against various cardiovascular risk factors. The liraglutide injection is administered subcutaneously once a day to provide 24-hour blood sugar control. Its pharmacokinetic properties are not affected by gender or age. Liraglutide is definitely a revolutionary drug in the treatment field of type 2 diabetes. Compared with metformin or sulfonylurea drugs, it has good efficacy, small side effects, and does not produce immune reactions. The sequence of liraglutide is H2N-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-COOH, and a palmitic acid and a glutamic acid are connected to the Lys at the 26th position. The market demand for liraglutide is large, and there are many synthesis methods. The genetic engineering method has high technical difficulty, is prone to produce by-products and other impurities, but has a stable source, high yield, and high biological activity. Therefore, it is a better choice to produce by fermentation and then modify the structure of the product, thus putting forward higher requirements for the detection methods of liraglutide and its Boc-liraglutide backbone.

[0004] In the process of obtaining liraglutide, the main chain of Boc-liraglutide is an important intermediate, and its content detection and control are essential processes. The sequence of the main chain of Boc-liraglutide is H2N-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-COOH, and there is a BocHN-protecting group on Lys at the 26th position, preparing for the next structural modification. Therefore, it is necessary to establish an analytical method for liraglutide and its main chain of Boc-liraglutide in the prior art to solve the above problems. Summary of the Invention

[0005] In view of the above technical needs, the present invention discloses a chromatographic method for simultaneously analyzing liraglutide and its main chain of Boc-liraglutide, aiming to provide a quality control method for liraglutide with good stability, good reproducibility, high sensitivity, simple operation, accuracy and rapidity.

[0006] The present invention provides an RP-HPLC chromatographic method for simultaneously analyzing liraglutide and its main chain of Boc-liraglutide, which is characterized in that a sample solution containing liraglutide and the main chain of Boc-liraglutide is injected into a high-performance liquid chromatograph, and the following high-performance liquid chromatographic conditions are used for detection;

[0007] Bonded silica gel chromatographic column;

[0008] Mobile phase: Mobile phase A is an aqueous solution of ammonium dihydrogen phosphate at 0.05 mol / mL - 0.15 mol / mL,

[0009] Mobile phase B is acetonitrile;

[0010] Among them, the volume ratio of mobile phase B is 15% → 95% for gradient elution.

[0011] In another preferred example, mobile phase A is an aqueous solution of ammonium dihydrogen phosphate at 0.1 mol / mL.

[0012] In another preferred example, the volume ratio of mobile phase B is 25% → 90%.

[0013] In another preferred example, the bonding group is selected from: C8, C18.

[0014] In another preferred example, the bonded silica gel chromatographic column is an octadecylsilane-bonded silica gel C8 column (4.6×250 mm).

[0015] In another preferred example, the chromatographic conditions further include:

[0016] Sample injection volume: 10 - 100 μL.

[0017] In another preferred example, the sample injection volume is 20 - 60 μL, preferably 50 μL.

[0018] In another preferred example, the chromatographic conditions further include:

[0019] Detection wavelength: 210 - 230 nm;

[0020] Flow rate: 0.5 - 1.0 mL / min;

[0021] Column temperature: 30 - 40 °C.

[0022] In another preferred example, the pH of the aqueous ammonium dihydrogen phosphate solution of mobile phase A is 3.5 - 4.0.

[0023] In another preferred example, the pH of the aqueous ammonium dihydrogen phosphate solution of mobile phase A is 3.7.

[0024] In another preferred example, the method is a quantitative, qualitative, and / or impurity detection method.

[0025] In another preferred example, the method is not an in vitro and / or auxiliary method.

[0026] In another preferred example, the method is a non-diagnostic method.

[0027] In another preferred example, the sample solution containing liraglutide and its Boc-liraglutide backbone is prepared by the following steps:

[0028] (s1) Fermenting recombinant bacteria to obtain bacterial cells,

[0029] (s2) Disrupting, centrifuging, and dissolving the bacterial cells to obtain macromolecular proteins,

[0030] (s3) Adding protease to cleave the macromolecular proteins to obtain the Boc-liraglutide backbone,

[0031] (s4) Chemically modifying the Boc-liraglutide backbone to obtain a sample solution containing liraglutide and its Boc-liraglutide backbone.

[0032] In another preferred example, the method further includes the step:

[0033] (s5) Preparation of the standard solution: Mixing a liraglutide standard solution with a known concentration and a Boc-liraglutide backbone solution obtained through purification to obtain a standard solution with a known concentration.

[0034] In another preferred example, in the step (s5), the purities of liraglutide and its Boc-liraglutide main chain reference substances are both above 98%.

[0035] In another preferred example, in the step (s5), the concentrations of the liraglutide and its Boc-liraglutide main chain reference substance solutions are 0.8 mg / mL and 0.5 mg / mL respectively.

[0036] In another preferred example, the injection concentration of the sample solution is 0.05 - 1.5 mg / mL.

[0037] In another preferred example, for the gradient elution, the elution time and the proportion of mobile phase B are as follows: from 25% → 60% in 0 - 20 min, from 60% → 90% in 20 - 35 min, from 90% → 25% again in 35 - 40 min, and then running at 25% for 10 min.

[0038] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. Description of the Drawings

[0039] Figure 1 It is the standard curve of the Boc-liraglutide main chain.

[0040] Figure 2 It is the standard curve of liraglutide.

[0041] Figure 3 It is the HPLC chromatogram of the liraglutide and its Boc-liraglutide main chain reference substances.

[0042] Figure 4 It is the HPLC chromatogram of the liraglutide and its Boc-liraglutide main chain sample solutions.

[0043] Figure 5 It is the HPLC chromatogram of the liraglutide and its Boc-liraglutide main chain sample solutions with mobile phase A being 0.05% trifluoroacetic acid aqueous solution.

[0044] Figure 6 It is the HPLC chromatogram of the liraglutide and its Boc-liraglutide main chain sample solutions with mobile phase A being 0.5% phosphoric acid aqueous solution. Detailed Embodiments

[0045] After extensive and in-depth research, the inventor of the present invention has discovered a chromatographic method for simultaneously analyzing liraglutide and its Boc-liraglutide backbone. This analytical method is fast, accurate, simple, and rapid, and can play a good role in quality control and analytical purification during the production process of liraglutide. On this basis, the present invention has been completed.

[0046] Liraglutide

[0047] Liraglutide was developed by Novo Nordisk, with the English name Liraglutide, molecular formula: C 172 H 265 N 43 O 51 , molecular weight: 3751.2, CAS number: 204656-20-2, is a human glucagon-like peptide-1 (GLP-1) analogue, with the sequence: H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Nε(Nα-PAL-γ-Glu))-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH (SEQ ID NO.: 1), and the sequence homology with human natural GLP-1 reaches 97%.

[0048] The structure of liraglutide is to replace the 28th lysine of the natural GLP-1(7-37) molecule with arginine, and at the same time, the ε-amino group of the side chain of the 20th lysine is acylated with hexadecanoic acid glutamate. Due to the presence of this fatty chain, it can reduce the degradation effect of DPP-4, extend the half-life, and the dosing frequency reaches once a day. It can significantly reduce the fasting or postprandial blood glucose of type 2 diabetes patients to regulate the blood glucose level in the body, and at the same time can reduce the patient's weight and the risk of death in patients with cardiovascular diseases.

[0049] Recombinant bacteria

[0050] The recombinant bacteria used in the present invention are prepared according to the method of patent application CN 202010066293.9, and preferably are recombinant Escherichia coli strains expressing the liraglutide backbone fusion protein FP-TEV-EK-GLP-1(20).

[0051] Boc-liraglutide backbone

[0052] The sequence of Boc-liraglutide backbone is H 2N-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-COOH (SEQ ID NO.: 1), which has a BocHN-protecting group on Lys at position 26, and is obtained through steps such as fermentation, disruption, washing, centrifugation, enzymatic cleavage, and purification of recombinant bacteria (such as recombinant Escherichia coli).

[0053] Liraglutide and its Boc-liraglutide backbone samples

[0054] In the present invention, liraglutide and its Boc-liraglutide backbone samples refer to products mainly containing liraglutide and its Boc-liraglutide backbone.

[0055] Liraglutide and its Boc-liraglutide backbone are prepared through the following steps:

[0056] Preparing Fmoc-modified compound 2 from the Boc-liraglutide backbone (compound 1), obtaining compound 3 after removing the Boc protection from compound 2, reacting compound 3 with the activated liraglutide side chain Pal-Glu-(OSu)-OtBu to obtain compound 4, then obtaining compound 5 through the Fmoc removal reaction, removing the tBu protecting group from the side chain, and finally obtaining liraglutide and its Boc-liraglutide backbone.

[0057]

[0058] Specifically, the method includes the steps:

[0059] (i) Providing the Boc-modified liraglutide backbone as described above;

[0060] (ii) Performing Fmoc modification on the Boc-modified liraglutide backbone to obtain the Fmoc- and Boc-modified liraglutide backbone;

[0061] (iii) Performing Boc removal on the Fmoc- and Boc-modified liraglutide backbone and reacting it with the liraglutide side chain to obtain Fmoc-modified liraglutide; and

[0062] (iv) Performing Fmoc removal and side chain tBu removal on the Fmoc-modified liraglutide to obtain liraglutide.

[0063] In another preferred example, in step (ii), Fmoc-Osu, NaHCO 3 and DMF / H 2O, so as to carry out Fmoc modification.

[0064] In another preferred embodiment, the molar ratio of the added Fmoc-Osu, NaHCO 3 to the Boc-modified liraglutide backbone is (0.8 - 1.5):(1.5 - 2.5):(0.8 - 1.2), preferably (1.0 - 1.2):(1.8 - 2.2):(0.8 - 1.2).

[0065] In another preferred embodiment, between step (ii) and step (iii), it further includes a step of purifying the prepared Fmoc- and Boc-modified liraglutide backbone. Preferably, it is purified using a C8 preparative column, and the mobile phase is an acetonitrile solution of TFA.

[0066] In another preferred embodiment, in step (iii), it further includes the steps:

[0067] (a) Add a TFA solution, stir at low temperature for de-Boc treatment to obtain a de-Boc product;

[0068] (b) Purify the de-Boc product, preferably by C8 reverse-phase purification;

[0069] (c) Optionally, add an organic solvent to the purified collection solution obtained from the purification treatment to obtain a solid de-Boc product. Preferably, the organic solvent is a mixed solution of methyl tert-butyl ether and petroleum ether;

[0070] (d) Mix the de-Boc product with the liraglutide side chain to obtain Fmoc-modified liraglutide.

[0071] In another preferred embodiment, in step (i), it includes the steps:

[0072] (ia) Use a recombinant bacterium to prepare the liraglutide backbone fusion protein described in the second aspect of the present invention,

[0073] (ib) Use enterokinase to carry out enzymatic cleavage on the liraglutide fusion protein to obtain the Boc-modified liraglutide backbone.

[0074] In another preferred embodiment, in step (ia), the liraglutide backbone fusion protein inclusion body is isolated from the fermentation broth of the recombinant bacterium. After denaturation-renaturation and enzymatic cleavage of the inclusion body, the Boc-liraglutide backbone fusion protein is obtained.

[0075] Main advantages of the present invention:

[0076] 1. It can simultaneously detect liraglutide and its Boc-liraglutide backbone, which is simple and convenient, can reduce the number of sample injections, and improve the monitoring efficiency.

[0077] 2. It is possible to simultaneously monitor the reaction efficiency and the product yield.

[0078] 3. It is possible to purify liraglutide and the main chain substance of Boc-liraglutide by this analysis method.

[0079] 4. Establish a good product quality control method and a production process fingerprint.

[0080] The following further elaborates the present invention in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0081] Example

[0082] Example 1 Preparation of Liraglutide and Its Main Chain of Boc-Liraglutide

[0083] Refer to the description in the embodiments of patent application CN 202010066293.9.

[0084] Step 1 Construction of Liraglutide Expression Strain

[0085] The DNA fragment of the fusion protein FP-TEV-EK-GLP-1(20) was cloned into the NcoI-XhoI site downstream of the araBAD promoter of the expression vector plasmid pBAD / His A (purchased from NTCC Company, kanamycin resistance) to obtain the plasmid pBAD-FP-TEV-EK-GLP-1(20). Then, the DNA sequence of pylRs was cloned into the SpeI-SalI site downstream of the araBAD promoter of the expression vector plasmid pEvol-pBpF (purchased from NTCC Company, chloramphenicol resistance), and at the same time, the DNA sequence of the tRNA (pylTcua) of lysyl-tRNA synthetase was inserted by PCR downstream of the proK promoter. The constructed plasmids pBAD-FP-TEV-EK-GLP-1(20) and pEvol-pylRs-pylT were co-transformed into Escherichia coli TOP10 strain, and a recombinant Escherichia coli strain expressing the main chain fusion protein FP-TEV-EK-GLP-1(20) of liraglutide was screened.

[0086] Step 2 Expression of the Main Chain of Boc-Liraglutide

[0087] Recombinant Escherichia coli was inoculated into the Escherichia coli seed solution (cultivated by the company) at an inoculation amount of 5% (volume ratio), at 37°C and pH 7.0. Fed-batch feeding was carried out until the pH rose to 7.05, and then separate feeding of carbon and nitrogen sources was started, and carbon source feeding was carried out according to the constant pH method. From 11 h after feeding to the end of fermentation, the mass ratio of carbon to nitrogen source was 1:1.0. After feeding, 7.5 M ammonia water was fed and automatically added to control the pH at 7.0 - 7.2. When cultured for about 4 - 6 hours, 2.5 g / L L-arabinose was added for induction, and the induction continued for 14 h until the end of fermentation. A fermentation broth containing the liraglutide backbone fusion protein was obtained.

[0088] Step 3 Preparation of Boc-liraglutide backbone inclusion bodies

[0089] After centrifuging the fermentation broth obtained in Step 2, the wet cells were mixed with the cell disruption buffer at a volume ratio of 1:1, suspended for 3 h, and the suspension was disrupted by a high-pressure homogenizer three times. After disruption, the inclusion bodies were collected by centrifugation and washed twice. The buffer composition was: 0.5% T-80, 1 mM EDTA-2Na, 100 mM NaCl, pH 7.5. After washing, the yield of the inclusion bodies was weighed to be 41 - 45 g / L. Boc-liraglutide backbone inclusion bodies were obtained after cell disruption, washing, and centrifugation.

[0090] Step 4 Renaturation and enzymatic cleavage of Boc-liraglutide backbone inclusion bodies

[0091] To the inclusion bodies obtained in Step 3, 7.5 mol / L urea dissolution buffer was added at a weight-to-volume ratio of 1:10, dissolved by stirring at room temperature, and the protein concentration was measured by the Bradford method. The total protein concentration of the inclusion body dissolution solution was controlled at about 25 mg / ml, and the pH was adjusted to 9.0 ± 0.1 with NaOH. The inclusion body dissolution solution was added dropwise to the renaturation buffer containing 5 - 10 mmol / L Tris, 10 mmol / L NaCl, 10 mmol / L Na 2 CO 3 0.3 - 0.5 mmol / L EDTA-2Na to dilute the inclusion body dissolution solution by 5 - 10 times for renaturation, maintaining the pH value of the fusion protein renaturation solution at 9.0 - 10.0, controlling the temperature at 4 - 8°C, and the renaturation time was 10 - 20 h.

[0092] Step 5 Preliminary purification of Boc-liraglutide backbone fusion protein

[0093] Take the fusion protein renaturation solution obtained in Step 4, filter it through a 0.45 μm filter membrane to remove undissolved substances; according to the difference in the isoelectric point of proteins, an anion exchange column was used to preliminarily purify the fusion protein.

[0094] Step 6 Enzymatic cleavage of Boc-liraglutide backbone fusion protein

[0095] The sample of the Boc-liraglutide backbone fusion protein preliminarily purified in step 5 is desalted through a hydrophobic column, eluted with pure water, and the elution volume is about 5 times the column volume. Adjust the pH value of the fusion protein solution to 7.5 - 8.5, control the temperature at 25°C, add enterokinase for enzymatic cleavage, and the enzymatic cleavage time is 5 - 16 h to obtain the Boc-liraglutide backbone.

[0096] Reverse-phase chromatography of the Boc-liraglutide backbone in step 7

[0097] According to the hydrophobicity differences between polypeptides and proteins, the Boc-liraglutide backbone is purified by polymer reverse-phase chromatography technology to remove some impurities.

[0098] The enzymatic cleavage solution of the Boc-liraglutide backbone fusion protein obtained in step 6 is filtered and clarified, and then subjected to reverse-phase chromatography for separation and purification. An aqueous solution containing 0.065% trifluoroacetic acid is used as mobile phase A; an acetonitrile solution containing 0.065% trifluoroacetic acid is used as mobile phase B. The Boc-liraglutide backbone binds to the packing material, control the loading amount of the Boc-liraglutide backbone < 10 mg / ml, and then perform gradient elution to collect the Boc-liraglutide backbone, which can be used as the Boc-liraglutide backbone standard.

[0099] Step 8 Preparation of liraglutide standard and Boc-liraglutide backbone sample solution using the Boc-liraglutide backbone

[0100] Take the Boc-liraglutide backbone compound 1 obtained in step 7, add Fmoc-Osu, NaHCO 3 and DMF / H 2 O according to the molar ratio in Table 1, and react for 8 - 12 hours to prepare Fmoc- and Boc-protected GLP-1. Purify using a C8 column, with an aqueous solution containing 0.065% (v / v) TFA as mobile phase A and acetonitrile containing 0.065% (v / v) TFA as mobile phase B for gradient elution. Add methyl tert-butyl ether to the purified collected solution, precipitate by centrifugation, and wash the precipitate with methyl tert-butyl ether 2 - 3 times to obtain the Fmoc-protected compound 2: DiFmoc-GLP-1 (Lys 20 Boc).

[0101] Table 1 Molar ratio of feedstock

[0102] Backbone of Boc-liraglutide Fmoc-OSu <![CDATA[NaHCO 3 > <![CDATA[DMF / H 2 O]]> Equivalent or volume 1.0 eq 1.1 eq 2.0 eq 30V / 30V

[0103] Take the purified compound 2, add TFA solution, stir at low temperature for 10 - 20 min, and purify the deprotected reactant by C8 reverse phase. Add 20 volumes of the mixed solution of methyl tert-butyl ether and petroleum ether (3:1) to the purified collected solution, precipitate by centrifugation, and wash the precipitate with the mixed solution 2 - 3 times to finally obtain the solid compound 3 with Boc removed: DiFmoc-GLP-1(Lys 20 NH 2 ).

[0104] Take the compound 3 after Boc removal, add 30 eq. of the mixture of EDPA, NMP and water (2:1), and stir gently at room temperature for 5 min. Dissolve the equivalent amount of Pal-Glu-(OSu)-OtBu (23.7 μmol) in NMP (303 μL) and add the resulting solution to the obtained mixture. Gently shake the reaction mixture at room temperature for 2 hours. Add 625 μL of 50% aqueous ethanol solution containing glycine (6.5 mg, 86.9 μmol) to terminate the reaction and obtain compound 4: DiFmoc-GLP-1-(Pal-Glu-(Lys 20 NH 2 )-OtBu).

[0105] Take the purified compound 4 (300 mg), add DMF solution containing 20% piperidine, and react at room temperature for 30 minutes. Add 10 volumes of the mixed solvent of methyl tert-butyl ether and petroleum ether to the reaction system, precipitate by centrifugation, and wash the solid with the mixed solvent of methyl tert-butyl ether and petroleum ether 3 - 5 times to obtain compound 5 (270 mg) after Fmoc removal: GLP-1-(Pal-Glu-(Lys 20 NH 2 )-OtBu).

[0106] Take compound 5 (270 mg), add 10 mL of the mixed solution of (TFA:TIS:H 2 O = 95:2.5:2.5):DCM (v:v = 1:1), shake and react at room temperature for 3 - 4 hours to remove the side chain tBu protecting group. Add 10 volumes of the mixed solvent of methyl tert-butyl ether and petroleum ether to the reaction system, precipitate by centrifugation, and wash the solid with the mixed solvent of methyl tert-butyl ether and petroleum ether 3 times to obtain 250 mg of the final product, i.e., liraglutide and its main chain solution of Boc-liraglutide. After HPLC purification, 120 mg of liraglutide with a purity greater than 98% is obtained, i.e., the liraglutide standard.

[0107] Example 2 Preparation of the standard curve and determination of the content of liraglutide and the main chain of Boc-liraglutide

[0108] Liquid chromatography conditions: Instrument: Waters 2695; Detector: Waters 2487 UV detector; Chromatographic column: Octyl bonded silica gel C 8 Column (4.6×250mm); mobile phase: mobile phase A is 0.1mol / mL ammonium dihydrogen phosphate aqueous solution, pH 3.7, mobile phase B is acetonitrile; detection wavelength: 215nm; flow rate: 1.0mL / min; column temperature: 30℃; injection volume: 50μL; gradient elution: elution time and volume proportion of mobile phase B are 0-20min from 25%→60%, 20-35min from 60%→90%, 35-40min from 90%→25%, and then 25% to 10min.

[0109] Preparation of standard solution: Mix the liraglutide standard solution of known concentration with the purified Boc-liraglutide main chain standard solution, accurately pipette the standard solution, dilute and fix the volume to obtain five concentration gradients (i.e., 0.01 mg / mL, 0.02 mg / mL, 0.04 mg / mL, 0.06 mg / mL, 0.08 mg / mL), inject six times continuously, and use the standard concentration as the abscissa and the peak area as the ordinate to obtain a standard curve of concentration and peak area. The standard curve of the Boc-liraglutide main chain is as follows: Figure 1 The standard curve of liraglutide is as follows Figure 2 The peak time of the main chain of the standard Boc-liraglutide is 15.742min, and the peak time of liraglutide is 22.106min. The chromatogram is shown in Figure 3 .

[0110] The reaction solution in step 8 of Example 1 for 30 min was taken as the test solution containing liraglutide and its Boc-liraglutide main chain, and the sample was diluted and fixed to an appropriate concentration to determine its content. The peak time of the Boc-liraglutide main chain in the sample solution was 15.806 min, and the peak time of liraglutide was 22.142 min. The chromatogram was as shown in FIG. Figure 4 .

[0111] Example 3 Precision Test

[0112] Liquid chromatography conditions: Instrument: Waters 2695; Detector: Waters 2487 UV detector; Chromatographic column: Octyl bonded silica gel C 8Column (4.6×250 mm); Mobile phase: Mobile phase A is 0.1 mol / mL ammonium dihydrogen phosphate aqueous solution with pH 3.7, and mobile phase B is acetonitrile; Detection wavelength: 215 nm; Flow rate: 1.0 mL / min; Column temperature: 30 °C; Injection volume: 50 μL; Gradient elution: The elution time and the volume percentage of mobile phase B are from 25% → 60% in 0 - 20 min, from 60% → 90% in 20 - 35 min, from 90% → 25% again in 35 - 40 min, and then run at 25% for 10 min.

[0113] Precisely pipette a certain amount of liraglutide (0.4 mg / mL) and its Boc - liraglutide main chain (0.25 mg / mL) standard solution, dilute to a suitable concentration, make up the volume, filter through a 0.45 μm filter membrane, and inject continuously for 6 times to examine the precision. The results are shown in Table 2.

[0114] Table 2

[0115]

[0116] As can be seen from Table 2, the RSD values of the peak areas for injecting the same sample 6 times are 0.75% and 0.91% respectively, meeting the requirements, indicating that the system precision is good and the test reliability is high.

[0117] Example 4 Repeatability test

[0118] Liquid chromatography conditions: Instrument: Waters 2695; Detector: Waters 2487 UV detector; Chromatographic column: Octadecylsilane bonded silica gel C 8 Column (4.6×250 mm); Mobile phase: Mobile phase A is 0.1 mol / mL ammonium dihydrogen phosphate aqueous solution with pH 3.7, and mobile phase B is acetonitrile; Detection wavelength: 215 nm; Flow rate: 1.0 mL / min; Column temperature: 30 °C; Injection volume: 50 μL; Gradient elution: The elution time and the volume percentage of mobile phase B are from 25% → 60% in 0 - 20 min, from 60% → 90% in 20 - 35 min, from 90% → 25% again in 35 - 40 min, and then run at 25% for 10 min.

[0119] Take 6 portions of the sample solution obtained by the small - scale production process, dilute them respectively, make up the volume to a suitable concentration (0.01 mg / mL - 0.1 mg / mL), filter through a 0.45 μm filter membrane, and obtain the contents of 6 samples. The results are shown in Table 3.

[0120] Table 3

[0121]

[0122] As can be seen from Table 3, the RSD values of the main chain of Boc-liraglutide and liraglutide are 1.24% and 1.06% respectively, meeting the requirements and showing good repeatability.

[0123] Example 5: Spiked Recovery Test

[0124] Liquid Chromatography Conditions: Instrument: Waters 2695; Detector: Waters 2487 UV Detector; Chromatographic Column: Octadecylsilyl Bonded Silica Gel C 8 Column (4.6×250 mm); Mobile Phase: Mobile Phase A is 0.1 mol / mL ammonium dihydrogen phosphate aqueous solution with a pH of 3.7, Mobile Phase B is acetonitrile; Detection Wavelength: 215 nm; Flow Rate: 1.0 mL / min; Column Temperature: 30 °C; Injection Volume: 50 μL; Gradient Elution: The elution time and the volume percentage of Mobile Phase B are from 25% → 60% in 0 - 20 min, from 60% → 90% in 20 - 35 min, from 90% → 25% again in 35 - 40 min, and then run at 25% for 10 min.

[0125] Take 6 portions of the sample solution, add standard solution with a concentration close to it respectively, shake well and make up the volume, filter through a 0.45 μm filter membrane, and measure the contents of 6 samples. The results are shown in Table 4.

[0126] Table 4

[0127]

[0128]

[0129] As can be seen from Table 4, the average recoveries of the main chain of Boc-liraglutide and liraglutide are 100.29% and 100.74% respectively; the RSD values are 1.99% and 1.23% respectively, meeting the requirements and having a high recovery rate.

[0130] In Comparative Example 1, HPLC tests were performed on the sample solutions of liraglutide and its main chain of Boc-liraglutide using 0.05% trifluoroacetic acid aqueous solution as Mobile Phase A.

[0131] Chromatographic Column: Octadecylsilyl Bonded Silica Gel Chromatographic Column;

[0132] Mobile Phase: Mobile Phase A is 0.05% trifluoroacetic acid aqueous solution with a pH of 2.5, Mobile Phase B is acetonitrile.

[0133]

[0134] Detection Wavelength: 215 nm;

[0135] Flow Rate: 1.0 mL / min;

[0136] Column Temperature: 30 °C;

[0137] Sample injection volume: 20 μL.

[0138] The experimental results are as Figure 5 shown. The main chain of Boc-liraglutide has a peak elution time of about 7.8 min. The results show that using mobile phase A as an aqueous solution of trifluoroacetic acid can only detect the main chain of Boc-liraglutide, and cannot detect liraglutide and the reaction yield.

[0139] In Comparative Example 2, HPLC testing was performed on a sample solution of liraglutide and its Boc-liraglutide main chain using mobile phase A as a 0.5% aqueous phosphoric acid solution

[0140] Chromatographic column: octadecylsilyl-bonded silica gel chromatographic column;

[0141] Mobile phase: Mobile phase A is a 0.5% aqueous phosphoric acid solution with a pH of 4.3, and mobile phase B is acetonitrile.

[0142]

[0143] Detection wavelength: 215 nm;

[0144] Flow rate: 1.0 mL / min;

[0145] Column temperature: 30 °C;

[0146] Sample injection volume: 20 μL.

[0147] The experimental results are as Figure 6 shown. Liraglutide has a peak elution time of about 16.0 min. The results show that using mobile phase A as a 0.5% aqueous phosphoric acid solution can only detect liraglutide, and cannot detect the main chain of Boc-liraglutide and the reaction efficiency.

[0148] The analytical method described in the present invention has the characteristics of high efficiency, good specificity, and high sensitivity, and can effectively separate liraglutide and the main chain of Boc-liraglutide.

[0149] The above embodiments are only used to illustrate the present invention, and are not intended to limit the present invention. Those skilled in the art can draw on the content of this article and appropriately modify the test parameters, which should all be included in the protection scope of the present invention. Sequence Listing <110> Ningbo Kunpeng Biotechnology Co., Ltd. <120> A chromatographic method for simultaneously analyzing liraglutide and its Boc-liraglutide main chain <130> P2020-0045 <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 31 <212> PRT <213> Artificial sequence() <400> 1 His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly 20 25 30

Claims

1. An RP-HPLC chromatographic method for simultaneously analyzing liraglutide and its Boc-liraglutide backbone, characterized in that, inject a sample solution containing liraglutide and the Boc-liraglutide backbone into a high-performance liquid chromatograph, and perform detection under the following high-performance liquid chromatography conditions; a bonded silica gel chromatographic column with a bonded group of C8; Mobile phase: Mobile phase A is an aqueous solution of ammonium dihydrogen phosphate with a concentration of 0.05 mol / L - 0.15 mol / L, Mobile phase B is acetonitrile; wherein, gradient elution is carried out with the volume percentage of mobile phase B being 15% → 95%; Detection wavelength: 210 - 230 nm; Injection volume: 20 - 100 μL; Flow rate: 0.5 - 1.0 mL / min; The Boc-liraglutide backbone has the following structure:

2. The method according to claim 1, characterized in that, the volume percentage of mobile phase B is 25% → 90%.

3. The method according to claim 1, characterized in that, the bonded silica gel chromatographic column is an octadecylsilane-bonded silica gel C8 column (4.6×250 mm).

4. The method according to claim 1, characterized in that, the injection volume is 20 - 60 μL.

5. The method according to claim 1, characterized in that, the chromatographic conditions further include: Column temperature: 30 - 40 °C.

6. The method according to claim 1, characterized in that, the pH of the aqueous solution of ammonium dihydrogen phosphate in mobile phase A is 3.5 - 4.

0.

7. The method according to claim 1, characterized in that, the sample solution containing liraglutide and its Boc-liraglutide backbone is prepared by the following steps: (s1) Ferment recombinant bacteria to obtain bacterial cells, (s2) Disrupt, centrifuge and dissolve the bacterial cells to obtain macromolecular proteins, (s3) Add protease to cleave the macromolecular proteins to obtain the Boc-liraglutide backbone, (s4) Chemically modify the Boc-liraglutide backbone to obtain a sample solution containing liraglutide and its Boc-liraglutide backbone.

8. The method according to claim 7, characterized in that, the method further includes the step: (s5) Preparation of standard solution: Mix a liraglutide standard solution with a known concentration and a Boc-liraglutide backbone solution obtained by purification to obtain a standard solution with a known concentration.

9. The method according to claim 1, characterized in that, the injection concentration of the sample solution is 0.05 - 1.5 mg / mL.

10. The method according to claim 1, characterized in that, mobile phase A is an aqueous solution of 0.1 mol / L ammonium dihydrogen phosphate.

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