CN113984911A - Chromatographic method for simultaneously analyzing liraglutide and Boc-liraglutide main chain thereof - Google Patents
Chromatographic method for simultaneously analyzing liraglutide and Boc-liraglutide main chain thereof Download PDFInfo
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- CN113984911A CN113984911A CN202010734182.0A CN202010734182A CN113984911A CN 113984911 A CN113984911 A CN 113984911A CN 202010734182 A CN202010734182 A CN 202010734182A CN 113984911 A CN113984911 A CN 113984911A
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- liraglutide
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Abstract
The invention discloses a chromatographic method for simultaneously analyzing liraglutide and Boc-liraglutide main chains thereof, and specifically comprises the steps of injecting a sample solution containing the liraglutide and the Boc-liraglutide main chains into a high performance liquid chromatograph, and detecting by adopting the following high performance liquid chromatography conditions: bonding a silica gel chromatographic column; the mobile phase A is 0.05mol/mL-0.15mol/mL ammonium dihydrogen phosphate aqueous solution, and the mobile phase B is acetonitrile; wherein, the mobile phase B is eluted in a gradient manner with a volume ratio of 15% → 95%. The analysis method has the characteristics of high analysis speed, accuracy, simplicity, convenience and quickness, good effects on quality control and analysis and purification in the production process of the liraglutide, good specificity, high sensitivity, low cost and the like.
Description
Technical Field
The invention belongs to the technical field of medicines, belongs to the establishment of quality standards and the development of detection methods, and relates to a chromatographic method for simultaneously analyzing liraglutide and Boc-liraglutide main chains thereof.
Background
Diabetes is a high incidence, the worldwide incidence is continuously increased, and the data published by the global health organization show that: the number of the Chinese diabetes patients is 1.75 hundred million in 2000, 2.39 million in 2010, 3 hundred million is expected to break through in 2025, the number of the Chinese diabetes patients approaches 6 hundred million by 2035, the global diabetes incidence is rapidly increasing, the number of the Chinese diabetes patients occupies 1/4 in the world, the prevalence rate is up to 11.6%, and the Chinese diabetes patients are at the top of the world. Diabetes mellitus relates to all life systems of human bodies, seriously influences the labor life of people and threatens the life safety of human beings.
The liraglutide is an incretin hormone peptide analogue secreted by intestinal cells, has high homology, almost has all physiological functions of endogenous substances, can be used for treating diabetes and cardiovascular complications thereof, is a long-acting glucagon-like peptide-1 analogue developed by Danish Nonoh and Node company, is used as a new generation of hypoglycemic drugs based on incretin, can safely and effectively reduce the blood sugar and can possibly protect various cardiovascular hazard factors. The liraglutide injection is administrated by subcutaneous injection once a day, provides 24h of blood sugar control, has pharmacokinetic characteristics which are not influenced by sex or age, and is a revolutionary medicine in the field of type 2 diabetes treatment. Compared with metformin or sulfonylureas drugs, the liraglutide has the advantages of good curative effect, small side effect, no immune reaction and the like, the sequence of the 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 26-Lys is connected with palmitic acid and glutamic acid. The demand of liraglutide on the market is large, and the synthesis method is more. The method of genetic engineering has great technical difficulty, is easy to produce byproducts and other impurities, but has stable source, high yield and high biological activity, so that the production by adopting a fermentation mode and then carrying out structural modification on the product is a better choice, thereby also providing higher requirements for the detection method of the liraglutide and the Boc-liraglutide main chain thereof.
In the process of obtaining the liraglutide, a Boc-liraglutide main chain is an important intermediate product, and is an indispensable process for content detection and control, the sequence of the Boc-liraglutide main chain is H2N-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-COOH, 26-Lys is provided with a BocHN-protecting group to prepare for next structural modification, so that an analysis method for the liraglutide and the Boc-liraglutide main chain in the prior art is necessary to establish, to solve the above problems.
Disclosure of Invention
The invention discloses a chromatographic method for simultaneously analyzing the liraglutide and the Boc-liraglutide main chain thereof aiming at the technical requirements, and aims to provide a method for controlling the quality of the liraglutide, which has the advantages of good stability, good reproducibility, high sensitivity, simple operation, accuracy and rapidness.
The invention provides an RP-HPLC chromatographic method for simultaneously analyzing liraglutide and Boc-liraglutide main chains thereof, which is characterized in that a sample solution containing the liraglutide and the Boc-liraglutide main chains is injected into a high performance liquid chromatograph, and the following high performance liquid chromatographic conditions are adopted for detection;
bonding a silica gel chromatographic column;
mobile phase: the mobile phase A is 0.05mol/mL-0.15mol/mL ammonium dihydrogen phosphate aqueous solution,
the mobile phase B is acetonitrile;
wherein, the mobile phase B is eluted in a gradient manner with a volume ratio of 15% → 95%.
In another preferred embodiment, the mobile phase A is 0.1mol/mL ammonium dihydrogen phosphate in water.
In another preferred embodiment, the mobile phase B volume ratio is 25% → 90%.
In another preferred embodiment, the bonding group is selected from: c8, C18.
In another preferred embodiment, the bonded silica gel chromatographic column is an octyl bonded silica gel C8 column (4.6X 250 mm).
In another preferred example, the chromatographic conditions further comprise:
sample introduction volume: 10-100 μ L.
In another preferred embodiment, the injection volume is 20-60. mu.L, preferably 50. mu.L.
In another preferred example, the chromatographic conditions further comprise:
detection wavelength: 210-230 nm;
flow rate: 0.5-1.0 mL/min;
column temperature: 30-40 ℃.
In another preferred embodiment, the mobile phase A ammonium dihydrogen phosphate aqueous solution has a pH of 3.5-4.0.
In another preferred embodiment, the mobile phase a ammonium dihydrogen phosphate aqueous solution has a pH of 3.7.
In another preferred embodiment, the method is a quantitative, qualitative and/or impurity detection method.
In another preferred embodiment, the method is a non-in vitro and/or adjuvant method.
In another preferred embodiment, the method is a non-diagnostic method.
In another preferred embodiment, the sample solution containing liraglutide and its Boc-liraglutide backbone is prepared by the following steps:
(s1) fermenting the recombinant bacteria to obtain bacteria,
(s2) crushing, centrifuging and dissolving the thalli to obtain macromolecular protein,
(s3) adding a protease to cleave the macromolecular protein to obtain a Boc-liraglutide backbone,
(s4) chemically modifying said Boc-liraglutide backbone to obtain a sample solution comprising liraglutide and its Boc-liraglutide backbone.
In another preferred example, the method further comprises the steps of:
(s5) preparation of Standard solution: and mixing the liraglutide standard solution with the known concentration with the Boc-liraglutide main chain solution obtained through purification to obtain the standard solution with the known concentration.
In another preferred example, in the step (s5), the purity of both liraglutide and its Boc-liraglutide backbone standard substance is above 98%.
In another preferred embodiment, in said step (s5), the concentrations of liraglutide and its Boc-liraglutide backbone standard solution are 0.8mg/mL and 0.5mg/mL, respectively.
In another preferred embodiment, the sample solution has a sample injection concentration of 0.05-1.5 mg/mL.
In another preferred embodiment, the ratio of gradient elution, elution time and mobile phase B volume is 0-20 min from 25% → 60%, 20-35 min from 60% → 90%, 35-40 min from 90% → 25%, and then 25% operation is carried out for 10 min.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 is a standard curve for the Boc-liraglutide backbone.
Fig. 2 is a standard curve for liraglutide.
FIG. 3 is an HPLC chromatogram of liraglutide and its Boc-liraglutide backbone standard.
FIG. 4 is an HPLC chromatogram of a sample solution of liraglutide and its Boc-liraglutide backbone.
FIG. 5 is an HPLC chromatogram of Liraglutide and its Boc-Liraglutide backbone sample solutions with mobile phase A being 0.05% trifluoroacetic acid in water.
FIG. 6 is an HPLC chromatogram of Liraglutide and its Boc-Liraglutide backbone sample solutions with mobile phase A being 0.5% phosphoric acid in water.
Detailed Description
The present inventors have extensively and intensively studied and found a chromatographic method for simultaneously analyzing liraglutide and its Boc-liraglutide backbone. The analysis method has the advantages of high analysis speed, accuracy, simplicity, convenience and quickness, and can play a good role in quality control and analysis and purification in the production process of the liraglutide. On the basis of this, the present invention has been completed.
Liraglutide
Liraglutide was developed by noh and knode corporation, having the english name Liraglutide, molecular formula: c172H265N43O51Molecular weight: 3751.2, CAS number: 204656-20-2, is a human glucagon-like peptide-1 (GLP-1) analogue, and has the sequence: H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys (N epsilon (N alpha-PAL-gamma-Glu)) -Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH (SEQ ID NO: 1), having a sequence homology of 97% to human native GLP-1.
The structure of liraglutide is that 28 th lysine of natural GLP-1(7-37) molecule is replaced by arginine, and epsilon amino group of 20 th lysine side chain is acylated by hexadecanoic acid glutamic acid. Due to the existence of the aliphatic chain, the degradation effect of DPP-4 can be reduced, the half-life period is prolonged, and the administration frequency reaches once a day. Can significantly reduce fasting or postprandial blood sugar of type 2 diabetes patients to achieve the regulation of blood sugar level in vivo, and simultaneously can reduce the weight of the patients and the death risk of the patients with cardiovascular diseases.
Recombinant bacterium
The recombinant strain used in the invention is prepared according to the method of patent application CN 202010066293.9, and is preferably a recombinant escherichia coli strain expressing liraglutide backbone fusion protein FP-TEV-EK-GLP-1 (20).
Boc-liraglutide backbone
Boc-liraglutide backbone with sequence 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 (SEQ ID NO: 1), wherein 26 Lys is provided with a BocHN-protecting group, and the recombinant strain is obtained by fermenting, crushing, washing, centrifuging, enzyme digestion, purifying and the like by recombinant bacteria (such as recombinant Escherichia coli).
Liraglutide and Boc-liraglutide backbone samples thereof
In the invention, the liraglutide and the Boc-liraglutide main chain sample thereof are products mainly containing liraglutide and Boc-liraglutide main chain thereof.
Liraglutide and its Boc-liraglutide backbone were prepared by the following steps:
preparing Fmoc modified compound 2 from Boc-liraglutide main chain (compound 1), removing Boc protection from the compound 2 to obtain compound 3, reacting the compound 3 with activated liraglutide side chain Pal-Glu- (OSu) -OtBu to obtain compound 4, removing Fmoc reaction to obtain compound 5, removing tBu protecting group from the side chain, and finally obtaining liraglutide and Boc-liraglutide main chain.
Specifically, the method comprises the steps of:
(i) providing a Boc modified liraglutide backbone as described above;
(ii) carrying out Fmoc modification on the Boc modified liraglutide main chain to prepare Fmoc and Boc modified liraglutide main chains;
(iii) carrying out Boc removal treatment on the Fmoc and Boc modified liraglutide main chain, and reacting the Fmoc and Boc modified liraglutide main chain with a liraglutide side chain to prepare Fmoc modified liraglutide; and
(iv) and (3) carrying out Fmoc removal and side chain tBu removal treatment on the Fmoc modified liraglutide to prepare the liraglutide.
In another preferred embodiment, Fmoc-Osu and NaHCO are added in step (ii)3And DMF/H2O, thereby performing Fmoc modification.
In another preferred embodiment, Fmoc-Osu and NaHCO are added3The molar ratio to Boc-modified liraglutide backbone was (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).
In another preferred embodiment, between step (ii) and step (iii), the step of purifying the Fmoc and Boc modified liraglutide backbone prepared is further included, preferably using a C8 preparation column with a mobile phase of TFA in acetonitrile.
In another preferred embodiment, in step (iii), the method further comprises the steps of:
(a) adding TFA solution, stirring at low temperature, and removing Boc to obtain a Boc-removed product;
(b) purifying the de-Boc product, preferably performing C8 reversed phase purification;
(c) optionally, adding an organic solvent to the purified collection from the purification treatment, preferably a tert-methyl ether: petroleum ether mixed liquor;
(d) and mixing the de-Boc product with the side chain of liraglutide to prepare the Fmoc modified liraglutide.
In another preferred example, in the step (i), the method comprises the steps of:
(ia) preparing the liraglutide backbone fusion protein of the second aspect of the present invention using the recombinant bacteria,
(ib) carrying out enzyme digestion treatment on the liraglutide fusion protein by using enterokinase, thereby obtaining the Boc modified liraglutide main chain.
In another preferred example, in the step (ia), liraglutide backbone fusion protein inclusion bodies are obtained by separating from the fermentation broth of the recombinant bacteria, and Boc-liraglutide backbone fusion protein is obtained after denaturation and enzyme digestion of the inclusion bodies.
The invention has the main advantages that:
1. the method can be used for simultaneously detecting the liraglutide and the Boc-liraglutide main chain thereof, is simple and convenient, can reduce the sampling times and improve the monitoring efficiency.
2. Reaction efficiency and product yield can be monitored simultaneously.
3. Liraglutide and Boc-liraglutide backbone species can be purified by this analytical method.
4. Establishing good product quality control method and production process fingerprint.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.
Examples
Example 1 preparation of liraglutide and its Boc-liraglutide backbone
See patent application CN 202010066293.9 for examples.
Step 1 construction of liraglutide expression strain
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, kanamycin resistance) to obtain plasmid pBAD-FP-TEV-EK-GLP-1 (20). The DNA sequence of pylRs was then cloned into the SpeI-SalI site downstream of the araBAD promoter of the expression vector plasmid pEvol-pBpF (available from NTCC for chloramphenicol resistance), while the DNA sequence of the tRNA (pylTcua) of lysyl-tRNA synthetase was PCR inserted downstream of the proK promoter. The constructed plasmid pBAD-FP-TEV-EK-GLP-1(20) and pEvol-pylRs-pylT are jointly transformed into an escherichia coli TOP10 strain, and a recombinant escherichia coli strain expressing liraglutide backbone fusion protein FP-TEV-EK-GLP-1(20) is obtained through screening.
Recombinant Escherichia coli was inoculated into an Escherichia coli seed solution (cultured by Co.) at 37 ℃ and pH7.0 in an inoculum size (volume ratio) of 5%, fed in batch until pH increased to 7.05, and then carbon-nitrogen source separate feeding was started, and carbon source feeding was performed according to the constant pH method. Feeding for 11h, and finishing fermentation, wherein the mass ratio of carbon to nitrogen is 1: 1.0. After the feed supplement, the pH value is controlled to be 7.0-7.2 by feed supplement and automatic fed-batch addition of 7.5M ammonia water. Culturing for 4-6 hr, adding 2.5g/L L-arabinose for induction, and continuing for 14 hr until fermentation is finished. Obtaining a fermentation broth comprising the liraglutide backbone fusion protein.
Step 3 preparation of Boc-liraglutide backbone inclusion body
And (3) centrifuging the fermentation liquor obtained in the step (2), and mixing the wet thalli according to the weight ratio of 1:1 volume and broken fungus buffer solution mix, and the suspension is 3h, and the suspension uses the broken fungus of high pressure homogenizer three times, and the inclusion body is collected in the centrifugation after breaking the fungus, washs it twice, and the buffer solution composition is: 0.5% T-80, 1mm EDTA-2Na, 100mm NaCl, pH 7.5. The yield of the inclusion bodies weighed after cleaning is 41-45 g/L. And obtaining the Boc-liraglutide main chain inclusion body after thallus crushing, cleaning and centrifuging.
Step 4, renaturation and enzyme digestion of Boc-liraglutide main chain inclusion body
Adding 7.5mol/L urea dissolving buffer solution into the inclusion body obtained in the step 3 according to the weight-volume ratio of 1:10, stirring and dissolving at room temperature, measuring the protein concentration by a Bradford method, controlling the total protein concentration of the inclusion body dissolving solution to be about 25mg/ml, and adjusting the pH value to be 9.0 +/-0.1 by NaOH. Dropping the inclusion body solution into a solution containing 5-10mmol/L Tris, 10mmol/L NaCl and 10mmol/L Na2CO30.3-0.5mmol/L EDTA-2Na in renaturation buffer solution, diluting the inclusion body dissolving 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 ℃, and controlling the renaturation time at 10-20 h.
Step 5 Primary purification of Boc-liraglutide backbone fusion proteins
Filtering the fusion protein renaturation solution obtained in the step 4 by a filter membrane of 0.45 mu m to remove undissolved substances; according to the difference of the isoelectric points of the proteins, the fusion protein is primarily purified by adopting an anion exchange column.
Step 6 enzyme digestion of Boc-liraglutide backbone fusion protein
And (4) desalting the Boc-liraglutide main chain fusion protein sample preliminarily purified in the step (5) through a hydrophobic column, and eluting with pure water, wherein the elution volume is about 5 times of the column volume. Adjusting the pH value of the fusion protein solution to 7.5-8.5, controlling the temperature to 25 ℃, adding enterokinase for enzyme digestion for 5-16h, and obtaining the Boc-liraglutide main chain.
Step 7 reverse phase chromatography of Boc-liraglutide backbone
And purifying the Boc-liraglutide main chain by adopting a polymer reverse phase chromatography technology according to the hydrophobicity difference of the polypeptide and the protein to remove a part of impurities.
And 6, filtering and clarifying the enzyme digestion solution of the Boc-liraglutide main chain fusion protein obtained in the step 6, and then carrying out reversed-phase chromatography separation and purification. Taking an aqueous solution containing 0.065% of trifluoroacetic acid as a mobile phase A; acetonitrile solution containing 0.065% trifluoroacetic acid was used as mobile phase B. And combining the Boc-liraglutide main chain with a filler, controlling the loading amount of the Boc-liraglutide main chain to be less than 10mg/ml, then carrying out gradient elution, and collecting the Boc-liraglutide main chain which can be used as a Boc-liraglutide main chain standard substance.
Taking Boc-liraglutide main chain compound 1 obtained in the step 7, and adding Fmoc-Osu and NaHCO according to the molar ratio of the mixture shown in the table 13And DMF/H2And O, reacting for 8-12 hours to obtain the GLP-1 protected by Fmoc and Boc. The purification was carried out using a C8 column, eluting with a gradient of 0.065% (v/v) TFA in water as mobile phase A and 0.065% (v/v) TFA in acetonitrile as mobile phase B. And (3) adding methyl tert-ether into the purified and collected solution, precipitating and centrifuging, and washing the precipitate for 2-3 times by using methyl tert-ether to obtain an Fmoc protected compound 2: DiFmoc-GLP-1 (Lys)20Boc)。
TABLE 1 molar ratio of the feeds
| Boc-liraglutide backbone | Fmoc-OSu | NaHCO3 | DMF/H2O | |
| Equivalent weight or volume | 1.0eq | 1.1eq | 2.0eq | 30V/30V |
And (3) adding a TFA solution into the purified compound 2, stirring at low temperature for 10-20 min, and performing reverse phase purification on the deprotection reaction product by C8. To the purified pool was added 20 volumes of methyl tert-ether: and (3) petroleum ether mixed liquor (3:1), precipitating and centrifuging, washing the precipitate for 2-3 times by using the mixed liquor, and finally obtaining a solid compound 3 without Boc: DiFmoc-GLP-1 (Lys)20NH2)。
The Boc-removed compound 3 was added with 30eq. EDPA, NMP and water mixture (2:1) and stirred gently at room temperature for 5 min. An equivalent amount of Pal-Glu- (OSu) -OtBu (23.7. mu. mol) dissolved in NMP (303. mu.L) was added to the resulting mixture, and the reaction mixture was gently shaken at room temperature for 2 hours. To this was added 625 μ L of 50% aqueous ethanol solution containing glycine (6.5mg, 86.9 μmol), thereby terminating the reaction to obtain compound 4: DiFmoc-GLP-1- (Pal-Glu- (Lys)20NH2)-OtBu)。
After purification, Compound 4(300mg) was added to a DMF solution containing 20% piperidine and reacted at room temperature for 30 minutes. Adding a mixed solvent of methyl tert-ether and petroleum ether with the volume 10 times that of the reaction system, precipitating and centrifuging, washing the solid with the mixed solvent of methyl tert-ether and petroleum ether for 3-5 times to obtain a compound 5(270mg) after Fmoc removal: GLP-1- (Pal-Glu- (Lys)20NH2)-OtBu)。
GLP-1 Compound 5(270mg) was taken in and added to (TFA: TIS: H)2And O95: 2.5:2.5) and DCM (v: 1) mixed solution 10mL, shaking and reacting at room temperature for 3-4 hours to remove a side chain tBu protecting group, adding a 10-fold volume of mixed solvent of methyl tert-ether and petroleum ether into a reaction system, precipitating and centrifuging, and washing the solid for 3 times by using the mixed solvent of methyl tert-ether and petroleum ether to obtain 250mg of final products, namely liraglutide and Boc-liraglutide main chain solution. After HPLC purification, 120mg of liraglutide with the purity of more than 98 percent, namely the standard liraglutide, is obtained.
Example 2 Standard Curve preparation and assay of Liraglutide and Boc-Liraglutide backbones
Liquid chromatography conditions: the instrument comprises the following steps: waters 2695; a detector: waters 2487 ultraviolet detector; a chromatographic column: octyl bonded silica gel C8Columns (4.6X 250 mm); mobile phase: mobile phaseA is 0.1mol/mL ammonium dihydrogen phosphate aqueous solution, the pH is 3.7, and the mobile phase B is acetonitrile; detection wavelength: 215 nm; flow rate: 1.0 mL/min; column temperature: 30 ℃; sample introduction volume: 50 mu L of the solution; the gradient elution was: the elution time and the volume ratio of the mobile phase B are 0-20 min from 25% → 60%, 20-35 min from 60% → 90%, 35-40 min from 90% → 25%, and then 25% and 10 min.
Preparation of standard solution: mixing a liraglutide standard solution with a known concentration with a Boc-liraglutide main chain standard solution obtained through purification, precisely transferring the standard solution, diluting and fixing the volume to obtain five concentration gradients (namely 0.01mg/mL, 0.02mg/mL, 0.04mg/mL, 0.06mg/mL and 0.08mg/mL), continuously feeding samples six times, taking the standard concentration as a horizontal coordinate and the peak area as a vertical coordinate to obtain a standard curve of concentration and peak area, wherein the standard curve of the Boc-liraglutide main chain is shown in figure 1, the standard curve of the liraglutide is shown in figure 2, the peak emergence time of the standard Boc-liraglutide main chain is 15.742min, the peak emergence time of the liraglutide is 22.106min, and the chromatogram is shown in figure 3.
Taking the reaction liquid reacted for 30min in the step 8 in the embodiment 1 as a solution to be detected containing liraglutide and the Boc-liraglutide main chain thereof, sampling, diluting, fixing the volume to a proper concentration, determining the content of the liraglutide, wherein the peak emergence time of the Boc-liraglutide main chain in the sample solution is 15.806min, the peak emergence time of the liraglutide is 22.142min, and a chromatogram is shown in figure 4.
Example 3 precision test
Liquid chromatography conditions: the instrument comprises the following steps: waters 2695; a detector: waters 2487 ultraviolet detector; a chromatographic column: octyl bonded silica gel C8Columns (4.6X 250 mm); mobile phase: the mobile phase A is 0.1mol/mL ammonium dihydrogen phosphate aqueous solution, the pH value is 3.7, and the mobile phase B is acetonitrile; detection wavelength: 215 nm; flow rate: 1.0 mL/min; column temperature: 30 ℃; sample introduction volume: 50 mu L of the solution; the gradient elution was: the elution time and the volume ratio of the mobile phase B are 0-20 min from 25% → 60%, 20-35 min from 60% → 90%, 35-40 min from 90% → 25%, and then 25% and 10 min.
Precisely transferring a certain amount of liraglutide (0.4mg/mL) and Boc-liraglutide main chain (0.25mg/mL) standard solution, diluting to a proper concentration, fixing the volume, passing through a 0.45-micrometer filter membrane, continuously injecting samples for 6 times, and inspecting the precision, wherein the results are shown in Table 2.
TABLE 2
As can be seen from Table 2, the RSD values of the peak areas of the same sample for 6 times are respectively 0.75% and 0.91%, which meets the requirements and shows that the system precision is good and the test reliability is high.
Example 4 repeatability test
Liquid chromatography conditions: the instrument comprises the following steps: waters 2695; a detector: waters 2487 ultraviolet detector; a chromatographic column: octyl bonded silica gel C8Columns (4.6X 250 mm); mobile phase: the mobile phase A is 0.1mol/mL ammonium dihydrogen phosphate aqueous solution, the pH value is 3.7, and the mobile phase B is acetonitrile; detection wavelength: 215 nm; flow rate: 1.0 mL/min; column temperature: 30 ℃; sample introduction volume: 50 mu L of the solution; the gradient elution was: the elution time and the volume ratio of the mobile phase B are 0-20 min from 25% → 60%, 20-35 min from 60% → 90%, 35-40 min from 90% → 25%, and then 25% and 10 min.
6 parts of sample solution obtained by the pilot plant production process are respectively diluted, the volume is determined to be proper concentration (0.01mg/mL-0.1mg/mL), and the sample solution is filtered through a 0.45 mu m filter membrane to obtain 6 sample contents, and the results are shown in Table 3.
TABLE 3
As can be seen from Table 3, the RSD values of the Boc-liraglutide main chain and liraglutide are 1.24% and 1.06%, respectively, which meet the requirements and have good repeatability.
EXAMPLE 5 sample recovery test
Liquid chromatography conditions: the instrument comprises the following steps: waters 2695; a detector: waters 2487 ultraviolet detector; a chromatographic column: octyl bonded silica gel C8Columns (4.6X 250 mm); mobile phase: the mobile phase A is 0.1mol/mL ammonium dihydrogen phosphate aqueous solution, the pH value is 3.7, and the mobile phase B is acetonitrile;detection wavelength: 215 nm; flow rate: 1.0 mL/min; column temperature: 30 ℃; sample introduction volume: 50 mu L of the solution; the gradient elution was: the elution time and the volume ratio of the mobile phase B are 0-20 min from 25% → 60%, 20-35 min from 60% → 90%, 35-40 min from 90% → 25%, and then 25% and 10 min.
Sampling 6 parts of sample solution, respectively adding standard solution with approximate concentration, shaking to constant volume, filtering with 0.45 μm filter membrane, and measuring 6 sample contents, the results are shown in Table 4
TABLE 4
As can be seen from table 4, the average recovery rates of Boc-liraglutide backbone and liraglutide were: 100.29%, 100.74%; RSD values are 1.99% and 1.23% respectively, and the method meets requirements and is high in recovery rate.
Comparative example 1 HPLC testing of liraglutide and its Boc-liraglutide backbone sample solutions using mobile phase A as 0.05% aqueous trifluoroacetic acid
A chromatographic column: octadecylsilane chemically bonded silica chromatographic column;
mobile phase: mobile phase A is 0.05% trifluoroacetic acid water solution, pH is 2.5, mobile phase B is acetonitrile,
detection wavelength: 215 nm;
flow rate: 1.0 mL/min;
column temperature: 30 ℃;
sample introduction volume: 20 μ L.
The experimental results are shown in fig. 5, where the peak time is around 7.8min, which is Boc-liraglutide backbone. The result shows that only Boc-liraglutide main chain can be detected and liraglutide cannot be detected by using trifluoroacetic acid aqueous solution as the mobile phase A, and the reaction yield is high.
Comparative example 2 HPLC testing of liraglutide and its Boc-liraglutide backbone sample solutions using mobile phase A as 0.5% aqueous phosphoric acid solution
A chromatographic column: octadecylsilane chemically bonded silica chromatographic column;
mobile phase: mobile phase A is 0.5% phosphoric acid water solution, pH is 4.3, mobile phase B is acetonitrile,
detection wavelength: 215 nm;
flow rate: 1.0 mL/min;
column temperature: 30 ℃;
sample introduction volume: 20 μ L.
The experimental results are shown in fig. 6, and the liraglutide with the peak-off time of about 16.0min is shown. The result shows that the use of 0.5% aqueous solution of phosphoric acid as mobile phase A can only detect liraglutide, but cannot detect Boc-liraglutide main chain and reaction efficiency.
The analysis method disclosed by the invention has the characteristics of high efficiency, good specificity and high sensitivity, and can be used for effectively separating the liraglutide and the Boc-liraglutide main chain.
The above examples are only for illustrating the present invention and are not to be construed as limiting the present invention. Those skilled in the art can use the present disclosure to appropriately modify the test parameters, which are all included in the scope of the present invention.
Sequence listing
<110> Ningbo spread Biotechnology Ltd
<120> chromatographic method for simultaneously analyzing liraglutide and Boc-liraglutide main chain thereof
<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 (10)
1. An RP-HPLC chromatographic method for simultaneously analyzing liraglutide and Boc-liraglutide main chains thereof is characterized in that a sample solution containing the liraglutide and the Boc-liraglutide main chains is injected into a high performance liquid chromatograph, and the following high performance liquid chromatographic conditions are adopted for detection;
bonding a silica gel chromatographic column;
mobile phase: the mobile phase A is 0.05mol/mL-0.15mol/mL ammonium dihydrogen phosphate aqueous solution,
the mobile phase B is acetonitrile;
wherein, the mobile phase B is eluted in a gradient manner with a volume ratio of 15% → 95%.
2. The method according to claim 1, wherein the mobile phase B volume fraction is 25% → 90%.
3. The method of claim 1, wherein the bonding group is selected from the group consisting of: c8, C18.
4. The method of claim 1, wherein the chromatographic conditions further comprise:
sample introduction volume: 10-100 μ L.
5. The method of claim 1, wherein the chromatographic conditions further comprise:
detection wavelength: 210-230 nm;
flow rate: 0.5-1.0 mL/min;
column temperature: 30-40 ℃.
6. The method of claim 1, wherein the mobile phase a aqueous ammonium dihydrogen phosphate solution has a pH of from 3.5 to 4.0.
7. The method of claim 1, wherein the sample solution comprising liraglutide and its Boc-liraglutide backbone is prepared by:
(s1) fermenting the recombinant bacteria to obtain bacteria,
(s2) crushing, centrifuging and dissolving the thalli to obtain macromolecular protein,
(s3) adding a protease to cleave the macromolecular protein to obtain a Boc-liraglutide backbone,
(s4) chemically modifying said Boc-liraglutide backbone to obtain a sample solution comprising liraglutide and its Boc-liraglutide backbone.
8. The method of claim 7, wherein the method further comprises the steps of:
(s5) preparation of Standard solution: and mixing the liraglutide standard solution with the known concentration with the Boc-liraglutide main chain solution obtained through purification to obtain the standard solution with the known concentration.
9. The method of claim 1, wherein the sample solution is injected at a concentration of 0.05 to 1.5 mg/mL.
10. The method according to claim 1, wherein the gradient elution, elution time and mobile phase B volume ratio is from 25% to 60% in 0-20 min, from 60% to 90% in 20-35 min, from 90% to 25% in 35-40 min, and then from 25% to 10 min.
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