CN113621046A - Use and method of polymer anion exchange filler in preparation of liraglutide - Google Patents
Use and method of polymer anion exchange filler in preparation of liraglutide Download PDFInfo
- Publication number
- CN113621046A CN113621046A CN202110954539.0A CN202110954539A CN113621046A CN 113621046 A CN113621046 A CN 113621046A CN 202110954539 A CN202110954539 A CN 202110954539A CN 113621046 A CN113621046 A CN 113621046A
- Authority
- CN
- China
- Prior art keywords
- liraglutide
- chromatographic column
- column
- modifier
- modification
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/605—Glucagons
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Endocrinology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to the field of protein engineering, in particular to application and a method of a polymer anion exchange filler in preparation of liraglutide, wherein the polymer anion exchange filler is selected from NanoQ-15L, Source 15Q or BestPoly 15Q; the preparation method comprises the following steps: 1) filling a polymer anion exchange filler on the chromatographic column, and replacing the chromatographic column by using a modification buffer solution I; 2) adding the liraglutide intermediate solution into a chromatographic column; 3) adding a modifying agent into a chromatographic column to carry out on-column modification reaction; 4) and eluting and collecting the liraglutide after the modification reaction is completed. The invention realizes the simultaneous implementation of modification reaction and chromatographic purification on the column, reduces the use of reaction kettles and pipeline facilities, reduces the use amount of organic reagents, reduces the discharge of hazardous wastes, obviously improves the production recovery rate and reduces the production cost. Meanwhile, the liraglutide prepared by the method is a protein crystal with a regular shape, and has an advantage in stability.
Description
Technical Field
The invention relates to the field of protein engineering, in particular to application and a method of polymer anion exchange filler in preparation of liraglutide.
Background
Liraglutide [ Arg34Lys26- (N-epsilon- (gamma-Glu (N-alpha-hexadecanoyl))) -GLP-1(7-37) ] is a GLP-1 (glucagon-like peptide-1) analogue, having the name Liraglutide and the sequence of H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys (Pal-g-Glu) -Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH. The liraglutide is formed by connecting a 31-amino acid main chain Arg34-GLP-1(7-37) and hexadecanopalmitic acid fatty acid by a glutamic acid linker, the connecting site is positioned at the amino group of a 26-position Lys side chain, and the structure is shown in the attached figure 1 in detail. Liraglutide was developed by norand nordred and was approved by FDA for marketing at 1 month 2010 and CFDA at 10 months 2011 for marketing in china for the treatment of type ii diabetes. The liraglutide serving as a new generation of hypoglycemic drugs based on incretins has long action time, fully retains multiple physiological activities of natural GLP-1, can safely and effectively reduce blood sugar and can protect multiple cardiovascular hazards. FDA approval was obtained 12 months in 2014 for weight loss in adults with a BMI of 27 or greater (overweight) accompanied by at least one condition associated with overweight (hypertension, type 2 diabetes, hyperlipidemia, etc.), or a BMI of 30 or greater (obesity).
The preparation of liraglutide can be divided into chemical synthesis and biological semisynthesis at present. Chemical synthesis is mainly a synthesis method of amino acid chains through solid phase synthesis, such as the synthesis methods disclosed in patents CN107960079A, CN104045706B, CN103304660A, CN106699871A and the like. However, chemical synthesis generally has the problems of environmental unfriendliness, low yield, complex reaction steps, high overall cost and the like.
The biological semi-synthetic liraglutide is prepared by preparing an intermediate Arg34-GLP-1(7-37) by a biological means, connecting a modifier on a side chain amino group Lys at the position 26 of the intermediate by a chemical modification reaction, and then obtaining the liraglutide by purification means such as chromatography and the like. Compared with chemical synthesis, the biological semi-synthesis method has the advantages of low cost, little environmental pollution, high yield, mild reaction conditions and the like. However, other modification sites exist on the intermediate, so that the intermediate modification process is difficult to control, the downstream purification process is complex, and the overall production process recovery rate is low. For example, in patents CN104592381A and CN1951965A, liraglutide is obtained by modifying an intermediate (Arg34-GLP-1(7-37)), the process routes both include chemical modification of the intermediate and chromatographic purification after modification, modification reactions are performed in a reaction kettle, and liraglutide is purified by transferring to a chromatographic apparatus after the reaction is completed, which has the problems of high equipment and facility cost, complex process, and low liraglutide production recovery rate. The liraglutide prepared is liquid or amorphous powder, and has poor stability and is not beneficial to transportation and storage.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, it is an object of the present invention to provide a use and a process of polymeric anion exchange filler for the preparation of liraglutide, for solving the problems of the prior art.
To achieve the above objects and other related objects, the present invention provides the use of a polymeric anion exchange filler in the preparation of liraglutide.
Preferably, the polymeric anionic filler is, for example, NanoQ-15L, Source 15Q or BestPoly 15Q.
Preferably, the use is in the preparation of liraglutide by reacting the liraglutide intermediate with a liraglutide modifier.
The application also provides a preparation method of liraglutide, which comprises the following steps:
1) filling a polymer anion exchange filler on the chromatographic column, and replacing the chromatographic column by using a modification buffer solution I;
2) adding the liraglutide intermediate solution into a chromatographic column;
3) adding a liraglutide modifier into a chromatographic column to perform modification reaction on the column;
4) and eluting and collecting the liraglutide after the modification reaction is completed.
Preferably, the polymeric anionic filler is, for example, NanoQ-15L, Source 15Q or BestPoly 15Q.
Preferably, the modification buffer I comprises 10-50mM sodium carbonate, 0.1-1.0 mM EDTA and 50-70% (v/v) acetonitrile aqueous solution based on the total volume of the modification buffer I.
Preferably, the liraglutide intermediate is Arg34-GLP-1(7-37), the structure of which is shown in FIG. 2.
Preferably, the preparation method further comprises crystallizing the liraglutide eluted and collected in the step (4).
Preferably, the method of crystallization comprises the steps of: and (4) adding a crystallization reagent into the liraglutide eluted and collected in the step (4) for crystallization, thereby obtaining the liraglutide crystal.
Preferably, the crystallization reagent comprises a basic amino acid, a phenolic compound and polyethylene glycol.
Preferably, the final concentration of the basic amino acid is 0.1-1.0M, the final concentration of the phenolic compound is 3-8 g/L, and the final concentration of the polyethylene glycol is 0.1-0.5g/L based on the total volume of the crystallization system.
Preferably, the basic amino acid is selected from one or more of arginine, lysine or histidine. The phenolic compound is phenol. The molecular weight of the polyethylene glycol is more than 1000; more preferably, the polyethylene glycol is PEG 5000.
As mentioned above, the use and process of the polymeric anion exchange filler of the present invention in the preparation of liraglutide has the following beneficial effects:
(1) compared with the traditional process, the method realizes that modification reaction and chromatographic purification are carried out on the column simultaneously, and reduces the use of reaction kettles and pipeline facilities, so that equipment facilities can be saved.
(2) The liraglutide is prepared into the protein crystal with a regular shape, and compared with the amorphous powder prepared by an isoelectric point precipitation process, the crystallized liraglutide has the advantage of stability.
Drawings
Fig. 1 shows a structural schematic diagram of liraglutide of the present invention.
FIG. 2 shows a schematic sequence of Arg34-GLP-1(7-37) of the present invention.
FIG. 3 shows a schematic diagram of the modification reaction.
Fig. 4 shows a microscopic image of liraglutide prepared in example 1.
Fig. 5 shows a microscopic image of liraglutide prepared in example 2.
Fig. 6 shows a microscopic image of liraglutide prepared in example 3.
Fig. 7 shows a microscopic image of liraglutide prepared in example 4.
Fig. 8 is a graph showing the general trend of the change in the liraglutide acceleration process prepared in examples 1, 2, 3 and 4.
Fig. 9 shows the kinetics curves for liraglutide prepared in example 1.
Fig. 10 shows the kinetics curves for liraglutide prepared in example 2.
Fig. 11 shows the kinetics curves for liraglutide prepared in example 3.
Fig. 12 shows the kinetics curves for liraglutide prepared in example 4.
Detailed Description
Aiming at the defects existing in the preparation of the biological semi-synthetic liraglutide, the application provides the application of the polymer anion exchange filler in the preparation of the liraglutide.
The polymer anion filler is a filler which takes a high molecular polymer as a matrix and is bonded with anion exchange groups.
The high molecular polymer is selected from polyacrylate, polystyrene-divinylbenzene, polystyrene and polymethacrylate. The polymerization degree range of the high molecular polymer is 2-25.
The anion exchange group is selected from-CH2-O-CHOHCH2-O-CHOH-CH2-N+(CH3)3、CH2N+(CH3)3、-O-CH2CHOHCH2N+(CH3)3、-O-CH2CH2-N+(C2H5)2H、-O-CH2CHOHCH2-N+(C2H5)2H。
The polymeric anionic filler is for example NanoQ-15L, Source 15Q or BestPoly 15Q.
In one embodiment, the polymeric anionic filler has a pore size of 15 to 30 μm.
In one embodiment, the use is in the preparation of liraglutide by reacting a liraglutide intermediate with a liraglutide modifier.
In one embodiment, the liraglutide intermediate is Arg34-GLP-1 (7-37).
The liraglutide modifier is a liraglutide fatty acid side chain. In one embodiment, the liraglutide modifier is palmitic acid-glutamic acid-OSU.
The application also provides a preparation method of liraglutide, which comprises the following steps:
1) filling a polymer anion exchange filler on the chromatographic column, and replacing the chromatographic column by using a modification buffer solution I;
2) adding the liraglutide intermediate solution into a chromatographic column;
3) adding a liraglutide modifier into a chromatographic column to perform modification reaction on the column;
4) and eluting and collecting the liraglutide after the modification reaction is completed.
In one embodiment, the polymeric anionic filler is a filler having a high molecular weight polymer as a matrix to which anion exchange groups are bonded.
The high molecular polymer is selected from polyacrylate, polystyrene-divinylbenzene, polystyrene and polymethacrylate, and is preferably polystyrene-divinylbenzene.
The anion exchange group is selected from-CH2-O-CHOHCH2-O-CHOH-CH2-N+(CH3)3、CH2N+(CH3)3、-O-CH2CHOHCH2N+(CH3)3、-O-CH2CH2-N+(C2H5)2H、-O-CH2CHOHCH2-N+(C2H5)2H。
The polymeric anionic filler is for example NanoQ-15L, Source 15Q or BestPoly 15Q.
In one embodiment, the polymeric anionic filler has a pore size of 15 to 30 μm.
In one embodiment, step 1) is packing the column as a dynamic axial compression column and replacing the column with the modification buffer i. After filling, the column pressure control range is 20-50 Bar, and the height of the chromatographic column is 23-27 cm.
In one embodiment, the column is rinsed with sodium hydroxide solution no less than 4 column volumes after packing, and the column is replaced with modification buffer I.
In one embodiment, the modification buffer I comprises an aqueous solution of 10-50mM sodium carbonate, 0.1-1.0 mM EDTA, 50-70% (v/v) Acetonitrile (ACN) based on the total volume of the modification buffer I.
In one embodiment, the final concentration of EDTA in the modification buffer i may be selected from any one of the following: 0.1 to 0.2mM, 0.2 to 0.3mM, 0.3 to 0.4mM, 0.4 to 0.5mM, 0.5 to 0.6mM, 0.6 to 0.7mM, 0.7 to 0.8mM, or 0.9 to 1.0 mM.
In one embodiment, the step 1) of replacing the chromatography column with the modification buffer i is carried out by rinsing the chromatography column with the modification buffer i for not less than 4 column volumes.
In one embodiment, in step 2), the liraglutide intermediate is dissolved in a modification buffer ii to obtain a liraglutide intermediate solution, wherein the modification buffer ii is a sodium carbonate solution of 10 to 50 mM.
In one embodiment, the concentration of the liraglutide intermediate solution is 5-20 mg/ml.
In one embodiment, the liraglutide intermediate solution is pumped into a chromatography column at a flow rate of 150-900 cm/h.
In one embodiment, the liraglutide intermediate is Arg34-GLP-1(7-37) (CAS No: 204521-68-6, available from Beijing Bernoulli Biotech, Inc., under the product number Bennu-G2002). Arg34-GLP-1(7-37) has a structure in which Lys34 of GLP-1(7-37) is replaced by Arg, and its sequence is shown in FIG. 2. The modification reaction refers to the reaction of the side chain amino Lys at the 26-position of Arg34-GLP-1(7-37) and the liraglutide modifier on a chromatographic column, and the reaction scheme is shown in figure 3.
In one embodiment, in step 3), the liraglutide modifier is dissolved in acetonitrile to obtain a solution of the liraglutide modifier, and the solution is added to the chromatographic column.
In one embodiment, the concentration of the liraglutide modifier in the solution is 20-100 mg/ml.
In one embodiment, in step 3), the ratio of the liraglutide intermediate: adding the liraglutide modifier into a chromatographic column according to the molar ratio of 1: 1-1: 1.2.
In one embodiment, in the step (3), the liraglutide modifier is pumped into the chromatographic column at a speed of 100-900 cm/h, the liraglutide modifier is pushed to flow in the chromatographic column, 2-6 times of column volume cyclic modification is completed, and the reaction time is controlled to be 0.5-2 hours.
In one embodiment, the liraglutide modifier is palmitic acid-glutamic acid-OSU.
In one embodiment, in step (4), the chromatographic column is eluted with eluent 1 after the modification reaction is completed, and then the liraglutide is collected by gradient elution with eluent 2.
In one embodiment, the eluent 1 comprises 10mM-20mM Tris, 3% to 7% acetonitrile solution. The elution refers to eluting the chromatography column with elution 1 for not less than 4 column volumes.
The eluent 2 is a group of solutions comprising solution A and solution B, wherein the solution A comprises 20mM Tris and 30% acetonitrile; solution B included 20mM Tris, 0.3M NaCl and 30% acetonitrile. The flow rate of the gradient elution is 100-900 cm/h. The collection refers to collecting the eluted liraglutide protein fraction according to ultraviolet absorption.
In one embodiment, the preparation method further comprises crystallizing the liraglutide eluted and collected in the step (4).
In one embodiment, the method of crystallization comprises the steps of: and (4) adding a crystallization reagent into the liraglutide eluted and collected in the step (4), thereby obtaining the liraglutide crystal.
In one embodiment, the crystallization reagent comprises a basic amino acid, a phenolic compound, and polyethylene glycol. The basic amino acid is selected from one or more of arginine, lysine or histidine. The phenolic compound is, for example, phenol. The polyethylene glycol has a molecular weight of more than 1000. The polyethylene glycol is, for example, PEG 1000, PEG 1500, PEG 2000, PEG 4000, PEG5000, PEG 8000, or the like.
In one embodiment, the final concentration of the basic amino acid is 0.1 to 1.0M based on the total volume of the crystallization system. And the final concentration of the phenolic compound is 3-8 g/L based on the total volume of the crystallization system. The final concentration of the polyethylene glycol is 0.1-0.5g/L based on the total volume of the crystallization system.
The final concentration of the basic amino acid is selected from any of the following ranges based on the total volume of the crystallization system: 0.1 to 0.3M, 0.3 to 0.5M, 0.5 to 0.7M or 0.7 to 1.0M.
The final concentration of the phenolic compound is selected from any of the following ranges based on the total volume of the crystallization system: 3-4 g/L, 4-5 g/L, 5-6 g/L, 6-7 g/L, 7-8 g/L.
The final concentration of the polyethylene glycol is selected from any range of the following, based on the total volume of the crystallization system: 0.1-0.2g/L, 0.2-0.3g/L, 0.3-0.4g/L or 0.4-0.5 g/L.
In one embodiment, the pH of the crystallization system is adjusted to 6.5-8.0 after the addition of the crystallization reagent. Preferably, the pH of the crystallization system is adjusted to 6.8 to 7.2.
In one embodiment, the pH is adjusted and the mixture is stirred for 60-90 min at 20-25 ℃. Stirring at a proper temperature can make the crystallization system uniform, and the generation of crystals is facilitated.
In one embodiment, the liraglutide is crystallized by standing for 8-12h at 2-8 ℃ after stirring is finished, and the liraglutide crystal is obtained by separation after crystallization.
In one embodiment, the liraglutide crystals obtained by the separation are washed and dried.
Washing refers to washing the liraglutide protein crystal with water to remove residual buffer salt, phenol, PEG5000 and organic solvent; and the drying means removing water in the liraglutide protein wet powder and harvesting the liraglutide crystal.
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Example 1
1.1 modifications
A mixture of 74mg of Arg34-GLP-1(7-37) (sequence scheme shown in FIG. 2), 80mg of EDPA, 5.2ml of 50% acetonitrile in water was gently shaken at room temperature until complete dissolution. Palmitic acid-glutamic acid-OSU (available from Doukang Biotechnology Co., Ltd., product No. PK000574) (36mg, CAS No. 294855-91-7, structural formula: see
) Dissolved in acetonitrile (884. mu.L), added to the protein solution, and shaken gently at room temperature for 1 hour and 20min, followed by addition of glycine (36mg) dissolved in 50% aqueous ethanol (36ml) to terminate the reaction. After the reaction was completed, the reaction mixture was diluted three times with water, the pH was adjusted to 4.5 with acetic acid, and the precipitate was collected by centrifugation, redissolved with 1M Tris, and then diluted 8 to 10 times with water to prepare C8 for purification. The schematic diagram of the modification reaction is shown in FIG. 3.
1.2 purification
Chromatography packing: c8-100-10(20mm, 80 ml); mobile phase A: 50mM Tris, pH 9.0; mobile phase B: ACN; the elution gradient is 0-100% of the B liquid within 60 min. And collecting liraglutide fractions according to the ultraviolet absorption value, precipitating at isoelectric points, and drying to obtain 25mg of liraglutide dry powder. A small amount of the suspension was microscopically examined and the liraglutide product was seen as an amorphous powder as shown in fig. 4. The recovery rate of Arg34-GLP-1(7-37) to liraglutide dry powder is 33.78%, and the calculation method of the recovery rate in the invention comprises the following steps: the liraglutide production was divided by the Arg34-GLP-1(7-37) input, i.e., 25/74-33.78% in this example.
Example 2
2.1 preparation of the solution
Modification buffer I: 20mM sodium carbonate, 60% acetonitrile in water, 0.5mM EDTA, pH 10.5.
Modification buffer II: 100mM sodium carbonate.
Preparation of Arg34-GLP-1(7-37) solution: taking 900mg of Arg34-GLP-1(7-37) freeze-dried powder, fully suspending with a modified buffer solution II according to 20mg/ml, supplementing acetonitrile with the same volume, stirring until the mixture is clear, and controlling the pH value to be 10.0.
Preparation of modifier solution: dissolving palmitic acid-glutamic acid-OSU (180mg) in acetonitrile (10 ml).
Eluent 1: 5% acetonitrile; eluent 2: a: 20mM Tris, 30% acetonitrile; b: 20mM Tris, 0.3M NaCl, 30% acetonitrile.
2.2 modification and purification
Filling NanoQ-15L filler into a chromatographic column with the diameter of 20mm and the height of 25cm, washing the chromatographic column with 0.5M sodium hydroxide for 2 times of the column volume, washing the chromatographic column with purified water for 2 times of the column volume, replacing the column volume for 4 times with modified buffer solution I, and pumping Arg34-GLP-1(7-37) protein solution. The column volume was re-equilibrated 2-3 times with modified buffer I. Injecting the modifier solution into the chromatographic column by using a syringe, and then circularly balancing by using the modification buffer solution I for reaction for 60 min.
After finishing the modification reaction, eluting 3 times of column volume by eluent 1, and then carrying out gradient elution: 10% of B to 30% of B, and eluting by 2 times of column volume; 30% B-50% B, eluting 10 times of column volume, and eluting at flow rate of 15 ml/min. According to UV280Collecting the liraglutide fraction.
2.3 crystallization of liraglutide
Supplementing 0.5M arginine, 5g/L phenol and PEG50000.2g/L into the liraglutide protein fraction, adjusting the pH to 7.0, standing for 10h at 4 ℃, and centrifugally collecting liraglutide protein crystal wet powder. And adding 20ml of water into each gram of wet protein powder, washing for three times, centrifuging, and drying in vacuum to obtain the liraglutide dry powder 506 mg. A small amount of the suspension was subjected to microscopic examination, and as shown in FIG. 5, it was found that the uniformity was good and the crystal shape was regular.
The recovery of Arg34-GLP-1(7-37) to liraglutide was 56.22%.
Example 3
3.1 solution preparation
Modification buffer I: 20mM sodium carbonate, 60% acetonitrile in water, 0.5mM EDTA, pH 10.5.
Modification buffer II: 100mM sodium carbonate.
Protein solution preparation: 5.55g of Arg34-GLP-1(7-37) -OH freeze-dried powder is taken, fully resuspended by modified buffer solution II according to 20mg/ml, and then equal volume of acetonitrile is added to be stirred until the mixture is clear, and the pH value is controlled to be 10.5.
Preparation of modifier solution: dissolving 1g of palmitic acid-glutamic acid-OSU in 50ml of acetonitrile.
Eluent 1: 5% acetonitrile; eluent 2: a-20mM Tris, 30% acetonitrile; b-20mM Tris, 0.3M NaCl, 30% acetonitrile.
3.2 modification and purification
Packing Source 15Q filler in a chromatographic column with the diameter of 50mm and the height of the column of 25cm, washing 2 column volumes with 0.5M sodium hydroxide, washing 2 column volumes with purified water, regenerating 3 column volumes with solution B, balancing 4 column volumes with modified buffer solution I, and loading protein. The column volume was re-equilibrated 2-3 times with modified buffer I. Pumping the modifier solution into the chromatographic column by a pump, circularly balancing by using a modification buffer solution I, and controlling the reaction time for 90 min.
After finishing the modification reaction, eluting 3 times of column volume by eluent 1, and then carrying out gradient elution: 10% of B to 30% of B, and eluting by 2 times of column volume; 30% B-50% B, eluting 10 times of column volume, and eluting flow rate of 50 ml/min. According to UV280Collecting the liraglutide fraction.
3.3 crystallization of liraglutide
Supplementing 0.5M arginine, 5g/L phenol and PEG50000.2g/L into the liraglutide protein fraction, adjusting the pH to 7.0, standing for 10h at 4 ℃, and centrifugally collecting liraglutide protein crystal wet powder. And adding 20ml of water into each gram of wet protein powder, washing for three times, centrifuging, and drying in vacuum to obtain 3.34g of liraglutide. A small amount of the suspension was subjected to microscopic examination, and as shown in FIG. 6, it was found that the uniformity was good and the crystal shape was regular.
The recovery of Arg34-GLP-1(7-37) to liraglutide was 60.18%.
Example 4
4.1 solution preparation
Modification buffer I: 20mM sodium carbonate, 0.5mM EDTA, 65% acetonitrile in water, pH 10.5.
Modification buffer II: 100mM sodium carbonate.
Protein solution preparation: 32.00g of Arg34-GLP-1(7-37) -OH freeze-dried powder is taken, fully resuspended by modified buffer solution II according to 20mg/ml, and then equal volume of acetonitrile is added to be stirred until the mixture is clear, and the pH value is controlled to be 9.0.
Preparation of modifier solution: dissolving palmitic acid-glutamic acid-OSU (6.4g) in acetonitrile (320 ml).
Eluent 1: 5% acetonitrile; eluent 2: a-20mM Tris, 30% acetonitrile; b-20mM Tris, 0.3M NaCl, 30% acetonitrile.
4.2 modification and purification
BestPoly 15Q filler is filled in a chromatographic column with the diameter of 150mm, and the column height is 25 cm. Washing 2 times of column volume with 0.5M sodium hydroxide, washing 2 times of column volume with purified water, regenerating 3 times of column volume with solution B, balancing 4 times of column volume with modified buffer solution I, and pumping protein solution. The column volume was re-equilibrated 2-3 times with modified buffer I. Pumping the modifier solution into the chromatographic column by a pump, circularly balancing by using a modification buffer solution I, and controlling the reaction time for 120 min.
After finishing the modification reaction, eluting 3 times of column volume by eluent 1, and then carrying out gradient elution: 10% of B to 30% of B, and eluting by 2 times of column volume; 30 percent of B to 50 percent of B, eluting 10 times of column volume, and eluting at the flow rate of 500 ml/min. According to UV280Collecting the liraglutide fraction.
4.3 crystallization of liraglutide
Supplementing 0.5M arginine, 5g/L phenol and PEG50000.2g/L into the liraglutide protein fraction, adjusting the pH to 7.0, standing for 10h at 4 ℃, and centrifugally collecting liraglutide protein crystal wet powder. And adding 20ml of water into each gram of wet protein powder, washing for three times, centrifuging, and drying in vacuum to obtain 20.65g of liraglutide. A small amount of the suspension was subjected to microscopic examination, and as shown in FIG. 7, it was found that the uniformity was good and the crystal shape was regular.
The recovery of Arg34-GLP-1(7-37) to liraglutide was 64.53%.
Comparative summary
1. A comparison of the recovery rates of liraglutide of the different examples is shown in table 1:
TABLE 1 comparison of recovery of liraglutide for different examples
| Examples | Example 1 | Example 2 | Example 3 | Example 4 |
| Arg34-GLP-1(7-37) -OH input | 74mg | 900mg | 5.55g | 32.00g |
| Liraglutide yield | 25mg | 506mg | 3.34g | 20.65g |
| Liraglutide production recovery rate | 33.78％ | 56.22％ | 60.18％ | 64.53％ |
As can be seen from the above table, liraglutide prepared by the manufacturing process of the present invention (examples 2, 3, and 4) has an advantage in production recovery rate as compared to the conventional manufacturing process of liraglutide (example 1). And the invention has stable recovery rate in the scale-up production of chromatographic scale 20mm (example 2), 50mm (example 3) and 150mm (example 4).
2. Comparison of the stability of liraglutide prepared in different examples
Accelerated test
Liraglutide prepared in different examples was placed in a stability incubator in the dark, incubated at 4 ℃ for 12 weeks, and total heteroproteins and high molecular proteins were measured using High Performance Liquid Chromatography (HPLC) for 0 week, 4 weeks, 8 weeks, and 12 weeks, respectively.
The total impurity detection method comprises the following steps: a chromatographic column Kromasil C4 column or other equivalent columns, a mobile phase A of ammonium phosphate acetonitrile buffer solution with the pH value of 8.5, and a mobile phase B of 80% acetonitrile water solution with the detection wavelength of 215 nm.
Detection method of HMWP: chromatographic column Waters Insulin HWMP gel column or other equivalent column, mobile phase is glacial acetic acid-isopropanol-water solution, and detection wavelength is 276 nm.
The liraglutide accelerated test polymer proteins prepared in the different examples and the total hybridization results are shown in table 2 and fig. 8. Wherein the high molecular protein is less than 0.1% in the accelerating process of 12 weeks, and the high molecular protein does not increase obviously in each embodiment. Overall, example 1 increased by about 0.123%/week, and examples 2, 3 and 4 increased by 0.004%/week, 0.007%/week and 0.021%/week, respectively, all far less than example 1.
TABLE 2 summary of data from various examples of the accelerated Liraglutide test
The above accelerated tests confirmed that the crystalline liraglutide prepared in examples 2, 3 and 4 was superior in stability compared to the amorphous powdery liraglutide of example 1.
ThT fluorescence method
Amyloid fibrils in GLP-1 (mainly β -sheet labile proteins) affect their stability, and different manufacturing processes lead to different growth rates of amyloid fibrils, which in turn lead to inconsistent stability during storage. ThT (thioflavin T) can bind to amyloid fibres in a sample, by increasing the incubation temperature, the ThT can rapidly react with increased amyloid fibres in the preparation, the growth rate of the amyloid fibres can be recorded by detecting fluorescence emitted during the reaction, and the lag time T can be calculated by curve fittinglag(time required to achieve rapid increase in fluorescence)) The stability of different samples can be compared by the residence time, with greater lag times representing better stability.
Liraglutide prepared in each example was prepared as a 2mg/ml solution, 200. mu.L of each solution was put in a 96-well plate, 4. mu.L of each 50mM ThT solution was added, mixed in the 96-well plate, incubated in an microplate reader at a constant temperature of 40 ℃ and then fluorescence intensity (excitation wavelength: 440nm, emission wavelength: 480nm) was continuously measured. Calculating t from the kinetic curves1/2(time to reach half of the maximum of fluorescence absorption), when T is calculated by the formulalag＝t1/2-2/k. Where k is the slope of the growth-period curve.
The kinetics curves of examples 1, 2, 3 and 4 are shown in fig. 9 to 12, and the corresponding lag times are shown in table 3 below.
TABLE 3 different examples liraglutide lag times TlagComparison of
| Examples | Example 1 | Example 2 | Example 3 | Example 4 |
| K | 28.25 | 49.61 | 48.27 | 52.76 |
| t1/2 | 2.32 | 4.39 | 4.19 | 4.83 |
| Tlag | 2.24 | 4.35 | 4.15 | 4.79 |
The crystalline liraglutide prepared in examples 2, 3 and 4 had a lag time greater than that of the amorphous powder liraglutide of example 1, demonstrating that crystalline liraglutide was more excellent in stability.
The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the invention set forth herein, as well as variations of the methods of the invention, will be apparent to persons skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.
Claims (13)
1. Use of a polymeric anion exchange filler in the preparation of liraglutide.
2. Use according to claim 1, characterized in that the polymeric anionic filler is selected from NanoQ-15L, Source 15Q or BestPoly 15Q.
3. Use according to claim 1, wherein the use is of a polymeric anion exchange filler in the preparation of liraglutide by reacting a liraglutide intermediate with a liraglutide modifier.
4. The use according to claim 3, wherein the liraglutide intermediate is Arg34-GLP-1(7-37) and/or the liraglutide modifier is a liraglutide fatty acid side chain.
5. Use according to claim 3, wherein the liraglutide modifier is palmitic acid-glutamic acid-OSU.
6. A preparation method of liraglutide is characterized by comprising the following steps:
1) filling a polymer anion exchange filler on the chromatographic column, and replacing the chromatographic column by using a modification buffer solution I;
2) adding the liraglutide intermediate solution into a chromatographic column;
3) adding a liraglutide modifier into a chromatographic column to perform modification reaction on the column;
4) and eluting and collecting the liraglutide after the modification reaction is completed.
7. The method of claim 6, wherein the polymeric anionic filler is selected from the group consisting of NanoQ-15L, Source 15Q and BestPoly 15Q.
8. The method according to claim 6, wherein the modification buffer I comprises 10-50mM sodium carbonate, 0.1-1.0 mM EDTA, and 50-70% acetonitrile in water by volume based on the total volume of the modification buffer I.
9. The method of claim 6, further comprising one or more of:
a) in the step 2), the liraglutide intermediate is Arg34-GLP-1 (7-37);
b) in the step 3), the modification reaction is that the side chain amino Lys at the 26-position of Arg34-GLP-1(7-37) reacts with a liraglutide modifier on a chromatographic column;
c) in step 3), according to the liraglutide intermediate: adding the liraglutide modifier into a chromatographic column according to the molar ratio of 1: 1-1: 1.2;
d) in the step 3), the liraglutide modifier is palmitic acid-glutamic acid-OSU;
e) in step 3), the reaction time of the modification reaction is 0.5 to 2 hours.
10. The preparation method of claim 6, further comprising adding a crystallization reagent to the liraglutide eluted and collected in the step (4) for crystallization, thereby obtaining the liraglutide crystal.
11. The method of claim 10, wherein the crystallization reagent comprises a basic amino acid, a phenolic compound, and polyethylene glycol.
12. The method of claim 11, further comprising one or more of:
1) the basic amino acid is selected from one or more of arginine, lysine or histidine;
2) the phenolic compound is phenol;
3) the molecular weight of the polyethylene glycol is more than 1000; preferably, the polyethylene glycol is PEG 5000.
13. The method according to claim 11, wherein the final concentration of the basic amino acid is 0.1 to 1.0M, the final concentration of the phenolic compound is 3 to 8g/L, and the final concentration of the polyethylene glycol is 0.1 to 0.5g/L, based on the total volume of the crystal system.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110954539.0A CN113621046B (en) | 2021-08-19 | 2021-08-19 | Use and method of polymer anion exchange filler in preparation of liraglutide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110954539.0A CN113621046B (en) | 2021-08-19 | 2021-08-19 | Use and method of polymer anion exchange filler in preparation of liraglutide |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113621046A true CN113621046A (en) | 2021-11-09 |
| CN113621046B CN113621046B (en) | 2022-05-27 |
Family
ID=78386629
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110954539.0A Active CN113621046B (en) | 2021-08-19 | 2021-08-19 | Use and method of polymer anion exchange filler in preparation of liraglutide |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113621046B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000055203A1 (en) * | 1999-03-15 | 2000-09-21 | Novo Nordisk A/S | Ion exchange chromatographic separation of glp-1 and related peptides |
| CN101279999A (en) * | 2008-05-21 | 2008-10-08 | 大连理工大学 | Method for modifying hirudin by polyethyleneglycol assisted by anion exchange column |
| US20170283478A1 (en) * | 2014-07-11 | 2017-10-05 | Dr. Reddy's Laboratories Limited | Process for preparation of liraglutide |
| WO2019082138A1 (en) * | 2017-10-27 | 2019-05-02 | Lorven Biologics Private Limited | Process for preparation of liraglutide using recombinant saccharomyces cerevisiae |
| WO2021130645A1 (en) * | 2019-12-23 | 2021-07-01 | Shilpa Medicare Limited | An improved process for preparation of liraglutide |
-
2021
- 2021-08-19 CN CN202110954539.0A patent/CN113621046B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000055203A1 (en) * | 1999-03-15 | 2000-09-21 | Novo Nordisk A/S | Ion exchange chromatographic separation of glp-1 and related peptides |
| CN101279999A (en) * | 2008-05-21 | 2008-10-08 | 大连理工大学 | Method for modifying hirudin by polyethyleneglycol assisted by anion exchange column |
| US20170283478A1 (en) * | 2014-07-11 | 2017-10-05 | Dr. Reddy's Laboratories Limited | Process for preparation of liraglutide |
| WO2019082138A1 (en) * | 2017-10-27 | 2019-05-02 | Lorven Biologics Private Limited | Process for preparation of liraglutide using recombinant saccharomyces cerevisiae |
| WO2021130645A1 (en) * | 2019-12-23 | 2021-07-01 | Shilpa Medicare Limited | An improved process for preparation of liraglutide |
Non-Patent Citations (1)
| Title |
|---|
| 李东华等: "超氧化物歧化酶吸附分离-化学修饰的耦合过程", 《化工进展》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113621046B (en) | 2022-05-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP4519646B2 (en) | Purification method of preproinsulin | |
| Hochuli | Large-scale chromatography of recombinant proteins | |
| EP3048109A1 (en) | Novel antibody purification method and antibody obtained therefrom, and novel antibody purification method using cation exchanger and antibody obtained therefrom | |
| CN112661815B (en) | Method for purifying Tirzepatide | |
| CN103421079A (en) | Modification method of polyethyleneglycol of protein | |
| CN111606978B (en) | Method for removing endotoxin in escherichia coli recombinant expression porcine parvovirus VP2 protein | |
| CN113621046B (en) | Use and method of polymer anion exchange filler in preparation of liraglutide | |
| EP0446582B1 (en) | Method for producing of recombinant human cysteineless gamma-interferon free of methionine at n-terminal | |
| CN109833648B (en) | Vomitoxin and derivative aptamer affinity column thereof, and preparation method and application thereof | |
| JP5274771B2 (en) | Chromatographic stationary phase regeneration | |
| CN101519445A (en) | Urine follicle-stimulating hormone with high specific activity and method for preparing same | |
| CN112661864B (en) | Purification method of recombinant human GLP-1-Fc fusion protein | |
| CN111057138A (en) | Method for separating and purifying recombinant human growth hormone from genetically engineered rice seeds | |
| CN105017401B (en) | Purification method of ziconotide | |
| CN112341535A (en) | Method for preparing insulin by separation and purification through ion exchange chromatography | |
| CN112279895B (en) | Preparation method of chemically synthesized acidic polypeptide | |
| CN107698676B (en) | Method for extracting and preparing high-purity menotrophin | |
| CN113683663A (en) | Purification method of organism-protected polypeptide crude product | |
| CN112500473A (en) | Separation and purification method of exenatide acetate | |
| CN109608516B (en) | Rapid purification method of royal jelly major proteins 1, 2 and 3 | |
| CN112986428A (en) | N-glycosylation liquid phase analysis method of pegylated recombinant human erythropoietin | |
| CN111208242A (en) | Method for detecting GLP-1 or analog thereof by using high performance liquid chromatography | |
| CN111138523A (en) | Method for purifying and preparing recombinant chicken interferon α from recombinant chicken interferon α renaturation solution | |
| RU2126690C1 (en) | Method of purification of crude insulin obtained from pig pancreas | |
| CN114957405B (en) | Protease degradation resistant oral absorption protein and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |