CN105017381A - Purification method of liraglutide - Google Patents
Purification method of liraglutide Download PDFInfo
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- CN105017381A CN105017381A CN201510425791.7A CN201510425791A CN105017381A CN 105017381 A CN105017381 A CN 105017381A CN 201510425791 A CN201510425791 A CN 201510425791A CN 105017381 A CN105017381 A CN 105017381A
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Abstract
The invention relates to a purification method of liraglutide obtained by solid phase synthesis. The purification method mainly solves the technical problem that the prior art produces low purity liraglutide and has a low yield. The purification method comprises dissolving crude peptide obtained by solid phase synthesis in a dilute ammonia water solution with mass percentage concentration of 10%, and carrying out purification by processes corresponding to two different systems according to difference of sample adsorption separation capabilities of polystyrene-divinylbenzene and octadecylsilane chemically bonded silica base fillers so that a high-purity, high-quality and high-yield liraglutide bulk drug is obtained.
Description
Technical field
The present invention relates to a kind of purification process of polypeptide compound, mainly relate to and use different substrates filler to be the chromatographic column of stationary phase and to combine from different purification system, the method for a kind of solid phase synthesis Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] of purifying.
Background technology
Diabetes (Diabetes Mellitus, DM) are the morbidities of a kind of global height, and about there are 400,000,000 2 diabetes mellitus types in the whole world, and by 2035, this numeral close to 600,000,000, wherein 80% will live in middle and low income country.According to epidemiology statistics, China diabetic subject nearly 9,200 ten thousand in 2010.Because diabetes relate to each system of whole body, even bring out the more causing property complication, have a strong impact on the work capacity of appointing, and threaten the life security of people, great harm is formed to your health.Although the clinical guidelines of existing multiple 2 patients with type Ⅰ DM management, still lacks the related data of country variant and regional case control's pattern, the factor affecting Treatment decsion and corresponding treatment final result at present.The DISCOVER(NCT02322762 that professor Ji Linong etc. carry out) related data that will provide from 25 countries in 5 continent, 12000 2 diabetes mellitus types of research project.
Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] to be first be also the high plain sample peptide-1 of a unique people GLP-1(pancreas at present) analogue, can long-acting treatment diabetes, obtain widespread use at European u.s.a. and japan.The high plain sample peptide-1 of pancreas is a kind of secretin, and when oral absorption nutritive substance, the high plain sample peptide-1 of pancreas can promote that dividing of Regular Insulin is secret, but the GLP-1 of human body can be degraded rapidly by dipeptidyl peptidase-4 (DPP-4).Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] is GLP-1 receptor full agonists, and its aminoacid sequence of 97% is identical with people GLP-1 molecule.People's structure is said, two intermolecular only has two places to there is difference, and first C16 lipid acid is connected on Methionin by L-glutamic acid on the 26th.Secondly, the 34th on Methionin substitute by arginine, to guarantee that C16 side chain can only be combined on the 26th.Fatty acid side chain can make Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] reversibly be combined with albumin in blood night, make the extended durations of action of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], and the opposing strengthened DPP-4 enzyme liberating, fatty acid side chain can also make Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] molecule, and in injection site, selfing is unified into heptamer, thus delay it from subcutaneous attraction, make to be its action time close to 24 hours, every day injects once and can inject at any time, irrelevant with dining.Be applicable to adults with type 2 diabetes; regulate blood sugar in the mode that glucose concn relies on, directly protect B cell, increase insulin secretion and reduce the glicentin secretion improperly of a cell; by improving satiety and reducing energy intake and reduce body weight, reduce systolic pressure. more early application is better.Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] is definitely the revolutionary medicine in diabetes B treatment field.
Within 2008, Novo Nordisk Co., Ltd develops the new drug for the treatment of diabetes, glucagon-like peptide-1 (GLP-1) analogue-Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] (Liraglutide).Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] is glucagon sample peptide 1 (GLP-1) derivative after fatty acid modifying, can reduce the HbA1c level of diabetes B (T2DM) patient, reduces body weight and improve β cell function.Because it only just stimulates insulin secretion higher than during normal value at blood sugar for human body, glucagon suppression is secreted, and therefore can not cause severe hypoglycemia untoward reaction.On January 25th, 2010, Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] is used for the treatment of diabetes B by FDA approval, can separately as the second line treatment medicine of diabetes B, also can with other oral antidiabetic drug conbined usage.Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] injection is isotonic injection liquid, subcutaneous injection administration, and peak reaching time of blood concentration is 10 ~ 14h, and the transformation period is 11 ~ 13h, and injection every day once, provides the glycemic control of 24h, and its pharmacokinetic properties is not by sex or age effects.
Also have much for method and the relevant patent of purifying Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] at present, but there is a problem inside, complex steps and yield is very low, also has that the packing variety of the chromatogram pillar related to inside some patents is more and some filler is also very expensive.Because Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] is 31 very hydrophobic peptides, not only separating effect is bad but also the damage of yield very low pillar is also very large to adopt general purification process.
Summary of the invention
The object of the present invention is to provide a method being suitable for purifying Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], mainly solve prior art and be separated and obtain the lower and technical problem that yield is low of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] purity.
For achieving the above object, technical scheme of the present invention is as follows: a kind of method of purifying Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], is characterized in that, comprise the steps:
1) the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] study of solid phase synthesis being dissolved in mass percentage concentration is in the weak ammonia dissolving of 10%, obtains thick liquid;
2) thick liquid is got, with the filler of polystyrene divinyl phenyl matter for stationary phase, the ammonia aqueous solution that configuration is 0.1% containing mass percentage concentration is B phase, be 0.1% ammoniacal liquor acetonitrile solution containing mass percentage concentration be A phase, gradient is 20-70%, determined wavelength 245nm carries out HPLC linear gradient elution, obtains the first step sample solution, and sample solution is meta-alkalescence;
3) get the first step sample solution mass percentage concentration be 20% bicarbonate of ammonia adjust sample solution pH value to partial neutral, then about 50-500ml (removing too much acetonitrile solution, for second step purifying is prepared) is evaporated to sample solution;
4) sample solution of partial neutral is carried out second step purifying, take octadecylsilane chemically bonded silica as stationary phase, the aqueous hydrochloric acid that configuration is 0.01% containing mass percentage concentration is B phase, the hydrochloric acid acetonitrile being 0.01% containing mass percentage concentration is dissolved as A phase, gradient is that 40-65% carries out HPLC linear gradient elution, obtain second step sample dissolution, sample solution is slant acidity;
5) get second step sample solution weak anion resin and turn salt to alkalescence on the weak side, obtain containing weakly alkaline sample solution, the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] product under weak basic condition is relatively more stable;
6) concentrating under reduced pressure is carried out by turning the sample dissolution after salt, freezing, dry, obtain the finished product.
Beneficial effect of the present invention: find that polystyrene divinyl benzene can bear strong acid and strong base after deliberation, with the chromatographic column that this filler is stationary phase, add alkaline mass percentage concentration be 0.1% ammonia-water systems do the first step purifying, and then be the chromatographic column of stationary phase in order to common octadecylsilane chemically bonded silica, add mass percentage concentration be 0.05% hydrochloric acid system do second step purifying.Two large steps finally can obtain highly purified Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] finished product, and yield is up to 64.5%.
Embodiment
A method for purifying Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], comprises the steps:
1. sample preparation: the weak ammonia being about 10% by the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] mass percentage concentration of solid phase synthesis gained dissolves (concentration of ordinary dissolution is about 15mg/ml) is that to collect filtrate after 0.45um membrane filtration for subsequent use with aperture;
2. purifying
The first step purification condition: chromatographic column: the chromatographic column being stationary phase with the filler of polystyrene divinyl phenyl matter, pillar diameter and length are: 3cm × 25cm. moving phase: A phase: proportioning contains the ammonia aqueous solution that mass percentage concentration is 0.1%; B phase: proportioning contains the ammoniacal liquor acetonitrile solution that mass percentage concentration is 0.1%.Flow velocity: 25-30ml/min.Determined wavelength: 245nm.Gradient: the mass percentage concentration of Mobile phase B: 20-65%, gradient treatment time 40-55min.Sample size is 0.8g;
Purge process: rinse the acetonitrile of chromatographic column mass percentage concentration more than 90% well rear loading, applied sample amount is the sample solution after dissolution filter.Linear gradient elution, collect object peak, purity is about 92%, places in receiving flask for subsequent use by the peptide solution gathered;
3. by the peptide solution collected after purifying with 20% Ammonium bicarbonate food grade adjust sample solution pH value to partial neutral, be evaporated to certain volume 50-100ml except too much acetonitrile, in order to second step purifying is prepared)
4. second step purification condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 3cm × 25cm, moving phase: A matched contains the aqueous hydrochloric acid that mass percentage concentration is 0.01%; B phase: trifluoroacetic acid aqueous solution solution; Flow velocity: 25-30ml/min; Determined wavelength: 245nm. gradient: the mass percentage concentration of Mobile phase B: 40-60%, gradient treatment time 45-60min; Sample size be the first step purified concentration after the sample solution of content 92%;
Purge process: rinse the acetonitrile of chromatographic column mass percentage concentration more than 90% well rear loading, applied sample amount be the first step purified concentration after the sample solution of content 92%, linear gradient elution, collects object peak, purity is about 98%, places in receiving flask for subsequent use by the peptide solution gathered;
5. turn salt: get 100g anionite-exchange resin Lewatit MP60 and be placed in and sizeablely turn salt glass column, with ultrapure water, ethanol, alkali cleaning, pickling, then the series of steps such as alkali cleaning to be rinsed to neutrality loading again and used, pours into the peptide solution after concentrated and turns in salt glass column, reach by the flow velocity controlling liquid sample the object turning salt, collect the peptide solution after turning salt simultaneously;
6. will turn all peptide solutions of gained after salt, carry out being evaporated to 1g/50ml, thickening temperature is no more than 40 DEG C, and then lyophilize obtains the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] that purity is greater than 98.0%, and purification yield can obtain more than 60%.
embodiment 2
1. sample preparation: the weak ammonia being about 10% by the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] mass percentage concentration of solid phase synthesis gained dissolves (concentration of ordinary dissolution is about 30mg/ml) is that to collect filtrate after 0.45um membrane filtration for subsequent use with aperture;
2. purifying
The first step purification condition: chromatographic column: the chromatographic column being stationary phase with the filler of polystyrene divinyl phenyl matter, pillar diameter and length are: 5cm × 25cm. moving phase: A phase: proportioning contains the ammonia aqueous solution that mass percentage concentration is 0.1%; B phase: proportioning contains the ammoniacal liquor acetonitrile solution that mass percentage concentration is 0.1%.Flow velocity: 45-50ml/min.Determined wavelength: 245nm.Gradient: the mass percentage concentration of Mobile phase B: 30-70%, gradient treatment time 50-60min.Sample size is 2.785g;
Purge process: rinse the acetonitrile of chromatographic column mass percentage concentration more than 90% well rear loading, applied sample amount is the sample solution after dissolution filter.Linear gradient elution, collect object peak, purity is about 90%, places in receiving flask for subsequent use by the peptide solution gathered;
3. by the peptide solution mass percentage concentration of collecting after purifying be 20% Ammonium bicarbonate food grade adjust sample solution pH value to partial neutral, be evaporated to certain volume 100-150ml except too much acetonitrile, in order to second step purifying is prepared)
4. second step purification condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 5cm × 25cm, moving phase: A matched contains the aqueous hydrochloric acid that mass percentage concentration is 0.01%; B phase: trifluoroacetic acid aqueous solution solution; Flow velocity: 45-50ml/min; Determined wavelength: 245nm. gradient: the mass percentage concentration of Mobile phase B: 40-65% gradient treatment time 55-65min; Sample size be the first step purified concentration after the sample solution of content 90%;
Purge process: rinse the acetonitrile of chromatographic column mass percentage concentration more than 90% well rear loading, applied sample amount be the first step purified concentration after the sample solution of content 90%, linear gradient elution, collects object peak, purity is about 98%, places in receiving flask for subsequent use by the peptide solution gathered;
5. turn salt: get 500g anionite-exchange resin Diaion WA-30 and be placed in and sizeablely turn salt glass column, with ultrapure water, ethanol, alkali cleaning, pickling, then the series of steps such as alkali cleaning to be rinsed to neutrality loading again and used, pours into the peptide solution after concentrated and turns in salt glass column, reach by the flow velocity controlling liquid sample the object turning salt, collect the peptide solution after turning salt simultaneously;
6. will turn all peptide solutions of gained after salt, carry out being evaporated to 1g/50ml, thickening temperature is no more than 40 DEG C, and then lyophilize obtains the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] that purity is greater than 98.0%, and purification yield can obtain more than 61.5%.
embodiment 3
1. sample preparation: the weak ammonia being about 10% by the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] mass percentage concentration of solid phase synthesis gained dissolves (concentration of ordinary dissolution is about 50mg/ml) is that to collect filtrate after 0.45um membrane filtration for subsequent use with aperture;
2. purifying
The first step purification condition: chromatographic column: the chromatographic column being stationary phase with the filler of polystyrene divinyl phenyl matter, pillar diameter and length are: 10cm × 100cm. moving phase: A phase: proportioning contains the ammonia aqueous solution that mass percentage concentration is 0.1%; B phase: proportioning contains the ammoniacal liquor acetonitrile solution that mass percentage concentration is 0.1%.Flow velocity: 60-80ml/min.Determined wavelength: 245nm.Gradient: the mass percentage concentration of Mobile phase B: 25-70%, gradient treatment time 60-70min.Sample size is 8.9g;
Purge process: rinse the acetonitrile of chromatographic column mass percentage concentration more than 90% well rear loading, applied sample amount is the sample solution after dissolution filter.Linear gradient elution, collect object peak, purity is about 88%, places in receiving flask for subsequent use by the peptide solution gathered;
3. by the peptide solution mass percentage concentration of collecting after purifying be 20% Ammonium bicarbonate food grade adjust sample solution pH value to partial neutral, be evaporated to certain volume 200-300ml except too much acetonitrile, in order to second step purifying is prepared)
4. second step purification condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 10cm × 100cm, moving phase: A matched contains the aqueous hydrochloric acid that mass percentage concentration is 0.01%; B phase: trifluoroacetic acid aqueous solution solution; Flow velocity: 60-80ml/min; Determined wavelength: 245nm. gradient: the mass percentage concentration of Mobile phase B: 45-60%, gradient treatment time 70-80min; Sample size be the first step purified concentration after the sample solution of content 88%;
Purge process: rinse the acetonitrile of chromatographic column mass percentage concentration more than 88% well rear loading, applied sample amount be the first step purified concentration after the sample solution of content 88%, linear gradient elution, collects object peak, purity is about 98%, places in receiving flask for subsequent use by the peptide solution gathered;
5. turn salt: get 1000g anionite-exchange resin Lewatit MP60 and be placed in and sizeablely turn salt glass column, with ultrapure water, ethanol, alkali cleaning, pickling, then the series of steps such as alkali cleaning to be rinsed to neutrality loading again and used, pours into the peptide solution after concentrated and turns in salt glass column, reach by the flow velocity controlling liquid sample the object turning salt, collect the peptide solution after turning salt simultaneously;
6. will turn all peptide solutions of gained after salt, carry out being evaporated to 1g/50ml, thickening temperature is no more than 40 DEG C, and then lyophilize obtains the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] that purity is greater than 98.0%, and purification yield can obtain more than 62.8%.
embodiment 4
1. sample preparation: the weak ammonia being about 10% by the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] mass percentage concentration of solid phase synthesis gained dissolves (concentration of ordinary dissolution is about 80mg/ml) is that to collect filtrate after 0.45um membrane filtration for subsequent use with aperture;
2. purifying
The first step purification condition: chromatographic column: the chromatographic column being stationary phase with the filler of polystyrene divinyl phenyl matter, pillar diameter and length are: 15cm × 100cm. moving phase: A phase: proportioning contains the ammonia aqueous solution that mass percentage concentration is 0.1%; B phase: proportioning contains the ammoniacal liquor acetonitrile solution that mass percentage concentration is 0.1%.Flow velocity: 80-100ml/min.Determined wavelength: 245nm.Gradient: the mass percentage concentration of Mobile phase B: 25-65%, gradient treatment time 80-100min.Sample size is 15g;
Purge process: rinse the acetonitrile of chromatographic column mass percentage concentration more than 90% well rear loading, applied sample amount is the sample solution after dissolution filter.Linear gradient elution, collect object peak, purity is about 85%, places in receiving flask for subsequent use by the peptide solution gathered;
3. by the peptide solution mass percentage concentration of collecting after purifying be 20% Ammonium bicarbonate food grade adjust sample solution pH value to partial neutral, be evaporated to certain volume 400-500ml except too much acetonitrile, in order to second step purifying is prepared)
4. second step purification condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 15cm × 100cm, moving phase: A matched contains the aqueous hydrochloric acid that mass percentage concentration is 0.01%; B phase: trifluoroacetic acid aqueous solution solution; Flow velocity: 80-100ml/min; Determined wavelength: 245nm. gradient: the mass percentage concentration of Mobile phase B: 53-60% gradient treatment time 100-120min; Sample size be the first step purified concentration after the sample solution of content 85%;
Purge process: rinse the acetonitrile of chromatographic column mass percentage concentration more than 90% well rear loading, applied sample amount be the first step purified concentration after the sample solution of content 85%, linear gradient elution, collects object peak, purity is about 98%, places in receiving flask for subsequent use by the peptide solution gathered;
5. turn salt: get 1500g anionite-exchange resin Diaion WA-30 and be placed in and sizeablely turn salt glass column, with ultrapure water, ethanol, alkali cleaning, pickling, then the series of steps such as alkali cleaning to be rinsed to neutrality loading again and used, pours into the peptide solution after concentrated and turns in salt glass column, reach by the flow velocity controlling liquid sample the object turning salt, collect the peptide solution after turning salt simultaneously;
6. will turn all peptide solutions of gained after salt, carry out being evaporated to 1g/50ml, thickening temperature is no more than 40 DEG C, and then lyophilize obtains the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] that purity is greater than 98.0%, and purification yield can obtain more than 64.5%.
Claims (5)
1. a method for purifying Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], is characterized in that, comprises the steps:
1) the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] study of solid phase synthesis is dissolved in weak ammonia dissolving, obtains thick liquid;
2) get thick liquid, with the filler of polystyrene divinyl phenyl matter for stationary phase, ammonia aqueous solution is B phase, ammoniacal liquor acetonitrile solution is A phase, and gradient is 33-65%, and determined wavelength 245nm carries out HPLC linear gradient elution, obtain the first step sample solution, sample solution is meta-alkalescence;
3) getting the first step sample solution bicarbonate of ammonia adjusts sample solution pH value to partial neutral, is then evaporated to 50-100ml to sample solution;
4) sample solution of partial neutral being carried out second step purifying, take octadecylsilane chemically bonded silica as stationary phase, and aqueous hydrochloric acid is B phase, hydrochloric acid acetonitrile is dissolved as A phase, gradient is that 43-60% carries out HPLC linear gradient elution, obtains second step sample dissolution, and sample solution is slant acidity;
5) get second step sample solution weak anion resin and turn salt to alkalescence on the weak side, obtain containing weakly alkaline sample solution;
6) concentrating under reduced pressure is carried out by turning the sample dissolution after salt, freezing, dry, finally obtain the high-quality Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] of high purity.
2. the method for a kind of purifying Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] according to claim 1, is characterized in that, the mass percentage concentration of described weak ammonia is 10%.
3. the method for a kind of purifying Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] according to claim 1, is characterized in that, described ammonia aqueous solution mass percentage concentration is 0.1%; Ammoniacal liquor acetonitrile solution mass percentage concentration is 0.1%.
4. the method for a kind of purifying Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] according to claim 1, is characterized in that, described bicarbonate of ammonia mass percentage concentration is 20%.
5. the method for a kind of purifying Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] according to claim 1, is characterized in that, described aqueous hydrochloric acid mass percentage concentration is 0.01%; Hydrochloric acid acetonitrile solution mass percentage concentration is 0.01%.
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| WO2021017793A1 (en) * | 2019-07-27 | 2021-02-04 | 深圳市健元医药科技有限公司 | Method for preparing chemically synthesized acidic polypeptide |
| WO2021123228A1 (en) | 2019-12-18 | 2021-06-24 | Krka, D.D., Novo Mesto | Pharmaceutical composition comprising glp-1 analogue |
| WO2021135765A1 (en) * | 2019-12-31 | 2021-07-08 | 翰宇药业（武汉）有限公司 | Salt conversion method for glp-1 analogue |
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| CN107903318A (en) * | 2017-12-29 | 2018-04-13 | 江苏诺泰澳赛诺生物制药股份有限公司 | A kind of method for purifying Liraglutide |
| CN108794618A (en) * | 2018-06-25 | 2018-11-13 | 杭州诺泰澳赛诺医药技术开发有限公司 | A method of purifying Liraglutide |
| CN108794618B (en) * | 2018-06-25 | 2021-08-17 | 杭州诺泰澳赛诺医药技术开发有限公司 | Method for purifying liraglutide |
| CN109438569A (en) * | 2018-09-28 | 2019-03-08 | 苏州纳微科技股份有限公司 | A kind of process for separation and purification of Liraglutide |
| CN111269309A (en) * | 2018-12-04 | 2020-06-12 | 翰宇药业（武汉）有限公司 | Purification method of GLP-1 analog polypeptide |
| WO2020127476A1 (en) | 2018-12-19 | 2020-06-25 | Krka, D.D., Novo Mesto | Pharmaceutical composition comprising glp-1 analogue |
| CN109503705A (en) * | 2018-12-26 | 2019-03-22 | 苏州天马医药集团天吉生物制药有限公司 | A kind of isolation and purification method of Liraglutide |
| WO2021017793A1 (en) * | 2019-07-27 | 2021-02-04 | 深圳市健元医药科技有限公司 | Method for preparing chemically synthesized acidic polypeptide |
| WO2021123228A1 (en) | 2019-12-18 | 2021-06-24 | Krka, D.D., Novo Mesto | Pharmaceutical composition comprising glp-1 analogue |
| WO2021135765A1 (en) * | 2019-12-31 | 2021-07-08 | 翰宇药业（武汉）有限公司 | Salt conversion method for glp-1 analogue |
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