CN109503705A - A kind of isolation and purification method of Liraglutide - Google Patents
A kind of isolation and purification method of Liraglutide Download PDFInfo
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- CN109503705A CN109503705A CN201811603717.XA CN201811603717A CN109503705A CN 109503705 A CN109503705 A CN 109503705A CN 201811603717 A CN201811603717 A CN 201811603717A CN 109503705 A CN109503705 A CN 109503705A
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- liraglutide
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- 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
Abstract
The invention discloses a kind of isolation and purification methods of Liraglutide, belong to separating and purifying technology field.Liraglutide crude product is dissolved in the weak aqua ammonia containing 4~6% organic solvents by the present invention obtains thick peptide solution, filtering, filtrate passes through polymethacrylate polymer filler and eight alkyl silane bonded silica gel fillers respectively, and the purification condition of different systems is combined to be isolated and purified, purpose product is collected, is finally obtained total pure 99.5% or more, product of the largest single impurity less than 0.1%, the method is simple, high income, and purity is high is very suitable for producing in batches.
Description
Technical field
The present invention relates to a kind of isolation and purification methods of Liraglutide, belong to separating and purifying technology field.
Background technique
Liraglutide is a kind of glucagon-like peptide 1 (GLP-1) analog of long-acting treatment type-2 diabetes mellitus, is belonged to
GLP-1 receptor stimulating agent is first people's glucagon-like polypeptide-1 (GLP-1) analog for type-2 diabetes mellitus treatment exploitation.
It is developed by Novo Nordisk Co., Ltd, and obtains FDA approval listing January 25 in 2010, SFDA batches are obtained on March 4th, 2011
Standard is in Discussion on Chinese Listed.Liraglutide is as hypoglycemic drug of a new generation based on incretin, not only long action time,
And be sufficiently reserved the multinomial physiological activity of natural GLP-1, can safely and effectively hypoglycemic and may to a variety of cardiovascular dangers because
It is known as protective effect, brings new selection for the treatment of diabetes B.Clinical therapeutic efficacy is encouraging.
Also there are many methods and relevant patent for purifying Liraglutide at present, but complex steps and yield are very
It is low.The invention proposes a kind of available Liraglutide purification process, product purity is high and yield is good and is easy to industrialization.
Summary of the invention
The present invention provides a kind of isolation and purification method of Liraglutide, mainly solves and isolates and purifies Li La in the prior art
The technical problem that Shandong peptide purity is low, yield is low and desalination is difficult.
The first purpose of the invention is to provide a kind of isolation and purification methods of Liraglutide, include the following steps:
(1) Liraglutide crude product is dissolved in the ammonium hydroxide containing 4~6% organic solvents, obtains thick peptide solution, then filters；
(2) filtrate of step (1) is isolated and purified by polymethacrylate polymer filler, wherein A phase is
10~30mmol/L sodium carbonate liquor, B phase are acetonitrile, collect main peak sample；
(3) step (2) are collected to obtained sample by eight alkyl silane bonded silica gel fillers, it is pure to carry out secondary separation
Change, wherein A phase is 90~110mmol/L metabisulfite solution, and B phase is acetonitrile, collects main peak sample；
(4) step (3) are collected to obtained sample, desalination, concentration is carried out using ultrafiltration, obtain Liraglutide.
Further, in step (1), organic solvent is methanol or acetonitrile.It is preferred that methanol.
Further, the pH of the ammonium hydroxide containing 4~6% organic solvents is 8.5~9.5.It is preferred that pH is 9.
Further, in step (2), the pH of sodium carbonate liquor is 8.5~9.5.It is preferred that pH is 9.
Further, in step (2), the gradient of B phase is 30~45%, and the gradient elution time is 35~45min.
Preferred gradient elution time is 40min.
Further, in step (3), the pH of metabisulfite solution is 3~4.It is preferred that pH is 3.2.
Further, in step (3), the gradient of B phase is 35~50%, and the gradient elution time is 35~45min.
Preferred gradient elution time is 40min.
Further, in step (2) or (3), when gradient elution, using 220nm as Detection wavelength.
Further, the molecule interception for the filter membrane that the ultrafiltration uses is 800~1200D.
Further, the method include thes steps that freezing, drying.
The beneficial effects of the present invention are:
The present invention using pH 9 or so and containing 4~6% methanol weak aqua ammonia as dissolution system.PH makes to get profit 9 or so
Shandong peptide is drawn to be completely dissolved, filler is effectively protected in the methanol organic solvent for being added 4~6%.The present invention uses polymethylacrylic acid
Ester polymer filler, more resistant to alkaline flushing filler, filler separating degree is good, filler longer life expectancy.The present invention is used and is purified twice,
Liraglutide is separated using carbanion in first time purification process, impurity effect is more preferable behind removal main peak, receives
Rate is higher.It in second of purification process, is purified using metabisulfite solution, and improves sulfuric acid more concentration and obviously increase separation
Degree optimizes optimum condition of the pH value 3.2, so that Liraglutide purity is higher.
In addition, the present invention uses ultrafiltration desalination, concentration, advantage is: during isolating and purifying, sample nondestructive consumption, full mistake
It is 5-10 times of revolving that journey low temperature, which guarantees sample stability, thickening efficiency faster, and desalting effect is significant, reaches sample and sulfate
Absolutely separation.
Using method of the invention, Liraglutide purifying total recovery is up to 65-70%, and purity is maximum 99.5% or more
List is miscellaneous to be no more than 0.1%.
Detailed description of the invention
Fig. 1 is the high-efficient liquid phase chromatogram of the thick peptide of synthesis in solid state Liraglutide；
Fig. 2 is the high-efficient liquid phase chromatogram for isolating and purifying rear Liraglutide；
Fig. 3 is that the MS of Liraglutide product schemes.
Specific embodiment
The present invention will be further explained below with reference to the attached drawings and specific examples, so that those skilled in the art can be with
It more fully understands the present invention and can be practiced, but illustrated embodiment is not as a limitation of the invention.
Embodiment 1: Liraglutide isolates and purifies
The isolation and purification method of Liraglutide of the invention, includes the following steps:
1, sample dissolves: by 10 grams of crude products, (wherein purpose peptide is 4 grams of Liraglutides, molten also comprising impurity and some solvents
Agent is not shown in HPLC testing result) it is 9 with pH and the weak aqua ammonia containing 5% methanol dissolves ultrasound, it is organic with 0.22um
Membrane filtration obtains crude product solution.
2, primary purifying: step (1) filtrate is isolated and purified by polymethacrylate polymer filler, A phase
Are as follows: 20mmol/L sodium carbonate liquor (pH is adjusted to 9 with phosphoric acid), B phase are as follows: acetonitrile, gradient 30-45%, gradient elution time is
40min, Detection wavelength 220nm, flow velocity 80ml/min, room temperature, sample introduction are the crude product containing 4 grams of purpose products.
Purification process: balancing loading after chromatographic column is rinsed well with 95% acetonitrile, applied sample amount is 4g purpose object.Linearly
Gradient elution 40min collects purpose peak, obtains purity greater than 95% or more fraction, as second step purification of samples, a step yield
About 85% or so.
3, secondarily purified: will once to purify obtained sample by eight alkyl silane bonded silica gel fillers, carry out further
It isolates and purifies, A phase are as follows: 100mmol/L metabisulfite solution (pH is adjusted to 3.2 with sulfuric acid), B phase are as follows: acetonitrile, gradient 35-50% ladder
Degree elution time is 40min, Detection wavelength 220nm, flow velocity 80ml/min, and room temperature, sample introduction is the sample of 3.4 grams of purpose products
Product.
Purification process: balancing loading after chromatographic column is rinsed well with 95% acetonitrile, applied sample amount is 3.4g purpose object.Line
Property gradient elution 40min, collect purpose peak, obtain purity greater than 99.5% or more, largest single impurity is less than 0.1% fraction, as super
Sample before filter, secondarily purified step yield are about 80%.Two step yields are about 68% or so.
4, ultrafiltration desalination: two samples after pure is subjected to desalination by ultrafiltration membrane, low temperature concentration, removes removing sulfate, is returned
Receipts obtain enriched sample.
5, it freezes, is dry: putting sample into lyophilized plate, be freeze-dried, can be obtained 99.5%, largest single impurity is less than
2.7 grams of 0.1% Liraglutide, purifying total recovery are 67.5%.
Embodiment 2: Liraglutide isolates and purifies
The isolation and purification method of Liraglutide of the invention, includes the following steps:
1, sample dissolves: 10 grams of crude products (wherein purpose peptide is 4 grams of Liraglutides, also comprising impurity and some solvents) are used
PH is 9 and the weak aqua ammonia containing 6% methanol dissolves ultrasound, obtains crude product solution with organic membrane filtration of 0.22um.
2, primary purifying: step (1) filtrate is isolated and purified by polymethacrylate polymer filler, A phase
Are as follows: 30mmol/L sodium carbonate liquor (pH is adjusted to 9 with phosphoric acid), B phase are as follows: acetonitrile, gradient 30-45%, gradient elution time is
40min, Detection wavelength 220nm, flow velocity 80ml/min, room temperature, sample introduction are the crude product containing 4 grams of purpose products.
Purification process: balancing loading after chromatographic column is rinsed well with 95% acetonitrile, applied sample amount is 4g purpose object.Linearly
Gradient elution 40min collects purpose peak, obtains purity greater than 95% or more fraction, as second step purification of samples, a step yield
About 81% or so.
3, secondarily purified: will once to purify obtained sample by eight alkyl silane bonded silica gel fillers, carry out further
It isolates and purifies, A phase are as follows: 90mmol/L metabisulfite solution (pH is adjusted to 3.2 with sulfuric acid), B phase are as follows: acetonitrile, gradient 35-50% gradient
Elution time is 40min, Detection wavelength 220nm, flow velocity 80ml/min, and room temperature, sample introduction is the sample of 3.24 grams of purpose products
Product.
Purification process: balancing loading after chromatographic column is rinsed well with 95% acetonitrile, applied sample amount is 3.24g purpose object.
Linear gradient elution 40min collects purpose peak, obtains purity greater than 99% or more, largest single impurity is less than 0.1% fraction, as super
Sample before filter, secondarily purified step yield are about 80%.Two step yields are about 64% or so.
4, ultrafiltration desalination: two samples after pure is subjected to desalination by ultrafiltration membrane, low temperature concentration, removes removing sulfate, is returned
Receipts obtain enriched sample.
5, it freezes, is dry: putting sample into lyophilized plate, be freeze-dried, can be obtained 99%, largest single impurity is less than
2.59 grams of 0.1% Liraglutide, purifying total recovery are 64.8%.
Embodiment described above is only to absolutely prove preferred embodiment that is of the invention and being lifted, protection model of the invention
It encloses without being limited thereto.Those skilled in the art's made equivalent substitute or transformation on the basis of the present invention, in the present invention
Protection scope within.Protection scope of the present invention is subject to claims.
Claims (10)
1. a kind of isolation and purification method of Liraglutide, which comprises the steps of:
(1) Liraglutide crude product is dissolved in the ammonium hydroxide containing 4~6% organic solvents, obtains thick peptide solution, then filters；
(2) filtrate of step (1) is isolated and purified by polymethacrylate polymer filler, wherein A phase be 10~
30mmol/L sodium carbonate liquor, B phase are acetonitrile, collect main peak sample；
(3) step (2) are collected to obtained sample by eight alkyl silane bonded silica gel fillers, carry out secondary separation purifying,
Middle A phase is 90~110mmol/L metabisulfite solution, and B phase is acetonitrile, collects main peak sample；
(4) step (3) are collected to obtained sample, desalination, concentration is carried out using ultrafiltration, obtain Liraglutide.
2. the method according to claim 1, wherein organic solvent is methanol or acetonitrile in step (1).
3. the method according to claim 1, wherein the pH of the ammonium hydroxide for containing 4~6% organic solvents is 8.5
~9.5.
4. the method according to claim 1, wherein in step (2), the pH of sodium carbonate liquor is 8.5~
9.5。
5. the gradient of B phase is 30~45% the method according to claim 1, wherein in step (2),
The gradient elution time is 35~45min.
6. the method according to claim 1, wherein the pH of metabisulfite solution is 3~4 in step (3).
7. the gradient of B phase is 35~50% the method according to claim 1, wherein in step (3),
The gradient elution time is 35~45min.
8. the method according to claim 1, wherein in step (2) or (3), when gradient elution, use
220nm is as Detection wavelength.
9. the method according to claim 1, wherein the molecule interception for the filter membrane that the ultrafiltration uses is 800
~1200D.
10. the method according to claim 1, wherein the method include thes steps that freezing, drying.
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| CN201811603717.XA CN109503705B (en) | 2018-12-26 | 2018-12-26 | Method for separating and purifying liraglutide |
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| CN201811603717.XA CN109503705B (en) | 2018-12-26 | 2018-12-26 | Method for separating and purifying liraglutide |
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| CN109503705B CN109503705B (en) | 2021-04-27 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110845602A (en) * | 2019-11-29 | 2020-02-28 | 苏州天马医药集团天吉生物制药有限公司 | Method for separating and purifying somaglutide |
| CN113049690A (en) * | 2019-12-27 | 2021-06-29 | 翰宇药业（武汉）有限公司 | Polypeptide desalting method |
| CN113121675A (en) * | 2019-12-31 | 2021-07-16 | 翰宇药业（武汉）有限公司 | Salt conversion method of GLP-1 analogue |
| CN114763371A (en) * | 2021-01-13 | 2022-07-19 | 深圳市健元医药科技有限公司 | Salifying preparation method of polypeptide |
| WO2023123591A1 (en) * | 2021-12-28 | 2023-07-06 | 深圳翰宇药业股份有限公司 | Method for purifying glp-1 analog and use thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102584982A (en) * | 2012-02-10 | 2012-07-18 | 深圳翰宇药业股份有限公司 | Method for purifying solid-phase synthetic coarse liraglutide |
| CN104055797A (en) * | 2014-06-17 | 2014-09-24 | 安徽华润金蟾药业股份有限公司 | Detection and identification of two composition components in toad skin extract |
| CN105017381A (en) * | 2015-07-20 | 2015-11-04 | 吉尔生化（上海）有限公司 | Purification method of liraglutide |
| 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 |
-
2018
- 2018-12-26 CN CN201811603717.XA patent/CN109503705B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102584982A (en) * | 2012-02-10 | 2012-07-18 | 深圳翰宇药业股份有限公司 | Method for purifying solid-phase synthetic coarse liraglutide |
| CN104055797A (en) * | 2014-06-17 | 2014-09-24 | 安徽华润金蟾药业股份有限公司 | Detection and identification of two composition components in toad skin extract |
| CN105017381A (en) * | 2015-07-20 | 2015-11-04 | 吉尔生化（上海）有限公司 | Purification method of liraglutide |
| 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 |
Non-Patent Citations (1)
| Title |
|---|
| VINÍCIUS ESTEVES SALGADO等: "Degradation of Optical and Surface Properties of Resin-Based Composites With Distinct Nanoparticle Sizes but Equivalent Surface Area", 《J DENT》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110845602A (en) * | 2019-11-29 | 2020-02-28 | 苏州天马医药集团天吉生物制药有限公司 | Method for separating and purifying somaglutide |
| CN113049690A (en) * | 2019-12-27 | 2021-06-29 | 翰宇药业（武汉）有限公司 | Polypeptide desalting method |
| CN113049690B (en) * | 2019-12-27 | 2022-07-22 | 翰宇药业（武汉）有限公司 | Polypeptide desalting method |
| CN113121675A (en) * | 2019-12-31 | 2021-07-16 | 翰宇药业（武汉）有限公司 | Salt conversion method of GLP-1 analogue |
| CN113121675B (en) * | 2019-12-31 | 2023-03-31 | 翰宇药业（武汉）有限公司 | Salt conversion method of GLP-1 analogue |
| CN114763371A (en) * | 2021-01-13 | 2022-07-19 | 深圳市健元医药科技有限公司 | Salifying preparation method of polypeptide |
| WO2023123591A1 (en) * | 2021-12-28 | 2023-07-06 | 深圳翰宇药业股份有限公司 | Method for purifying glp-1 analog and use thereof |
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| CN109503705B (en) | 2021-04-27 |
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