CN113049690B - Polypeptide desalting method - Google Patents
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Abstract
The invention belongs to the technical field of pharmaceutical chemistry, and discloses a polypeptide desalting method. The polypeptide desalting method includes loading the polypeptide sample to the preparation column, washing with mobile phases A1 and B, washing with mobile phases A2 and B, performing gradient elution with A2 and B, collecting the sample, performing reduced pressure rotary evaporation, and freeze-drying to obtain desalted fine peptide. Compared with the prior art, the method for desalting the polypeptide can be used for transferring the salt on a reversed-phase high-efficiency preparative chromatography system which takes the alkyl bonded silica gel filler as a fixed phase, solves the problem that the sample is difficult to elute during desalting, does not need to additionally increase equipment and materials, can further remove impurities while transferring the salt, improves the purity of the sample, reduces the volume of the sample after transferring the salt and increases the concentration of the sample, has simple operation in the whole process and short production period, and is beneficial to large-scale industrial production.
Description
Technical Field
The invention belongs to the technical field of pharmaceutical chemistry, and relates to a polypeptide desalting method.
Background
The polypeptide is a compound with an amphoteric structure, wherein some polypeptide compounds have both acidic groups and basic groups, and some polypeptide compounds have only acidic groups or only basic groups. Purification of synthetic polypeptides can be accomplished using a variety of buffer salts, such as trifluoroacetic acid, ammonium phosphate (or sodium, potassium), ammonium sulfate (or sodium, potassium), ammonium acetate (or sodium, potassium), ammonium formate (or sodium, potassium), sodium perchlorate (or ammonium, potassium), ammonium bicarbonate (or sodium, potassium), or ammonium chloride (or sodium, potassium), among others. When the purity of the polypeptide is satisfactory and the impurities are reduced to below the required limit, it is necessary to remove the excess buffer salt from the fraction purified in the previous step to obtain the peptide in the form of a salt, i.e., the peptide is bound with a certain amount of acid groups or a certain amount of cations or is not bound with any free state of ions, so that the polypeptide drug can maintain high pharmaceutical activity, good stability or good solubility.
Generally, the peptide is in the form of acetate, trifluoroacetate, hydrochloride or no salt, and part of the polypeptide is combined with a cation to prepare an ammonium, sodium or potassium salt. In the purification process of the synthetic polypeptide, an alkyl bonded silica gel filler is generally adopted as a reversed-phase high-efficiency preparation chromatographic system of a stationary phase, most of salt conversion operations also use the purified chromatographic system, and extra equipment and consumables are prevented from being added due to system replacement. A small part of the polypeptide is subjected to salt conversion by adopting a reversed-phase polymer packing, or adopting an ion exchange system, or adopting a membrane filtration mode. Among the four salt conversion modes, the reversed-phase high-efficiency preparation chromatography system using the alkyl bonded silica gel filler as the stationary phase is the preferred mode, and has the advantages of simple process, low production cost, capability of further removing impurities and the like. However, the reversed-phase polymer packing, the ion system and the membrane filtration method need additional equipment and materials, and have the disadvantages of long treatment period, incomplete salt conversion, low purity, increased impurities and the like, so that the reversed-phase high-efficiency preparation chromatography system is preferred for salt conversion.
However, the reversed-phase high-performance preparative chromatography system generally uses an alkyl-bonded silica gel filler as a stationary phase, and due to the special properties of some polypeptides, the sample cannot be eluted in the process of desalting with pure water and acetonitrile, which leads to serious loss of the sample.
Disclosure of Invention
In view of the above, the present invention provides a method for desalting polypeptide, which can perform salt conversion on a reverse-phase high-performance preparative chromatography system using an alkyl-bonded silica gel filler as a stationary phase, and solve the problem that a sample is difficult to elute during desalting.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
a method for desalting polypeptide comprises loading polypeptide sample onto preparative column, washing with mobile phases A1 and B, washing with mobile phases A2 and B, gradient eluting with A2 and B, collecting sample, rotary steaming under reduced pressure, and lyophilizing to obtain desalted refined peptide;
wherein the mobile phase A1 is selected from pure water or dilute buffer salt solution; the mobile phase A2 is pure water or carbonic acid water solution; the mobile phase B is selected from any one or mixture of several solvents of acetonitrile, methanol, ethanol or isopropanol in any proportion.
The polypeptide desalting method uses the same preparation column for purification and salt conversion, can complete desalting without replacing the filler or adding additional equipment, has simple desalting operation and is beneficial to improving the production efficiency.
In the invention, the method also comprises the step of balancing the preparation column by the mobile phase A1 and the mobile phase B before loading.
The method for desalting the polypeptide improves the yield of the desalting step by improving the sample loading amount in the desalting process. In the invention, the loading amount of the polypeptide sample is 1.0-10.0% of the mass of the prepared column filler. Further, in some embodiments, the polypeptide sample is loaded in an amount of 1.5% to 6.0% by weight of the filler.
In the present invention, the polypeptide is a peptide which is difficult to elute on a reverse phase preparative chromatography. Such as liraglutide, somaglutide.
In the present invention, the polypeptide sample is dissolved in mobile phase A1 and then loaded.
Further, the method of the present invention may further comprise a step of filtering with a filter membrane after the polypeptide is solubilized. In some embodiments, the filter is a 0.45 μm filter.
In the invention, the packing in the preparation column is any one of reversed-phase C18, C8, C4, C1, phenyl, amino or cyano.
In the invention, the particle size of the filler filled in the preparation column is 5-100 mu m, and the aperture of the filler is 5-100 nm, namely
In the invention, the salt used in the dilute buffer salt solution is at least one of ammonium acetate, sodium acetate, potassium acetate, ammonium carbonate, ammonium bicarbonate, sodium carbonate, sodium bicarbonate, potassium carbonate or potassium bicarbonate
Further, the concentration of salt in the dilute buffer salt solution in the method is 5-500 mmol/L. In some embodiments, the salt concentration is 20 to 200 mmol/L.
The method for desalting the polypeptide uses carbonic acid water solution for elution, solves the problem that a sample is difficult to elute on a reversed-phase filler, and improves the yield of the sample in the desalting process.
In the invention, the carbon dioxide concentration of the carbonic acid aqueous solution is 5-500 mmol/L. In some embodiments, the carbon dioxide concentration of the aqueous carbonic acid solution is 10 to 30 mmol/L.
In the invention, the ratio of the mobile phases A1 to B is 90% when the mobile phases A1 and B are washed: 10 percent, and the washing time is 5-15 min.
In the invention, the ratio of the mobile phases A2 to B is 90% when the mobile phases A2 and B are washed: 10 percent, and the washing time is 5-15 min.
In the invention, the ratio gradient of the mobile phase B is 10-60% when the mobile phases A2 and B are subjected to gradient elution, and the elution time is 25-35 min.
Compared with the prior art, the method for desalting the polypeptide can be used for transferring the salt on a reversed-phase high-efficiency preparative chromatography system which takes the alkyl bonded silica gel filler as a fixed phase, solves the problem that the sample is difficult to elute during desalting, does not need to additionally add equipment and materials, can further remove impurities while transferring the salt, improves the purity of the sample, reduces the volume of the sample after transferring the salt and increases the concentration of the sample, has simple operation in the whole process and short production period, and is beneficial to large-scale industrial production.
Drawings
FIG. 1 shows a Liraglutide detection profile prior to desalting;
FIG. 2 shows a detection profile of liraglutide desalinization protamine of example 1;
FIG. 3 shows a detection profile of liraglutide desalinization protamine of example 2;
FIG. 4 shows a detection chromatogram before desalting of Somalrubin;
FIG. 5 shows a somalglutide desalinization depsipeptide detection chromatogram of example 3;
FIG. 6 shows a somalglutide desalinized depsipeptide detection chromatogram of example 4;
FIG. 7 shows a liraglutide desalinization protamine detection profile of comparative example 1;
FIG. 8 shows a detection profile of liraglutide desalted protamine of comparative example 2;
FIG. 9 shows a Somalutide desalinized protamine detection profile of comparative example 3;
fig. 10 shows a somalglutide desalinization protamine assay profile of comparative example 4.
Detailed Description
The embodiment of the invention discloses a method for desalting polypeptide. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is specifically noted that all such substitutions and modifications will be apparent to those skilled in the art and are intended to be included herein. While the methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the methods described herein, as well as appropriate variations and combinations, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.
For a further understanding of the present invention, reference will now be made in detail to the following examples.
Example 1: preparation of liraglutide desalted and refined peptide
Liraglutide 45.0g (purity 99.16%, see FIG. 1 for chromatography) and TFA salt (TFA content 2.9%) were weighed out, dissolved in 1L of 200mmol/L aqueous ammonium bicarbonate solution, and then filtered through a 0.45 μm filter.
A preparation column with an internal diameter of 15cm contains 10 μm of C4 packing (the pore diameter of the packing is 10nm, i.e.Weight 3.0kg), mobile phase a1 was 200mmol/L ammonium bicarbonate and mobile phase B was acetonitrile. 90% A1+ 10% B was equilibrated for 5min, 620mL/min, and then the sample was loaded in its entirety, at an amount of 1.5% of the filler weight. After loading, the sample was washed with 90% A1+ 10% B for 8min, 620 mL/min. Switching A1 to A2, A2 to pure water, washing with 90% A2+ 10% B for 10min, and washing with 620 mL/min; then gradient elution is carried out, the gradient of B is 10-60% (25min), fractions are collected in sections, the purity is detected by UPLC, and samples with the purity more than or equal to 99.0% and the single impurity less than or equal to 0.10% are combined. Then, the mixture is reduced in pressure and rotary evaporated, and freeze-dried to obtain 40.0g of salt-free liraglutide, wherein the yield of the salt-free preparation process is 88.9%, and TFA is not detected in a freeze-dried sample. The detection diagram of the salt-free protamine is shown in figure 2.
Example 2: preparation of liraglutide desalted and refined peptide
Liraglutide 180.0g (purity 99.16%, see FIG. 1 for chromatography) and TFA salt (TFA content 2.9%) were weighed out and dissolved in 4L of 200mmol/L aqueous ammonium bicarbonate solution, followed by filtration through a 0.45 μm filter.
A preparation column with an internal diameter of 15cm contains 10 μm of C4 packing (the pore diameter of the packing is 10nm, i.e.Weight 3.0kg), mobile phase a1 was 150mmol/L ammonium bicarbonate and mobile phase B was 90% ethanol + 10% isopropanol. The 90% A1+ 10% B was equilibrated for 10min, 450mL/min, and then the sample was loaded in its entiretyThe loading was 6.0% by weight of the filler. After loading, the sample was rinsed with 90% A1+ 10% B for 12min, 450 mL/min. Switching A1 to A2, A2 to 30mmol/L carbon dioxide water solution, and washing with 90% A2+ 10% B for 12min and 450 mL/min; then gradient elution is carried out, the gradient of B is 10% -60% (35min), fractions are collected in sections, the purity is detected by UPLC, and samples with the purity more than or equal to 99.0% and the single impurity less than or equal to 0.10% are combined. Then, the mixture was rotary-evaporated under reduced pressure and lyophilized to obtain 173.7g of salt-free liraglutide, the yield of the salt-free preparation process was 96.5%, and no TFA was detected in the lyophilized sample. The detection diagram of the salt-free refined peptide is shown in figure 3.
Example 3: preparation of somaglutide desalinized fine peptide
100.0g of somaglutide (purity 99.23%, see FIG. 4 for chromatography) and TFA salt (TFA content 3.3%) were weighed, dissolved in 2L of 200mmol/L aqueous ammonium bicarbonate solution, and filtered through a 0.45 μm filter.
A preparation column with an internal diameter of 15cm contains 10 μm of C18 packing (the pore diameter of the packing is 10nm, i.e.Weight 3.0kg), mobile phase a1 was 100mmol/L ammonium bicarbonate and mobile phase B was methanol. 90% A1+ 10% B was equilibrated for 10min, 550mL/min, and then the samples were all loaded at 3.3% of the filler weight. After loading, the sample was rinsed with 90% A1+ 10% B for 12min, 550 mL/min. Switching A1 to A2, A2 to 10mmol/L carbon dioxide water solution, and flushing with 90% A2+ 10% B for 12min, 550 mL/min; then gradient elution is carried out, the gradient of B is 10% -60% (35min), fractions are collected by sections, the purity is detected by UPLC, and samples with the purity more than or equal to 99.0% and the single impurity less than or equal to 0.10% are combined. And then carrying out reduced pressure rotary evaporation, and freeze-drying to obtain 95.3g of salt-free somaglutide, wherein the yield of the salt-free preparation process is 95.3%, and TFA is not detected in a freeze-dried sample. The detection diagram of the salt-free refined peptide is shown in figure 5.
Example 4: preparation of somagluteptide desalinized fine peptide
150.0g of somaglutide (purity 99.23%, see FIG. 4 for chromatography) and TFA salt (TFA content 3.3%) were weighed, dissolved in 3L of 200mmol of aqueous ammonium bicarbonate solution and filtered through a 0.45 μm filter.
The inner diameter of the preparation column is 15cm10 μm of C18 filler (filler pore size 10nm, i.e.Weight 3.0kg), mobile phase a1 was 100mmol ammonium bicarbonate and mobile phase B was 80% methanol + 20% isopropanol. 90% A1+ 10% B was equilibrated for 10min, 500mL/min, and then the samples were all loaded at 5.0% of the filler weight. After loading, the sample was rinsed with 90% A1+ 10% B for 10min, 500 mL/min. Switching A1 to A2, A2 to 20mmol/L carbon dioxide water solution, and washing with 90% A2+ 10% B for 10min, 500 mL/min; then gradient elution is carried out, the gradient of B is 10% -60% (35min), fractions are collected in sections, the purity is detected by UPLC, and samples with the purity more than or equal to 99.0% and the single impurity less than or equal to 0.10% are combined. And then carrying out reduced pressure rotary evaporation, and freeze-drying to obtain 145.5g of salt-free somaglutide, wherein the yield of the salt-free preparation process is 97.0%, and TFA is not detected in a freeze-dried sample. The detection diagram of the salt-free protamine is shown in FIG. 6.
Comparative example 1: preparation of liraglutide desalted and refined peptide
Liraglutide 100.0g (purity 99.16%, chromatography see FIG. 1) was weighed out to a TFA content of 2.9% as TFA salt, dissolved in 2L of 200mmol/L aqueous ammonium bicarbonate solution and filtered through a 0.45 μm filter.
A preparation column with an internal diameter of 15cm is filled with 60 μm of polymer filler (Nanmini UniPSN, filler pore diameter 30nm, i.e.Weight 3.0kg), samples were loaded in five times, 20.0g each, 0.67% of the weight of the packing, mobile phase a1 was 200mmol/L ammonium bicarbonate, and mobile phase B was acetonitrile. 90% A1+ 10% B was equilibrated for 5min, 620 mL/min. After loading, the sample was rinsed with 90% A1+ 10% B for 8min, 620 mL/min. Switching A1 to A2, switching A2 to pure water, washing with 90% A2+ 10% B for 20min, and washing with 620 mL/min; then gradient elution is carried out, the gradient of B is 10% -60% (25min), fractions are collected in sections, the purity is detected by UPLC, and samples with the purity more than or equal to 99.0% and the single impurity less than or equal to 0.10% are combined. And then carrying out reduced pressure rotary evaporation, and freeze-drying to obtain 80.5g of salt-free liraglutide, wherein the yield of the salt-free preparation process is 80.5%, and TFA is not detected in a freeze-dried sample. The detection pattern of the salt-free refined peptide is shown in FIG. 7.
Comparative example 2: preparation of liraglutide desalted and refined peptide
Liraglutide 100.0g (purity 99.16%, chromatography see FIG. 1) was weighed out, the TFA salt TFA content was 2.9%, dissolved in 2L of 200mmol/L aqueous ammonium bicarbonate solution, and then filtered through a 0.45 μm filter.
A preparation column with an internal diameter of 15cm is filled with 10 μm of C4 filler (the pore diameter of the filler is 10nm, i.e.Weight 3.0kg) and samples were loaded in five times, 20.0g each, with a loading of 0.67% of the weight of the packing, mobile phase a1 of 200mmol/L ammonium bicarbonate and mobile phase B of acetonitrile. 90% A1+ 10% B was equilibrated for 5min, 620mL/min, and then the samples were all loaded. After loading, the sample was rinsed with 90% A1+ 10% B for 8min, 620 mL/min. Switching A1 to A2, switching A2 to pure water, washing with 90% A2+ 10% B for 10min, and washing with 620 mL/min; then gradient elution is carried out, the gradient of B is 10-60% (25min), fractions are collected in sections, the purity is detected by UPLC, and samples with the purity more than or equal to 99.0% and the single impurity less than or equal to 0.10% are combined. Then, the mixture is reduced in pressure and rotary evaporated, and freeze-dried to obtain 68.0g of salt-free liraglutide, wherein the yield of the salt-free preparation process is 68.0%, and TFA is not detected in a freeze-dried sample. The detection pattern of the salt-free protamine is shown in FIG. 8.
Comparative example 3: preparation of somaglutide desalinized fine peptide
Somaltulipide 100.0g (purity 99.23%, see FIG. 4 for chromatography) and TFA salt (TFA content 3.3%) were weighed out and dissolved in 2L of 200mmol/L aqueous ammonium bicarbonate solution, followed by filtration through a 0.45 μm filter.
A preparation column with an internal diameter of 15cm was filled with 60 μm of a polymer filler (Nami UniPSN, filler pore diameter 30nm, i.e.Weight 3.0kg), the sample was loaded in four times, 25.0g each, 0.83% of the weight of the packing, mobile phase a1 was 200mmol/L ammonium bicarbonate, and mobile phase B was methanol. 90% A1+ 10% B was equilibrated for 10min, 550 mL/min. After loading, the sample was rinsed with 90% A1+ 10% B for 10min, 550 mL/min. The A1 is switched to be A2, and the A2 is switched to be pure waterWashing with 90% A2+ 10% B for 20min, 550 mL/min; then gradient elution is carried out, the gradient of B is 10% -60% (35min), fractions are collected in sections, the purity is detected by UPLC, and samples with the purity more than or equal to 99.0% and the single impurity less than or equal to 0.10% are combined. And then carrying out rotary evaporation under reduced pressure, and freeze-drying to obtain 81.0g of salt-free somaglutide, wherein the yield of the salt-free preparation process is 81.0%, and TFA is not detected in a freeze-dried sample. The detection pattern of the salt-free protamine is shown in FIG. 9.
Comparative example 4: preparation of somagluteptide desalinized fine peptide
Somaltulipide 150.0g (purity 99.23%, see FIG. 4 for chromatography) and TFA salt (TFA content 3.3%) were weighed out and dissolved in 3L of 200mmol/L aqueous ammonium bicarbonate solution, followed by filtration through a 0.45 μm filter.
A10 cm internal diameter preparative column contains 10 μm of C18 packing (packing pore size 10nm, i.e.Weight 1.2kg), the sample was loaded in total, the loading was 12.5% of the weight of the filler, mobile phase a1 was 200mmol/L ammonium bicarbonate, and mobile phase B was acetonitrile. 90% A1+ 10% B was equilibrated for 5min, 620mL/min, and then the samples were all loaded. After loading, the sample was rinsed with 90% A1+ 10% B for 8min, 620 mL/min. When the washing time reaches 3min, a large amount of samples are washed out, the washed-out samples are collected, and the washing is continued for 8min at 90% A1+ 10% B, and the volume is 620 mL/min. Switching A1 to A2, switching A2 to pure water, and washing with 90% A2+ 10% B for 8min and 620 mL/min; then gradient elution is carried out, the gradient of B is 10-60% (25min), fractions are collected in sections, the purity is detected by UPLC, and samples with the purity more than or equal to 99.0% and the single impurity less than or equal to 0.10% are combined. And then carrying out reduced pressure rotary evaporation, and freeze-drying to obtain 65.0g of salt-free somaglutide, wherein the yield of the salt-free preparation process is 43.3%, and TFA is not detected in a freeze-dried sample. The detection pattern of the salt-free protamine is shown in FIG. 10.
The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core idea. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.
Claims (8)
1. A method for desalting polypeptide comprises loading polypeptide sample onto preparative column, washing with mobile phases A1 and B, washing with mobile phases A2 and B, gradient eluting with A2 and B, collecting sample, rotary steaming under reduced pressure, and lyophilizing to obtain desalted refined peptide;
wherein the mobile phase a1 is ammonium bicarbonate; the mobile phase A2 is pure water or carbonic acid water solution; the mobile phase B is selected from any one or mixture of several solvents of acetonitrile, methanol, ethanol or isopropanol in any proportion;
the method also comprises the step of balancing the preparation column with mobile phase A1 and mobile phase B before loading; the ratio of the mobile phases a1 and B was 90%: 10 percent;
the packing in the preparation column is reversed-phase C18 or C4;
the polypeptide sample is liraglutide or somaglutide;
the loading amount of the polypeptide sample is 1.5-6.0% of the mass of the prepared column filler;
the ratio of the mobile phase A1 to the mobile phase B is 90% when the mobile phases A1 and B are washed: 10 percent, and the washing time is 5-15 min; the ratio of the mobile phase A2 to the mobile phase B is 90% when the mobile phases A2 and B are washed: 10 percent, and the washing time is 5-15 min;
the gradient of the mobile phase B is 10% -60% when the mobile phases A2 and B are subjected to gradient elution, and the elution time is 25-35 min.
2. The method of claim 1, wherein the polypeptide sample is loaded after solubilization with mobile phase a 1.
3. The method according to any one of claims 1 to 2, wherein the particle diameter of the packing material for the preparative column is 5 to 100 μm, and the pore diameter of the packing material is 5 to 100 nm.
4. The method according to any one of claims 1-2 wherein the concentration of ammonium bicarbonate is 5 to 500 mmol/L.
5. The method of claim 3, wherein the concentration of ammonium bicarbonate is 5-500 mmol/L.
6. The method according to any one of claims 1-2 or 5, wherein the carbon dioxide concentration of the aqueous carbonic acid solution is 5 to 500 mmol/L.
7. The method according to claim 3, wherein the carbon dioxide concentration of the carbonic acid aqueous solution is 5 to 500 mmol/L.
8. The method according to claim 4, wherein the concentration of carbon dioxide in the aqueous carbonic acid solution is 5 to 500 mmol/L.
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Citations (3)
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 |
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 |
CN110540587A (en) * | 2019-08-30 | 2019-12-06 | 杭州诺泰澳赛诺医药技术开发有限公司 | Chromatographic method for effectively improving purification yield of synthetic peptide |
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US10690635B2 (en) * | 2016-03-23 | 2020-06-23 | Bachem Holding Ag | Purification of glucagon-like peptide 1 analogs |
CN107525880A (en) * | 2017-08-15 | 2017-12-29 | 浙江美华鼎昌医药科技有限公司 | A kind of ultra performance liquid chromatography analysis method of Liraglutide |
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-
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- 2019-12-27 CN CN201911380280.2A patent/CN113049690B/en active Active
-
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- 2020-09-28 WO PCT/CN2020/118280 patent/WO2021129016A1/en active Application Filing
Patent Citations (3)
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 |
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 |
CN110540587A (en) * | 2019-08-30 | 2019-12-06 | 杭州诺泰澳赛诺医药技术开发有限公司 | Chromatographic method for effectively improving purification yield of synthetic peptide |
Non-Patent Citations (3)
Title |
---|
2D-LC as an on-line desalting tool allowing peptide identification directly from MS unfriendly HPLC methods;Luo, Hao 等;《JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS》;20170415;第137卷;第139-145页 * |
基因重组胰高血糖素样肽-1衍生多肽的表达和产物纯化与鉴定;马义 等;《中国生物工程杂志》;20090630;第29卷(第06期);第1-6页 * |
大米肽中压离子交换色谱分离及其免疫活性评价;程云辉 等;《食品与机械》;20180331;第34卷(第03期);第151-155页 * |
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