Purification method of glucagon-like peptide-1 analogue
阅读说明：本技术 一种胰高血糖素样肽-1类似物的纯化方法 (Purification method of glucagon-like peptide-1 analogue ) 是由 翟鹏 张媛媛 赵新宇 曾伶俐 金耀光 陈旭 吴博 于 2020-08-14 设计创作，主要内容包括：本发明公开了一种胰高血糖素样肽-1类似物的纯化方法,所述方法包括使用聚合物反相填料纯化GLP-1类似物。所述方法具有单位体积填料载量高的特点,能够减少样品损失,提高纯化产率,降低生产成本。(The invention discloses a purification method of glucagon-like peptide-1 analog, which comprises purifying GLP-1 analog by using polymer reversed phase packing. The method has the characteristic of high filler loading capacity per unit volume, can reduce sample loss, improves the purification yield and reduces the production cost.)
1. A method of purifying a GLP-1 analogue represented by formula (I), the method comprising purifying the GLP-1 analogue using a polymeric reverse phase packing;
X7X8X9X10TFTSDVSSYLEX22QAAX26X27FIAWLVX34GX36G (I)
wherein:
X7is H or absent;
X8is A, Aib, G, S, V or absent;
X9is E or absent;
X10is G or absent;
X22is G or E;
X26r, K or K with a side chain modified with a fatty acid or fatty acid derivative;
X27e, K or K with a side chain modified with a fatty acid or fatty acid derivative;
X34r, K or K with a side chain modified with a fatty acid or fatty acid derivative;
X36r, G, K or K with a side chain modified with a fatty acid or fatty acid derivative.
2. The method of claim 1, wherein the polymeric reverse phase filler is a polystyrene-divinylbenzene polymer.
3. The method of claim 1 or 2, wherein X is deleted7X8。
4. The process of claim 1 or 2, wherein the reverse phase packing is selected from NM100, diameter HP20SS and/or Super 100.
5. The method of claim 1 or 2, wherein X8Is A, Aib, G, S or V.
6. The method of claim 1 or 2, wherein X22Is G or E.
7. The method of claim 1 or 2, wherein X26R, K or K with a side chain modified with a fatty acid or fatty acid derivative.
8. The method of claim 1 or 2, wherein X27E, K or K with a side chain modified with a fatty acid or fatty acid derivative.
9. The method of claim 1 or 2, wherein X34R, K or K with a side chain modified with a fatty acid or fatty acid derivative.
10. The method of claim 1 or 2, wherein X36R, G, K or K with a side chain modified with a fatty acid or fatty acid derivative.
11. The method of claim 1 or 2, wherein the fatty acid is selected from C12-C22 fatty acids.
12. The method of claim 11, wherein the fatty acid is palmitic acid or stearic acid.
13. The method of claim 1 or 2, wherein the fatty acid derivative is selected from the group consisting of:
Wherein, denotes the terminal as a site linked to a lysine side chain on the polypeptide chain.
14. The method of claim 1 or 2, wherein the GLP-1 analogue of formula (I) is selected from the group consisting of:
(1) GLP1(7-37, 34R), the amino acid sequence of which is:
HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG；
(2) GLP1(9-37,34R), the amino acid sequence of which is:
EGTFTSDVSSYLEGQAAKEFIAWLVRGRG；
(3) GLP1(7-37, 8G, 22E, 36G), the amino acid sequence of which is:
HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG；
(4) liraglutide (Liraglutide) having the structure:
(5) semaglutide (Semaglutide), having the structure:
。
15. the method of claim 1 or 2, wherein the pH of the mobile phase is in the range of 8.5-9.5.
16. The method of claim 15, wherein the mobile phase has a pH of 8.5 to 9.0.
17. The method of claim 1 or 2, wherein the pH is adjusted using an acid or a base; the acid is hydrochloric acid or acetic acid; the alkali is sodium hydroxide.
18. The process of claim 1 or 2, wherein an alcohol is used as the mobile phase; the alcohol is isopropanol, ethanol, butanol or propanol.
19. The method of claim 18, wherein the mobile phase is: 5-50mM Tris, 1-10% w/w isopropanol, pH 8.5-9.5.
20. The method of claim 19, wherein the mobile phase is: 20mM Tris, 5% w/w isopropanol, pH 9.0.
Technical Field
The invention relates to a peptide purification method, in particular to a purification method of glucagon-like peptide-1 analogues.
Background
Diabetes is a group of metabolic diseases characterized by hyperglycemia. Hyperglycemia is caused by a defect in insulin secretion or an impaired biological action, or both. Hyperglycemia occurring in the long term of diabetes results in chronic damage to, and dysfunction of, various tissues, particularly the eyes, kidneys, heart, blood vessels, nerves. In recent years, the prevalence rate of diabetes in China has increased rapidly, and the number of people with diabetes is over one hundred million in the world.
Glucagon-like peptide 1 (GLP-1) acts on pancreatic beta cells in a glucose-dependent manner, promotes the transcription of insulin genes, and increases the biosynthesis and secretion of insulin; stimulating the proliferation and differentiation of beta cells, inhibiting beta cell apoptosis, increasing the number of pancreatic beta cells, inhibiting glucagon secretion, suppressing appetite and ingestion, delaying gastric content emptying, etc. These functions are all beneficial in lowering postprandial blood glucose and maintaining blood glucose at a constant level.
The main form of glucagon-like peptide 1 (GLP-1) that is biologically active in humans is GLP-1 (7-37), but it is susceptible to hydrolysis by dipeptidyl peptidase IV (DPP-IV) with a half-life of less than 5 minutes. Thus, the half-life of the pharmaceutical GLP-1 receptor agonist must be extended. Representative mature GLP1 drugs in the market at present are Liraglutide (Liraglutide) and Semaglutide (Semaglutide) developed by Novonide, and the molecular structure of the GLP1 drugs is a derivative formed by adding a fatty acid side chain on a GLP-1 analogue molecule. The once-a-day administration mode is realized by the functions of shielding (or mutating) DPP-IV enzyme cutting sites and increasing the in-vivo retention time of albumin binding.
Currently, Novonide is produced by adopting a yeast recombinant expression mode, and domestic imitations are mostly realized by adopting chemical synthesis or escherichia coli recombinant expression. The chemical synthesis method mostly adopts the reversed phase chromatography of silica gel with small particle size (below 10 mu m) C8 or C18 for purification; GLP-1 analogues produced by recombinant expression are captured and purified by anion (such as CN110498849A, CN 04592381A) cation (such as CN110128552A, CN 110724187A) exchange chromatography.
The silica gel C8/C18 reverse phase chromatography with small particle size has higher requirements on the sample, and is not suitable for capturing and purifying the recombinant protein sample containing more impurities; meanwhile, the filler mostly uses harmful organic solvents such as acetonitrile and the like as an elution phase, and the environmental protection pressure is large. In addition, such chromatographic packing materials have poor alkali resistance, which is not favorable for on-site cleaning and reuse of the packing materials. GLP-1 analogue produced by recombinant expression is greatly influenced by impurities such as host cell DNA, endotoxin and the like if anion exchange chromatography is adopted for capture and purification; the method for cation exchange capture purification needs to adjust the pH value of a sample to be captured to be below the isoelectric point of protein, and in the case of GLP-1 samples, due to the existence of host protein and nucleic acid, the GLP-1 samples are often co-precipitated in the process of crossing the isoelectric point and are difficult to redissolve, so that a large amount of samples are lost.
Disclosure of Invention
In order to reduce sample loss and improve purification yield, the invention provides a method for purifying GLP-1 analogues, which uses polystyrene-divinylbenzene polymer as reversed phase packing, can realize yield close to or over 90 percent, and can make the loading of the GLP-1 analogues reach more than 30g/L of chromatographic packing.
Specifically, the invention provides the following technical scheme:
a method of purifying a GLP-1 analogue represented by formula (I), the method comprising purifying the GLP-1 analogue using a polymeric reverse phase packing; preferably, the polymeric reverse phase filler is a polystyrene-divinylbenzene polymer,
X7X8X9X10TFTSDVSSYLEX22QAAX26X27FIAWLVX34GX36G (I)
wherein:
X7is H or absent;
X8is A, Aib, G, S, V or absent;
X9is E or absent;
X10is G or absent;
X22is G or E;
X26r, K or K with a side chain modified with a fatty acid or fatty acid derivative;
X27e, K or K with a side chain modified with a fatty acid or fatty acid derivative;
X34r, K or K with a side chain modified with a fatty acid or fatty acid derivative;
X36r, G, K or K with a side chain modified with a fatty acid or fatty acid derivative;
preferably, X is deleted7、X8、X9Or X10One or more of; more preferably, X is deleted7X8；
Preferably, the reverse phase packing is selected from NM100, diameter HP20SS and/or Super 100.
Compared with the prior common process, the method of the invention has the following characteristics:
1. the packing loading per unit volume in the purification process is high and can reach more than 30g GLP-1/L chromatographic packing.
2. The purification steps are simple, isocratic elution is used, the yield close to or over 90 percent can be realized, and the purity of the product can reach about 90 percent.
3. The filler has larger grain diameter, can bear higher content of impurities in a sample, has small back pressure, and does not need to use an expensive high-pressure chromatograph.
4. The filler resists strong acid and strong alkali, can be repeatedly cleaned by sodium hydroxide, effectively removes residual impurities and increases the number of using cycles of the filler.
5. Only a small amount of environment-friendly alcohol organic solvent (such as ethanol, isopropanol and the like) is used in the purification.
6. The price of the filler is lower, and the production cost is low.
Drawings
FIG. 1 is a schematic representation of a GLP-1 fusion protein.
Figure 2 shows the measurement of the loading of GLP1 analogues (a. GLP1(7-37, 34R); b. GLP1(9-37, 34R)) on polystyrene-divinylbenzene polymer reverse phase chromatography NM100 at different pH values.
Figure 3 shows the load measurements of GLP1 analogue (GLP 1(7-37, 8G, 22E, 36G)) on polystyrene-divinylbenzene polymer reverse phase chromatography DIAION HP20SS chromatography at different pH values.
Figure 4 shows the load measurements of GLP1 analogue (GLP 1(9-37, 34R)) on other brand polystyrene-divinylbenzene polymer reverse phase chromatograms at the optimum pH.
Figure 5 shows the measurement of the amount of acylated GLP1 analog on HP20SS on polystyrene-divinylbenzene polymer reverse phase chromatography DIAION at pH 9.0.
Detailed Description
The technical scheme of the disclosure is clearly and completely described in the following with reference to the accompanying drawings. Obviously, all other embodiments obtained by a person of ordinary skill in the art without making creative efforts based on the specific embodiments in the present disclosure belong to the protection scope of the present disclosure.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present disclosure.
The term "GLP-1 analog" as used herein is intended to designate GLP-1 (7-37), GLP-1 (7-36) amides and analogs and derivatives thereof. Such GLP-1 peptides include, but are not limited to, native glucagon-like peptide-1, such as those peptide fragments comprising GLP-1 (7-37) and functional derivatives thereof disclosed in WO 87/06941; these peptide fragments comprising GLP-1 (7-36) and functional derivatives thereof disclosed in WO 90/11296; the active GLP-1 peptides 7-34, 7-35, 7-36 and 7-37 disclosed in WO 91/11457; such GLP-1 derivatives are disclosed in WO 98/08871, wherein a lipophilic substituent is attached to at least one amino acid residue; n-terminally truncated fragments of these GLP-1 disclosed in EP 0699686-A2; and EP 0708179-a2 discloses such GLP-1 analogues and derivatives comprising an N-terminal imidazole group.
As described above, the present disclosure is directed to provide a method for purifying a GLP-1 analog to solve the technical problems of large sample loss, many influencing factors, and the like, and to improve process stability and efficiency.
The purification method of the GLP-1 analogue shown in the formula (I) comprises the steps of purifying the GLP-1 analogue by using a polymer reversed phase packing; preferably, the polymeric reverse phase filler is a polystyrene-divinylbenzene polymer,
X7X8X9X10TFTSDVSSYLEX22QAAX26X27FIAWLVX34GX36G (I)
wherein:
X7is H or absent;
X8is A, Aib, G, S, V or absent;
X9is E or absent;
X10is G or absent;
X22is G or E;
X26r, K or K with a side chain modified with a fatty acid or fatty acid derivative;
X27e, K or K with a side chain modified with a fatty acid or fatty acid derivative;
X34r, K or K with a side chain modified with a fatty acid or fatty acid derivative;
X36r, G, K or K with a side chain modified with a fatty acid or fatty acid derivative.
In a preferred embodiment of the invention, X is deleted7、X8、X9Or X10One or more of the above.
In a particular embodiment of the invention, X is deleted7X8。
In a particular embodiment of the invention, X is deleted7X8X9X10。
In a preferred embodiment of the invention, the inverse filler is selected from nam microsciences, Suzhou NM100, Mitsubishi chemical DIAION HP20SS, and/or Suzhou Qiangyao Biotechnology, Inc.
In a preferred embodiment of the invention, X8Is A, Aib, G, S, V or deleted.
In one embodiment of the present invention, X8Is A.
In one embodiment of the present invention, X8Is Aib. Aib is 2-aminoisobutyric acid.
In one embodiment of the present invention, X8Is G.
In one embodiment of the present invention, X8Is S.
In one embodiment of the present invention, X8Is V.
In one embodiment of the present invention, X22Is G.
In one embodiment of the present invention, X22Is E.
In one embodiment of the present invention, X26Is R.
In one embodiment of the present invention, X26Is K.
In one embodiment of the present invention, X26Is K with a side chain modified by a fatty acid or a fatty acid derivative.
In one embodiment of the present invention, X27Is E.
In one embodiment of the present invention, X27Is K.
In one embodiment of the present invention, X27Is K with a side chain modified by a fatty acid or a fatty acid derivative.
In one embodiment of the present invention, X34Is R.
In one embodiment of the present invention, X34Is K.
In one embodiment of the present invention, X34Is K with a side chain modified by a fatty acid or a fatty acid derivative.
In one embodiment of the present invention, X36Is R.
In one embodiment of the present invention, X36Is G.
In one embodiment of the present invention, X36Is K.
In one embodiment of the present invention, X36Is K with a side chain modified by a fatty acid or a fatty acid derivative.
In a particular embodiment of the invention, X is deleted7X8。
In a particular embodiment of the invention, X is deleted7X8X9X10。
In a preferred embodiment of the invention, the fatty acid is selected from C12-C22 fatty acids, more preferably the fatty acid is palmitic or stearic acid.
In a preferred embodiment of the invention, the fatty acid derivative is selected from:
or is or
Wherein, denotes the terminal as a site linked to a lysine side chain on the polypeptide chain.
GLP-1 analogs of formula (I) are selected from the group consisting of:
(1) GLP1(7-37, 34R), the amino acid sequence of which is:
HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG；
(2) GLP1(9-37,34R), the amino acid sequence of which is:
EGTFTSDVSSYLEGQAAKEFIAWLVRGRG；
(3) GLP1(7-37, 8G, 22E, 36G), the amino acid sequence of which is:
HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG；
(4) liraglutide (Liraglutide) having the structure:
(5) semaglutide (Semaglutide), having the structure:
in a preferred embodiment of the invention, the pH of the mobile phase is in the range of 8.5 to 9.5; more preferably from pH8.5 to 9.0.
In a preferred embodiment of the invention, the pH is adjusted using an acid or a base. The acid is selected from organic acid or inorganic acid, preferably, the acid is hydrochloric acid or acetic acid. The alkali is selected from organic alkali or inorganic alkali, and preferably, the alkali is sodium hydroxide.
In a preferred embodiment of the invention, alcohols are used as mobile phase; preferably, isopropanol, ethanol, butanol or propanol is used as the mobile phase.
In one embodiment of the invention, isopropanol is used as the mobile phase; preferably, the mobile phase is: 5-50mM Tris, 1-10% w/w isopropanol, pH 8.5-9.5; more preferably, the mobile phase is: 20mM Tris, 5% w/w (mass fraction) isopropanol, pH 8.5-9.0.
The chemical synthesis method and the genetic engineering method of the GLP-1 analogue can be produced by the chemical synthesis method, can also be produced by the genetic engineering method, and preferably use the genetic engineering method. Among them, the chemical method is preferably a solid phase synthesis method. Of course, they can be synthesized by other conventional methods known in the art.
In a preferred embodiment of the present invention, the purification is carried out using polystyrene-divinylbenzene polymer reverse phase fillers such as NM100 manufactured by Suzhou Nami micro technology Co., Ltd, DIAION HP20SS manufactured by Mitsubishi chemical corporation, Super100 manufactured by Suzhou Qiangyao Biotechnology Co., Ltd.
The experimental result of the invention shows that the polystyrene-divinylbenzene polymer reverse phase filler is obviously influenced by the pH of the sample, and can realize ideal loading capacity (about 30 g/L) only within the range of pH 8.5-9.5.
The technical solution of the present invention will be described below by way of specific examples.
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