Method for separating and purifying GLP-1 analogue in fermentation liquor by coupling aqueous two-phase extraction with reversed-phase column chromatography

Abstract

The invention relates to the technical field of biological separation and purification, and discloses a method for separating and purifying a GLP-1 analogue in fermentation liquor by coupling aqueous two-phase extraction with reversed-phase column chromatography. For the GLP-1 analogue fermentation liquor, two water phase layers are formed by adjusting the proportion of the hydrophilic organic solvent to the soluble inorganic salt, so that efficient enrichment of the GLP-1 analogue is realized, a brand-new technical approach is provided for separation and purification of the GLP-1 analogue, and further, the method has the advantages that the method is simple and convenient to operate, and the application prospect is wide. A crude product obtained after efficient enrichment can be purified through reverse preparative liquid chromatography, and the high-purity GLP-1 analogue is obtained. The method provided by the invention is simple and efficient, is suitable for industrial production, and fills the technical blank of separation and purification of the GLP-1 analogue in the fermentation liquor.

Description

Technical Field

[0001] The present invention relates to the technical field of biological separation and purification, and in particular to a method for separating and purifying a GLP-1 analogue in a fermentation broth by coupling a two-phase aqueous extraction method with a reversed-phase column chromatography. Background Art

[0002] Glucagon-like peptide-1 (GLP-1) is a peptide substance produced by gastrointestinal endocrine cells. It is an incretin that can control blood sugar through multiple pathways and plays a vital role in regulating appetite. However, due to the rapid degradation and short-lived biological activity of GLP-1 in the body, GLP-1 itself is not suitable for direct treatment of diabetes and obesity. In order to overcome this limitation, scientists have begun to devote themselves to the study of GLP-1 analogs, aiming to obtain more stable and lasting biological activity. The widespread use of GLP-1 analogs is expected to bring new hope to the treatment of metabolic diseases such as diabetes and obesity, thereby improving the quality of life of patients and reducing the global medical burden.

[0003] In recent years, with the rapid development of genetic engineering and biotechnology, the research on GLP-1 analogs has also made significant progress. Through genetic engineering technology, researchers have successfully constructed strains that can express GLP-1 analogs in large quantities, laying a solid foundation for increasing their production. However, since GLP-1 analogs coexist with other proteins and impurities in the fermentation broth, traditional separation and purification methods have problems such as complex operation, low yield and low purity, which restricts its further promotion in clinical applications.

[0004] At present, there is no effective and feasible technical method for the separation and purification of GLP-1 analogs in fermentation broth. Summary of the invention

[0005] In order to separate and purify GLP-1 analogs in fermentation broth, the present invention provides a method for separating and purifying GLP-1 analogs in fermentation broth by coupling aqueous two-phase extraction with reversed-phase column chromatography.

[0006] The specific technical scheme of the present invention is: The present invention provides a method for separating and purifying a GLP-1 analogue in a fermentation broth by coupling aqueous two-phase extraction with reversed-phase column chromatography, comprising the following steps: At 0-30°C, inorganic salts and hydrophilic organic solvents are added to the fermentation broth of the GLP-1 analog, the mixture is evenly suspended, the pH is adjusted to 8.5-10.5, the system is centrifuged to separate phases, the upper phase is taken, digested by enterokinase, and purified by reverse preparative liquid chromatography; wherein: Based on the total mass of the system, the amount of inorganic salt added is 10%~35%, and the amount of hydrophilic organic solvent added is 15%~40%; The inorganic salt is selected from one or more of sodium citrate, ammonium sulfate, dipotassium hydrogen phosphate, sodium bicarbonate, sodium carbonate, and sodium phosphate; The hydrophilic organic solvent is selected from one or more of ethanol, tert-butyl alcohol, n-propyl alcohol and isopropyl alcohol; The protein concentration of the GLP-1 analog fermentation broth is 9~13 mg / mL.

[0007] As a preferred embodiment of the above method, the inorganic salt is dipotassium hydrogen phosphate.

[0008] As a preferred embodiment of the above method, the hydrophilic organic solvent is ethanol.

[0009] As a preferred embodiment of the above method, the amount of inorganic salt added is 13% to 24% based on the total mass of the system.

[0010] As a preferred embodiment of the above method, the amount of inorganic salt added is 13% to 17% based on the total mass of the system.

[0011] As a preferred embodiment of the above method, the amount of the hydrophilic organic solvent added is 16% to 26% based on the total mass of the system.

[0012] As a preferred embodiment of the above method, the pH is adjusted to 9.5.

[0013] As a preferred embodiment of the above method, the purification method is: after the crude product of GLP-1 analog obtained by enterokinase enzymatic cleavage is dissolved in Tris buffer, it is loaded on a reverse preparative liquid chromatography column, and the peak product at the 21min peak position is collected, wherein: the mobile phase for reverse preparative liquid chromatography separation is: A (water containing 0.1% (volume fraction) TFA), B (acetonitrile with 0.1% TFA (volume fraction)); the sample loading solution is 10% (volume fraction) acetonitrile solution; the chromatographic column is Sepax Bio-C8 (10μm, 21.2*250mm); the elution conditions are: 0-5 min, 5%B; 5-5.5min, 5-30%B; 5.5-30 min, 30%-80%B; 30-30.5min, 80-100%B; 30.5-35min, 100%B; 35-35.5min, 100-5%B; 35.5-40 min, 5% B; then next batch cycle.

[0014] Compared with the prior art, the present invention has the following technical effects: (1) The present invention aims at the fermentation broth of GLP-1 analogs, and by adjusting the ratio between the hydrophilic organic solvent and the soluble inorganic salt, two aqueous phase layers are formed, thereby achieving efficient enrichment of GLP-1 analogs, and providing a new technical approach for the separation and purification of GLP-1 analogs. Furthermore, the crude product after efficient enrichment can be purified by reverse preparative liquid chromatography to obtain high-purity GLP-1 analogs. The method provided by the present invention fills the technical gap in the field of separation and purification of GLP-1 analogs in fermentation broth.

[0015] (2) The present invention also provides optimal extraction conditions for a two-phase aqueous extraction system that can achieve efficient enrichment of GLP-1 analogue fermentation broth, such as preferred hydrophilic organic solvents and soluble inorganic salts, preferred pH conditions, preferred temperature conditions, preferred protein concentration conditions, etc., so as to optimize the separation and purification effect of GLP-1 analogues in the fermentation broth. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a screen result diagram of the aqueous two-phase extraction system; Figure 2 This is a diagram of further screening results of the aqueous two-phase extraction system; Figure 3 This is the result diagram of preliminary screening of product purification factors by salt / alcohol content of the new aqueous two-phase extraction; Figure 4 This is a graph showing the recovery rate of products from the initial screening of salt / alcohol content in a novel aqueous two-phase extraction; Figure 5 This is the result diagram of the new aqueous two-phase extraction salt / alcohol content further screening product purification factors; Figure 6 This is a graph showing the recovery rate of the product further screened by the salt / alcohol content of the new aqueous two-phase extraction; Figure 7 This is the result diagram of the temperature screening of the product purification factor of the two-phase aqueous extraction system; Figure 8 This is the result diagram of product recovery rate of temperature screening of two-phase aqueous extraction system; Fig. 9 This is the result diagram of the pH screening of product purification factors in the two-phase aqueous extraction system; Fig.10 This is the result graph of product recovery rate of pH screening in two-phase aqueous extraction system; Fig.11 This is the result diagram of the protein concentration screening product purification factor of the two-phase aqueous extraction system; Fig.12 This is the result diagram of the product recovery rate of protein concentration screening in the two-phase aqueous extraction system; Fig.13 It is the liquid chromatogram of the original fermentation broth of GLP-1 analog; Fig.14 It is the liquid chromatogram of the upper phase after extraction by the two-phase aqueous extraction system; Fig.15 It is the electrophoresis diagram of the upper phase after extraction by the aqueous two-phase extraction system, the sample obtained by reverse preparative liquid phase separation coupled with the aqueous two-phase extraction system, and the GLP-1 analog standard; Fig.16 It is the liquid chromatogram after the GLP-1 analog is cleaved by enzyme; Fig.17 It is the reverse preparative liquid phase preparative chromatogram after enzymatic cleavage of GLP-1 analog; Fig.18 It is the chromatogram of the sample obtained by coupling the aqueous two-phase extraction system with reverse preparative liquid phase separation. DETAILED DESCRIPTION

[0017] The present invention provides a method for separating and purifying a GLP-1 analogue in a fermentation broth by coupling aqueous two-phase extraction with reversed-phase column chromatography, comprising the following steps: At 0-30°C, inorganic salts and hydrophilic organic solvents are added to the fermentation broth of the GLP-1 analog, the mixture is evenly suspended, the pH is adjusted to 8.5-10.5, the system is centrifuged to separate phases, the upper phase is taken, digested by enterokinase, and purified by reverse preparative liquid chromatography; wherein: Based on the total mass of the system, the amount of inorganic salt added is 10%~35%, and the amount of hydrophilic organic solvent added is 15%~40%; The inorganic salt is selected from one or more of sodium citrate, ammonium sulfate, dipotassium hydrogen phosphate, sodium bicarbonate, sodium carbonate, and sodium phosphate; The hydrophilic organic solvent is selected from one or more of ethanol, tert-butyl alcohol, n-propyl alcohol and isopropyl alcohol; The protein concentration of the GLP-1 analog fermentation broth is 9~13 mg / mL.

[0018] As a preferred embodiment of the above method, the inorganic salt is dipotassium hydrogen phosphate.

[0019] As a preferred embodiment of the above method, the hydrophilic organic solvent is ethanol.

[0020] As a preferred embodiment of the above method, the amount of inorganic salt added is 13% to 24% based on the total mass of the system.

[0021] As a preferred embodiment of the above method, the amount of inorganic salt added is 13% to 17% based on the total mass of the system.

[0022] As a preferred embodiment of the above method, the amount of the hydrophilic organic solvent added is 16% to 26% based on the total mass of the system.

[0023] As a preferred embodiment of the above method, the pH is adjusted to 9.5.

[0024] As a preferred embodiment of the above method, the purification method is: after the crude product of GLP-1 analog obtained by enterokinase enzymatic cleavage is dissolved in Tris buffer, it is loaded on a reverse preparative liquid chromatography column, and the peak product at the 21min peak position is collected, wherein: the mobile phase for reverse preparative liquid chromatography separation is: A (water containing 0.1% (volume fraction) TFA), B (acetonitrile with 0.1% TFA (volume fraction)); the sample loading solution is 10% (volume fraction) acetonitrile solution; the chromatographic column is Sepax Bio-C8 (10μm, 21.2*250mm); the elution conditions are: 0-5 min, 5%B; 5-5.5min, 5-30%B; 5.5-30 min, 30%-80%B; 30-30.5min, 80-100%B; 30.5-35min, 100%B; 35-35.5min, 100-5%B; 35.5-40 min, 5% B; then next batch cycle.

[0025] Since GLP-1 analogs coexist with other proteins and impurities in the fermentation broth, traditional separation and purification methods have problems such as complex operation, low yield and low purity, which restricts the further promotion of genetic engineering production of GLP-1 analogs in clinical applications.

[0026] The above method is for GLP-1 analog fermentation broth, and by adjusting the ratio between the hydrophilic organic solvent and the soluble inorganic salt, two aqueous phase layers are formed, thereby achieving efficient enrichment of GLP-1 analogs, providing a new technical approach for the separation and purification of GLP-1 analogs. Furthermore, the crude product after efficient enrichment can be purified by reverse preparative liquid chromatography to obtain high-purity GLP-1 analogs. The above method fills the technical gap in the field of separation and purification of GLP-1 analogs in fermentation broth.

[0027] The present invention will be further described below in conjunction with embodiments. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following description are generally only embodiments of a part of the present invention, rather than all embodiments. Therefore, based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work should belong to the scope of protection of the present invention.

[0028] The GLP-1 analog standard used in the examples of the present invention is from Lunan Pharmaceutical Group, with batch number F027-1-RS-221101; the GLP-1 analog fermentation broth (unextracted) used in the examples of the present invention is from Lunan Pharmaceutical Group, and its liquid chromatogram is shown in Fig.13The determination conditions of the liquid chromatogram are as follows: the chromatographic column is Sepax Bio-C8 (5μm, 4.6*250 mm); the mobile phases are A (water containing 0.1% (volume fraction) TFA) and B (acetonitrile containing 0.1% (volume fraction) TFA); the sample loading solution is 5% (volume fraction) acetonitrile solution; the elution conditions are: 0-5 min, 5% B; 5-5.5 min, 5-30% B; 5.5-30 min, 30%-80% B; 30-30.5 min, 80-100% B; 30.5-35 min, 100% B; 35-35.5 min, 100-5% B; 35.5-40 min, 5% B; and then the next batch is cycled.

[0029] The pH condition of the experiment in the embodiment of the present invention was adjusted by using 1 mol / L hydrochloric acid solution or sodium hydroxide solution; the protein concentration of the fermentation broth in the experiment in the embodiment of the present invention was adjusted by using deionized water, and the protein concentration of the original fermentation broth was 13 mg / mL.

[0030] Example 1 Preliminary screening of aqueous two-phase extraction system This example conducts a preliminary screening of the two-phase aqueous extraction system, and is carried out according to the following steps: Step 1: Taking the aqueous two-phase extraction system as the total system (100 g), take 60% (w / w) of the mixed GLP-1 analog fermentation broth accounting for the mass fraction of the total system into a centrifuge tube.

[0031] Step 2: Take soluble inorganic salts such as anhydrous sodium citrate, anhydrous ammonium sulfate, anhydrous potassium hydrogen phosphate, anhydrous sodium bicarbonate, anhydrous sodium carbonate, and anhydrous sodium phosphate buffer salt, and at the same time, take hydrophilic low molecular weight organic solvents such as anhydrous ethanol, isopropanol, tert-butanol, and n-propanol. Among them, the soluble inorganic salt accounts for 20% (w / w) of the total system mass percentage, and the hydrophilic low molecular weight organic solvent accounts for 20% (w / w) of the total system mass percentage.

[0032] Step 3: In an ice-water bath, the soluble inorganic salts in step 2 were mixed with the hydrophilic low molecular weight organic solvent combination, and each combination was fully mixed with the GLP-1 analog fermentation broth, and then centrifuged at 4500g for 20 minutes at 4°C to accelerate the formation of a two-phase aqueous phase, and the phase formation results were recorded, as shown in Table 1. Among the 11 systems with good phase formation effects, five groups could extract the GLP-1 analog into the upper phase. Combination of hydrophilic organic solvent and soluble inorganic salt Phase separation Phenomenon Isopropyl alcohol-sodium citrate Phase separation The upper phase is slightly yellow and transparent, and the lower phase is light yellow and turbid Isopropyl alcohol-ammonium sulfate Phase separation The upper phase is slightly yellow and turbid, and the lower phase is light yellow and clear Isopropyl alcohol-potassium hydrogen phosphate Phase separation The upper phase is light yellow and clear, the lower phase is milky white and turbid, and the middle layer is thin Ethanol-ammonium sulfate Phase separation The upper phase is slightly yellow and turbid, and the lower phase is light yellow and clear Ethanol-Dipotassium Hydrogen Phosphate Phase separation The upper phase is yellow and turbid, the lower phase is yellow and clear tert-Butyl alcohol-sodium citrate Phase separation The upper phase is light yellow and clear, while the lower phase is yellow and turbid Tert-Butyl Alcohol-Ammonium Sulfate Phase separation The upper phase is light yellow and clear, while the lower phase is yellow and turbid tert-Butyl alcohol-dipotassium hydrogen phosphate Phase separation After adding alcohol, it condenses, with less phase separation, the upper phase is colorless and clear, and the lower phase is yellow and clear n-Propanol-Sodium Citrate Phase separation The upper phase is colorless and clear, while the lower phase is yellow and turbid n-Propanol-Ammonium Sulfate Phase separation The upper phase is colorless and clear, the lower phase is yellow and clear n-Propanol-Dipotassium Hydrogen Phosphate Phase separation The upper phase is light yellow and clear, the lower phase is light yellow and slightly turbid Isopropyl alcohol-sodium bicarbonate No phase separation The liquid is yellow and clear, with a lot of insoluble salt at the bottom Isopropyl alcohol-sodium hydrogen phosphate buffer No phase separation The liquid is light yellow and turbid, with salt precipitation and a large amount of salt insoluble Isopropyl alcohol-sodium carbonate No phase separation The liquid is yellow and clear, with a lot of insoluble salt at the bottom Ethanol-sodium citrate No phase separation Yellow turbid liquid Ethanol-Sodium Bicarbonate No phase separation The liquid is yellow and clear, with a lot of insoluble salt at the bottom Ethanol-sodium hydrogen phosphate buffer No phase separation The liquid is light yellow and turbid, with salt precipitation and a large amount of salt insoluble Ethanol-Sodium Carbonate No phase separation Yellow turbid liquid tert-Butyl alcohol-sodium bicarbonate No phase separation The liquid is yellow and clear, with a lot of insoluble salt at the bottom tert-Butyl alcohol-disodium hydrogen phosphate buffer No phase separation The liquid is light yellow and turbid, with salt precipitation and a large amount of salt insoluble tert-Butyl alcohol-sodium carbonate No phase separation The liquid is yellow and clear, with a large amount of insoluble salt at the bottom and a thin layer in the middle n-Propanol-Sodium Bicarbonate No phase separation The liquid is yellow and clear, with a lot of insoluble salt at the bottom n-Propanol-disodium hydrogen phosphate buffer No phase separation The liquid is light yellow and turbid, with salt precipitation and a large amount of salt insoluble n-Propanol-Sodium Carbonate No phase separation The liquid is light yellow and clear, with salt precipitation and a large amount of salt insoluble

[0033] Step 4: Among the 11 systems with good phase formation effects, five of them can extract GLP-1 analogs into the upper phase. The recovery rate of GLP-1 analogs in the upper phase after liquid phase analysis of the new two-phase aqueous extraction is shown in the figure. Figure 1The method for determining the recovery rate of GLP-1 analogs is as follows: dilute the GLP-1 analog solution after the novel two-phase aqueous extraction and the original fermentation broth to the same concentration, and use reverse liquid chromatography to determine the concentration of GLP-1 analogs in the original fermentation broth and the GLP-1 analog solution after the novel two-phase aqueous extraction, respectively. Among them, the parameters of reverse liquid chromatography are: the chromatographic column is Sepax Bio-C8 (5μm, 4.6*250mm); the mobile phase is A (water containing 0.1% (volume fraction) TFA) and B (acetonitrile containing 0.1% (volume fraction) TFA); the sample loading solution is 5% (volume fraction) acetonitrile solution; the elution conditions are: 0-5 min, 5% B; 5-5.5min, 5-30% B; 5.5-30 min, 30%-80% B; 30-30.5min, 80-100% B; 30.5-35min, 100% B; 35-35.5min, 100-5% B; 35.5-40 min, 5% B; then the next batch is cycled.

[0034] The recovery rate determination results show that the recovery rate of the isopropanol-ammonium sulfate system is 54%, the recovery rate of the isopropanol-dipotassium hydrogen phosphate system is 45%, the recovery rate of the isopropanol-sodium citrate system is 43%, the recovery rate of the ethanol-ammonium sulfate system is 41%, and the recovery rate of the ethanol-dipotassium hydrogen phosphate system is the highest at 48%. It can be seen that in the preliminary screening of the two-phase aqueous extraction system, the recovery rate of the isopropanol-ammonium sulfate system is the highest, the recovery rate of the ethanol-dipotassium hydrogen phosphate system is second, and the recovery rate of the isopropanol-dipotassium hydrogen phosphate system is similar to that of the ethanol-dipotassium hydrogen phosphate system.

[0035] Example 2 Final screening of aqueous two-phase extraction system In this example, the final screening of the two-phase aqueous extraction system was carried out according to the following steps (in the extraction system, the pH was 9.5, the extraction temperature was 0° C. (ice-water bath condition), and the protein concentration in the fermentation broth was adjusted to 11 mg / mL): Step 1: Taking the aqueous two-phase extraction system as the total system (100 g), take 60% (w / w) of the mixed GLP-1 analog fermentation broth accounting for the mass fraction of the total system into a centrifuge tube.

[0036] Step 2: Take soluble inorganic salts anhydrous ammonium sulfate and anhydrous potassium hydrogen phosphate respectively, and at the same time, take hydrophilic low molecular weight organic solvents anhydrous ethanol and isopropanol respectively. Among them, the soluble inorganic salt accounts for 20% (w / w) of the total system mass percentage, and the hydrophilic low molecular weight organic solvent accounts for 20% (w / w) of the total system mass percentage.

[0037] Step 3: In an ice water bath, the soluble inorganic salts described in step 2 are mixed with the hydrophilic low molecular weight organic solvent combination, each combination is fully mixed with the GLP-1 analog fermentation broth, and then centrifuged at 4500g for 20 minutes at 4°C to accelerate the formation of a two-phase aqueous solution.

[0038] Step 4: Liquid phase analysis of the recovery rate of the GLP-1 analogue in the upper phase after the two-phase aqueous extraction, the method is as shown in step 4 of Example 1, and the results are shown in Figure 2 .

[0039] Step 5: Take the upper phase after the novel two-phase aqueous extraction and calculate the purification factor (PF). The results are shown in Figure 2 .

[0040] From the results of recovery rate and purification factor determination, it can be seen that the recovery rate of isopropanol-ammonium sulfate system is 18%, the recovery rate of isopropanol-dipotassium hydrogen phosphate system is 31%, the recovery rate of ethanol-ammonium sulfate system is 31%, and the recovery rate of ethanol-dipotassium hydrogen phosphate system is 48%; and the purification factor of ethanol-dipotassium hydrogen phosphate system is 3.41, which is better than other systems, so it is finally determined that the best two-phase aqueous extraction system for GLP-1 analogs is ethanol-dipotassium hydrogen phosphate.

[0041] Example 3 Optimization and screening of anhydrous potassium hydrogen phosphate content and anhydrous ethanol content In this example, the ethanol-dipotassium hydrogen phosphate system was used as the two-phase aqueous extraction system to optimize the content of anhydrous dipotassium hydrogen phosphate and anhydrous ethanol in the following steps (in the extraction system, the pH was 9.5, the extraction temperature was 0° C. (ice-water bath condition), and the protein concentration in the fermentation broth was adjusted to 11 mg / mL): Step 1: Taking the two-phase aqueous extraction system as the total system, the mass of the total system is controlled to be 30g, and taking 15%, 18%, 21%, 24%, 27%, and 30% of anhydrous dipotassium hydrogen phosphate in the total system mass percentage, respectively, and combining them with 14%, 18%, 22%, 26%, 30%, and 34% of anhydrous ethanol in the total system mass percentage to form a two-phase aqueous extraction system.

[0042] Step 2: The mass of the total system is controlled to be 100 g, and the uniformly mixed GLP-1 analog fermentation broth is mixed with the two-phase extraction system composed of different contents of anhydrous ethanol and anhydrous potassium hydrogen phosphate obtained in step 1.

[0043] Step 3: In an ice-water bath, the two-phase extraction system composed of different amounts of anhydrous ethanol and anhydrous potassium hydrogen phosphate was fully mixed with the GLP-1 analog fermentation broth, and centrifuged at 4500g for 20 minutes at 4°C to accelerate the formation of a two-phase solution. 36 groups of experiments were formed, each group of experiments was repeated three times, and the test results were averaged.

[0044] Step 4: Take the upper phase after extraction with the two-phase aqueous extraction system and measure the impurity removal effect. The results of the GLP-1 analog recovery rate determination are shown in Figure 3 The determination method is shown in step 4 of Example 1. Among them, the recovery rate of GLP-1 analogs is as high as 99%, which is a 15% dipotassium hydrogen phosphate-18% ethanol system.

[0045] Step 5: Take the upper phase after extraction with the aqueous two-phase extraction system and calculate the purification factor (PF). The results are shown in Figure 4 Among them, the highest recovery rate corresponds to a purification factor of 2.6.

[0046] Step 6: Take the upper phase after extraction by the two-phase aqueous extraction system and measure the pigment removal effect: dilute the extracted GLP-1 analog solution and the original GLP-1 analog fermentation broth to the same concentration, use water as a blank, and measure the absorbance at 350nm, 420nm, and 610nm. The results showed that the pigment removal rate of the 15% dipotassium hydrogen phosphate-18% ethanol system was 88%.

[0047] Step 7: Take the upper phase after extraction by the two-phase aqueous extraction system and determine the bacterial removal effect: take the supernatant of the extracted GLP-1 analog solution and the original GLP-1 analog fermentation broth, use the supernatant of the fermentation broth as a blank, and determine the absorbance at 600nm. The results showed that the bacterial removal rate of the 15% dipotassium hydrogen phosphate-18% ethanol system extraction was 99%.

[0048] Example 4 Final determination of the content of anhydrous dipotassium hydrogen phosphate and anhydrous ethanol In this example, the ethanol-dipotassium hydrogen phosphate system is used as the two-phase extraction system, and the optimal content ratio of the system is determined by the following steps (in the extraction system, the pH is 9.5, the extraction temperature is 0° C. (ice water bath condition), and the protein concentration in the fermentation broth is adjusted to 11 mg / mL): Step 1: Taking the two-phase aqueous extraction system as the total system, the mass of the total system is controlled to be 100g, and taking 13%, 15%, and 17% of anhydrous dipotassium hydrogen phosphate in the total system by mass percentage, respectively, and combining them with 16%, 18%, 20%, 22%, 24%, and 26% of anhydrous ethanol in the total system by mass percentage to form a two-phase aqueous extraction system.

[0049] Step 2: The mass of the total system is controlled to be 100 g, and the uniformly mixed GLP-1 analog fermentation broth is mixed with the two-phase extraction system composed of different contents of anhydrous ethanol and anhydrous potassium hydrogen phosphate obtained in step 1.

[0050] Step 3: In an ice-water bath, the two-phase extraction system composed of different amounts of anhydrous ethanol and anhydrous potassium hydrogen phosphate was fully mixed with the GLP-1 analog fermentation broth, and centrifuged at 4500g for 20 minutes at 4°C to accelerate the formation of a two-phase solution. 18 groups of two-two crossover experiments were formed, each group of experiments was repeated three times, and the test results were averaged.

[0051] Step 4: Take the upper phase after extraction with the two-phase aqueous extraction system and measure the impurity removal effect. The results of the GLP-1 analog recovery rate determination are shown in Figure 5 The method is the same as step 4 in Example 1. This step shows that the recovery rate of GLP-1 analogs is up to 99% in the 15% dipotassium hydrogen phosphate-20% ethanol system.

[0052] Step 5: Take the upper phase after extraction with the aqueous two-phase extraction system and calculate the purification factor (PF). The results are shown in Figure 6 The results showed that the highest purification factor was 4.0, which was the 15% dipotassium hydrogen phosphate-16% ethanol system; the purification factor of the 15% dipotassium hydrogen phosphate-20% ethanol system was 2.9.

[0053] Step 6: Take the upper phase after extraction by the two-phase aqueous extraction system and measure the pigment removal effect: the method is as shown in step 6 in Example 3. The results show that the pigment removal rates of the 15% dipotassium hydrogen phosphate-16% ethanol system, the 15% dipotassium hydrogen phosphate-18% ethanol system, and the 15% dipotassium hydrogen phosphate-20% ethanol system are all 89%.

[0054] Step 7: Take the upper phase after extraction by the two-phase aqueous extraction system and determine the bacterial removal effect. The method is shown in Step 7 in Example 3. The results show that the bacterial removal rates of the 15% dipotassium hydrogen phosphate-16% ethanol system, the 15% dipotassium hydrogen phosphate-18% ethanol system, and the 15% dipotassium hydrogen phosphate-20% ethanol system are 99%.

[0055] Example 5 Determination of the Optimum Temperature of the Aqueous Two-Phase Extraction System In this example, a 15% potassium dihydrogen phosphate-18% ethanol system was used as a two-phase aqueous extraction system to determine the optimal temperature for extraction, and the following steps were performed (in the extraction system, the pH was 9.5, and the protein concentration in the fermentation broth was adjusted to 11 mg / mL): Step 1: Take 5 portions of GLP-1 analog fermentation broth, 65 g each.

[0056] Step 2: In an ice water bath, add 15g of potassium dihydrogen phosphate to each portion of GLP-1 analog fermentation broth, use a vortex oscillator to mix thoroughly, and after the salt is completely dissolved, adjust the system temperature to: 0℃, 25℃, 30℃, 35℃, 40℃, and then add 20g of pre-cooled or pre-heated anhydrous ethanol, and mix thoroughly. Each group of experiments was repeated three times, and the test results were averaged.

[0057] Step 3: Centrifuge at 4500 g for 20 min at 4°C to accelerate phase separation.

[0058] Step 4: Take the upper phase after the two-phase extraction system, and perform the GLP-1 analog recovery rate and impurity removal effect determination. The results are shown in Figure 7 The method is as shown in step 4 of Example 1. The recovery rate of the GLP-1 analogue is as high as 92%, which is obtained under 0°C.

[0059] Step 5: Take the upper phase after extraction with the aqueous two-phase extraction system and calculate the purification factor (PF). The results are shown in Figure 8 The results showed that the highest purification factor was 3.4, which was obtained under 0°C conditions.

[0060] Step 6: Take the upper phase after extraction by the two-phase aqueous extraction system and measure the pigment removal effect. The method is as shown in Step 6 in Example 3. The results show that the pigment removal rate is 92% under 0°C conditions.

[0061] Step 7: Take the upper phase after extraction by the two-phase aqueous extraction system and measure the bacterial removal effect. The method is shown in Step 7 in Example 3. The results show that the bacterial removal rate is 99% under 0°C conditions.

[0062] Example 6 Determination of the Optimal pH of the Aqueous Two-Phase Extraction System In this example, a 15% potassium dihydrogen phosphate-18% ethanol system was used as a two-phase aqueous extraction system to determine the optimal pH of the system according to the following steps (in the extraction system, the extraction temperature was 0° C. (ice-water bath condition), and the protein concentration in the fermentation broth was adjusted to 11 mg / mL): Step 1: Take 5 portions of GLP-1 analog fermentation broth, 65 g each.

[0063] Step 2: In an ice water bath, add 15 g of potassium dihydrogen phosphate to each portion of GLP-1 analog fermentation broth, mix thoroughly using a vortex oscillator, and after all the salt is dissolved, add 20 g of pre-cooled or pre-heated anhydrous ethanol and mix thoroughly.

[0064] Step 3: Adjust the pH of the system to 8.5, 9.0, 9.5, 10.0, and 10.5 respectively. Each group of experiments was repeated three times, and the test results were averaged.

[0065] Step 4: Centrifuge at 4500 g for 20 min at 4°C to accelerate phase separation.

[0066] Step 5: Take the upper phase after the two-phase extraction system, and perform the GLP-1 analog recovery rate and impurity removal effect determination. The results are shown in Fig. 9The method is as shown in step 4 of Example 1. The recovery rate of GLP-1 analogue is as high as 97%, which is obtained under pH 9.5.

[0067] Step 6: Take the upper phase after the two-phase extraction system and calculate the purification factor (PF). The results are shown in Fig.10 The results showed that the highest purification factor was 2.8, which was obtained under pH 9.5.

[0068] Step 7: Take the upper phase after the two-phase extraction system is extracted, and measure the pigment removal effect: the method is as shown in step 6 in Example 3. The results show that the highest pigment removal rate is 88%, which is obtained under the condition of pH 9.5.

[0069] Step 8: Take the upper phase after extraction by the two-phase aqueous extraction system and measure the bacterial cell removal effect, the method is as shown in step 7 in Example 3. The results show that the highest bacterial cell removal rate is 99%, which is obtained under the condition of pH 9.5.

[0070] Example 7 Determination of the Optimal Protein Concentration in the Aqueous Two-Phase Extraction System In this example, a 15% potassium dihydrogen phosphate-18% ethanol system was used as a two-phase aqueous extraction system to determine the optimal protein concentration of the extraction system according to the following steps (in the extraction system, the pH was 9.5 and the extraction temperature was 0° C. (ice-water bath condition)): Step 1: Take 5 portions of 65 g of GLP-1 analog fermentation broth, each containing a different protein concentration, namely 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, and 13 mg / mL.

[0071] Step 2: In an ice water bath, add 15 g of potassium dihydrogen phosphate to each portion of GLP-1 analog fermentation broth, mix thoroughly using a vortex oscillator, and after all the salt is dissolved, add 20 g of pre-cooled anhydrous ethanol and mix thoroughly.

[0072] Step 3: Adjust the system pH to 9.5 and repeat each experiment three times.

[0073] Step 4: Centrifuge at 4500 g for 20 min at 4°C to accelerate phase separation.

[0074] Step 5: Take the upper phase after the two-phase extraction system, and perform the GLP-1 analog recovery rate and impurity removal effect determination. The results are shown in Fig.11 The method is shown in step 4 of Example 1. The results show that the recovery rate of the GLP-1 analogue is as high as 98%, which is obtained under the condition of a protein concentration of 11 mg / mL.

[0075] Step 6: Take the upper phase after the two-phase extraction system and calculate the purification factor (PF). The results are shown in Fig.12The results showed that the highest purification factor was 3.2, which was obtained at a protein concentration of 11 mg / mL.

[0076] Step 7: Take the upper phase after extraction by the two-phase aqueous extraction system and measure the pigment removal effect: the method is as shown in step 6 of Example 3. The results show that the highest pigment removal rate is 88%, which is obtained under the condition of a protein concentration of 11 mg / mL.

[0077] Step 8: Take the upper phase after extraction by the aqueous two-phase extraction system and measure the bacterial removal effect, the method is as shown in step 7 in Example 3. The results show that the highest bacterial removal rate is 99%, which is obtained under the condition of a protein concentration of 11 mg / mL.

[0078] Example 8 Further purification of GLP-1 analogs In this example, a 15% potassium dihydrogen phosphate-18% ethanol system was used as a two-phase aqueous extraction system to further purify the GLP-1 analogs according to the following steps: Step 1: Take 201 g of GLP-1 analog fermentation broth with a protein concentration of 11 mg / mL.

[0079] Step 2: Add 45 g of potassium dihydrogen phosphate to an ice water bath and mix thoroughly using a vortex oscillator. After all the salt is dissolved, add 54 g of pre-cooled anhydrous ethanol and mix thoroughly.

[0080] Step 3: Adjust the system pH to 9.5. Set up 3 parallel experiments and take the average value of the test results.

[0081] Step 4: Centrifuge at 4500 g for 20 min at 4°C to accelerate phase separation.

[0082] Step 5: Take the upper phase after extraction by the two-phase aqueous extraction system and test the liquid chromatogram. The result is shown in 14. Take the upper phase after extraction by the two-phase aqueous extraction system and perform the GLP-1 analog recovery rate determination and impurity removal effect determination. The method is shown in step 4 in Example 1. The recovery rate of the GLP-1 analog is measured to be 97%.

[0083] Step 6: Take the upper phase after extraction with the aqueous two-phase extraction system and calculate the purification factor (PF). The result shows that the purification factor is 3.4.

[0084] Step 7: Take the upper phase after extraction by the two-phase aqueous extraction system and measure the pigment removal effect: the method is as shown in step 6 in Example 3. The result shows that the pigment removal rate is 88%.

[0085] Step 8: Take the upper phase after extraction by the two-phase aqueous extraction system and measure the bacterial removal effect, the method is as shown in step 7 in Example 3. The result shows that the bacterial removal rate is 99%.

[0086] Step 9: Take the upper phase after the two-phase aqueous extraction system extraction and perform chromatographic analysis. The results are shown in Fig.14 ; and 15% reducing SDS-PAGE electrophoresis was used for determination, the sample volume was 10uL, the results are shown in Fig.15 The electrophoresis results showed that the impurity protein bands were significantly reduced. Fig.14 As shown in the figure, after chromatographic analysis, the impurity peaks were significantly reduced.

[0087] Step 10: After the upper phase is desalted, add 15 mL of enterokinase buffer, mix well, add 3.5 mg of enterokinase, and digest at 25°C for 16 hours.

[0088] Step 11: Adjust the pH of the solution after the fusion protein tag is removed to 4.2-4.5, precipitate at 10°C for 30 minutes, centrifuge at 5000r for 30 minutes, discard the supernatant, add 20mmol / L Tris buffer at pH 8.0 to the precipitate to fully dissolve it, and obtain the crude product of GLP-1 analog. The GLP-1 analog after enzyme cleavage was tested by liquid chromatography, and the results are shown in Fig.16 .

[0089] Step 12: Use reverse preparative liquid chromatography to further remove impurities and improve purity. Dissolve the crude GLP-1 analog and load it onto a reverse preparative liquid chromatography column to collect the target peak product: the mobile phase is A (water containing 0.1% (volume fraction) TFA) and B (acetonitrile with 0.1% TFA (volume fraction)); the sample loading solution is 10% (volume fraction) acetonitrile solution; the chromatographic column is Sepax Bio-C8 (10μm, 21.2*250mm); the elution conditions are: 0-5 min, 5% B; 5-5.5min, 5-30% B; 5.5-30 min, 30%-80% B; 30-30.5min, 80-100% B; 30.5-35min, 100% B; 35-35.5min, 100-5% B; 35.5-40 min, 5% B; then the next batch is cycled. The target peak collection time is selected as 21min. like Fig.17 , which is a reverse preparative liquid phase chromatogram of GLP-1 analog after enzymatic cleavage.

[0090] Step 13: Take the GLP-1 analog sample collected after purification and perform chromatographic analysis. The results are shown in Fig.18 ; and 15% reducing SDS-PAGE electrophoresis was used for determination, the sample volume was 10uL, the results are shown in Fig.15 The electrophoresis results showed that the purified GLP-1 analog sample was a single band. The product prepared by reverse preparative liquid chromatography had significantly fewer impurities and excellent purification effect. After chromatographic column analysis, a single peak was shown with a purity of 99.3% ( Fig.18 ).

[0091] The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0092] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for separating and purifying GLP-1 analogs in fermentation broth by aqueous two-phase extraction coupled with reverse phase column chromatography, characterized in that: The following steps are involved: At 0-30°C, inorganic salts and hydrophilic organic solvents are added to the fermentation broth of the GLP-1 analog, the mixture is evenly suspended, the pH is adjusted to 8.5-10.5, the system is centrifuged to separate phases, the upper phase is taken, digested by enterokinase, and purified by reverse preparative liquid chromatography; wherein: Based on the total mass of the system, the amount of inorganic salt added is 10%~35%, and the amount of hydrophilic organic solvent added is 15%~40%; The inorganic salt is selected from one or more of sodium citrate, ammonium sulfate, dipotassium hydrogen phosphate, sodium bicarbonate, sodium carbonate, and sodium phosphate; The hydrophilic organic solvent is selected from one or more of ethanol, tert-butyl alcohol, n-propyl alcohol and isopropyl alcohol; The protein concentration of the GLP-1 analog fermentation broth is 9~13 mg / mL.

2. The method for separating and purifying a GLP-1 analogue in a fermentation broth by aqueous two-phase extraction coupled with reverse phase column chromatography as claimed in claim 1, characterized in that: The inorganic salt is dipotassium hydrogen phosphate.

3. The method for separating and purifying a GLP-1 analogue in a fermentation broth by aqueous two-phase extraction coupled with reverse phase column chromatography as claimed in claim 1, characterized in that: The hydrophilic organic solvent is ethanol.

4. The method for separating and purifying a GLP-1 analogue in a fermentation broth by aqueous two-phase extraction coupled with reverse phase column chromatography as claimed in claim 1, characterized in that: Based on the total mass of the system, the amount of inorganic salt added is 13%~24%.

5. The method for separating and purifying a GLP-1 analogue in a fermentation broth by aqueous two-phase extraction coupled with reverse phase column chromatography as claimed in claim 4, characterized in that: Based on the total mass of the system, the amount of inorganic salt added is 13%~17%.

6. The method for separating and purifying a GLP-1 analogue in a fermentation broth by aqueous two-phase extraction coupled with reverse phase column chromatography as claimed in claim 1, characterized in that: Based on the total mass of the system, the amount of hydrophilic organic solvent added is 16%~26%.

7. The method for separating and purifying a GLP-1 analogue in a fermentation broth by aqueous two-phase extraction coupled with reverse phase column chromatography as claimed in claim 1, characterized in that: Adjust pH to 9.

5.

8. The method for separating and purifying a GLP-1 analogue in a fermentation broth by aqueous two-phase extraction coupled with reverse phase column chromatography as claimed in claim 1, characterized in that: The purification method is as follows: after the crude product of GLP-1 analogue obtained by enterokinase enzymatic cleavage is dissolved in Tris buffer, the product is loaded on a reverse preparative liquid chromatography column, and the peak product at the 21-minute peak position is collected, wherein: the mobile phase for reverse preparative liquid chromatography separation is: A (water containing 0.1% (volume fraction) TFA), B (acetonitrile with 0.1% TFA (volume fraction)); the sample loading solution is a 10% (volume fraction) acetonitrile solution; the chromatographic column is Sepax Bio-C8 (10 μm, 21.2*250 mm); the elution conditions are: 0-5 min, 5% B; 5-5.5 min, 5-30% B; 5.5-30 min, 30%-80% B; 30-30.5 min, 80-100% B; 30.5-35 min, 100% B; 35-35.5 min, 100-5% B; 35.5-40 min, 5% B; then the next batch of cycles.

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