A method for separating and purifying GLP-1 analogues in fermentation broth by coupling aqueous two-phase extraction with reversed-phase column chromatography

Abstract

The present application relates to the technical field of biological separation and purification, and discloses a method for separating and purifying GLP-1 analogues in fermentation liquor by using aqueous two-phase extraction coupled with reversed-phase column chromatography. For GLP-1 analogue fermentation liquor, the present application forms two aqueous phases by adjusting the ratio between a hydrophilic organic solvent and a soluble inorganic salt, thereby realizing efficient enrichment of GLP-1 analogues, providing a brand-new technical approach for the separation and purification of GLP-1 analogues. Further, the crude product after efficient enrichment can be purified by reversed-phase preparative liquid chromatography to obtain GLP-1 analogues with high purity. The method provided by the present application is simple and efficient, and is suitable for industrial production, thereby filling the technical gap in the separation and purification of GLP-1 analogues in fermentation liquor.

Description

TECHNICAL FIELD

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

[0002] Glucagon-like peptide-1 (GLP-1) is a peptide produced by gastrointestinal endocrine cells, which is a kind of intestinal insulin and can control blood glucose through multiple pathways, and plays a crucial role in regulating appetite. However, due to the characteristics of rapid degradation and short biological activity of GLP-1 in vivo, GLP-1 itself is not suitable for direct use in the treatment of diabetes and obesity. In order to overcome this limitation, scientists have begun to research GLP-1 analogues in order to obtain more stable and persistent biological activity. The wide application of GLP-1 analogues is expected to bring new hope for 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 analogues has also made significant progress. Through genetic engineering technology, researchers have successfully constructed strains that can express GLP-1 analogues in large quantities, laying a solid foundation for the improvement of their yield. However, due to the coexistence of GLP-1 analogues with other proteins and impurities in the fermentation broth, the traditional separation and purification methods have problems such as complex operation, low yield and low purity, which restrict the further promotion of their clinical application.

[0004] At present, in the field of separation and purification of GLP-1 analogues in fermentation broth, there is no effective and feasible technical method. SUMMARY

[0005] In order to realize the separation and purification of GLP-1 analogues in fermentation broth, the present application provides a method for separating and purifying GLP-1 analogues in fermentation broth by coupling aqueous two-phase extraction with reversed-phase column chromatography.

[0006] The specific technical scheme of the present application is as follows:

[0007] The present application provides a method for separating and purifying GLP-1 analogues in fermentation broth by coupling aqueous two-phase extraction with reversed-phase column chromatography, which comprises the following steps:

[0008] Under the condition of 0~30℃, inorganic salt and hydrophilic organic solvent are added to the GLP-1 analogue fermentation broth, and the mixture is uniformly suspended, the pH is adjusted to 8.5~10.5, and the system is centrifuged to separate the phases, and the upper phase is taken and subjected to enzyme digestion by enterokinase, and then purified by reversed-phase preparative liquid chromatography; wherein:

[0009] 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%;

[0010] The inorganic salt is selected from one or more of sodium citrate, ammonium sulfate, dipotassium hydrogen phosphate, sodium bicarbonate, sodium carbonate, and sodium phosphate;

[0011] The hydrophilic organic solvent is selected from one or more of ethanol, tert-butanol, n-propanol, and isopropanol;

[0012] The protein concentration of the fermentation broth of GLP-1 analogs is 9~13 mg / mL.

[0013] As a preferred embodiment of the above method, the inorganic salt is dipotassium hydrogen phosphate.

[0014] As a preferred embodiment of the above method, the hydrophilic organic solvent is ethanol.

[0015] As a preferred method, the amount of inorganic salt added is 13% to 24% based on the total mass of the system.

[0016] As a preferred method, the amount of inorganic salt added is 13% to 17% based on the total mass of the system.

[0017] As a preferred embodiment of the above method, the amount of hydrophilic organic solvent added is 16% to 26% based on the total mass of the system.

[0018] As a preferred method described above, the pH is adjusted to 9.5.

[0019] As a preferred embodiment of the above method, the purification process is as follows: the crude GLP-1 analog obtained by enterokinase digestion is dissolved in Tris buffer and loaded onto a reverse preparative liquid chromatography column. The peak product at the elution position at 21 min is collected. The mobile phase for reverse preparative liquid chromatography separation is: A (water containing 0.1% (v / v) TFA) and B (acetonitrile containing 0.1% (v / v) TFA); the sample loading solution is 10% (v / v) 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. min, 5% B; then cycle through the next batch.

[0020] Compared with the prior art, the present invention has the following technical effects:

[0021] (1) The present application is directed to GLP-1 analogue fermentation broth, by adjusting the ratio between hydrophilic organic solvent and soluble inorganic salt, forming two water phase layers, so as to realize the efficient enrichment of GLP-1 analogue, and provide a new technical way for the separation and purification of GLP-1 analogue, further, the crude product after efficient enrichment can be purified by reverse phase preparative liquid chromatography, and high-purity GLP-1 analogue is obtained. The method provided by the present application fills the technical blank in the field of separation and purification of GLP-1 analogue in fermentation broth.

[0022] (2) The present application also aims at the two-aqueous-phase extraction system which can realize the efficient enrichment of GLP-1 analogue fermentation broth, and gives the optimal extraction conditions, such as the preferred hydrophilic organic solvent and soluble inorganic salt, the preferred pH condition, the preferred temperature condition, the preferred protein concentration condition, etc., so that the separation and purification effect of GLP-1 analogue in fermentation broth is optimized. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is a two-aqueous-phase extraction system screening result graph;

[0024] Figure 2 is a two-aqueous-phase extraction system further screening result graph;

[0025] Figure 3 is a new two-aqueous-phase extraction salt / alcohol content preliminary screening product purification factor result graph;

[0026] Figure 4 is a new two-aqueous-phase extraction salt / alcohol content preliminary screening product recovery rate result graph;

[0027] Figure 5 is a new two-aqueous-phase extraction salt / alcohol content further screening product purification factor result graph;

[0028] Figure 6 is a new two-aqueous-phase extraction salt / alcohol content further screening product recovery rate result graph;

[0029] Figure 7 is a two-aqueous-phase extraction system temperature screening product purification factor result graph;

[0030] Figure 8 is a two-aqueous-phase extraction system temperature screening product recovery rate result graph;

[0031] Figure 9 is a two-aqueous-phase extraction system pH screening product purification factor result graph;

[0032] Figure 10 is a two-aqueous-phase extraction system pH screening product recovery rate result graph;

[0033] Figure 11is a product recovery rate result graph of the aqueous two-phase extraction system protein concentration screening;

[0034] Figure 12 is a product recovery rate result graph of the aqueous two-phase extraction system protein concentration screening;

[0035] Figure 13 is a liquid chromatogram of the original fermentation liquid of the GLP-1 analogue;

[0036] Figure 14 is a liquid chromatogram of the upper phase after extraction by the aqueous two-phase extraction system;

[0037] Figure 15 is an electropherogram of the sample obtained by the aqueous two-phase extraction system coupled reverse preparation liquid phase separation and the GLP-1 analogue standard;

[0038] Figure 16 is a liquid chromatogram of the GLP-1 analogue after enzyme cutting;

[0039] Figure 17 is a reverse preparation liquid chromatogram of the GLP-1 analogue after enzyme cutting;

[0040] Figure 18 is a chromatogram of the sample obtained by the aqueous two-phase extraction system coupled reverse preparation liquid phase separation. DETAILED DESCRIPTION

[0041] The application provides a method for separating and purifying a GLP-1 analogue in a fermentation liquid by coupling an aqueous two-phase extraction with a reverse column chromatography, comprising the following steps:

[0042] At 0-30℃, inorganic salts and hydrophilic organic solvents are added to the GLP-1 analogue fermentation liquid, the mixture is uniformly suspended, the pH is adjusted to 8.5-10.5, and the system is centrifuged to separate the phases, and the upper phase is subjected to enzyme cutting by enterokinase and then purified by a reverse preparation liquid chromatography; wherein:

[0043] The inorganic salts are added in an amount of 10%-35% and the hydrophilic organic solvents are added in an amount of 15%-40%, based on the total mass of the system;

[0044] The inorganic salts are selected from one or more of sodium citrate, ammonium sulfate, dipotassium hydrogen phosphate, sodium bicarbonate, sodium carbonate and sodium phosphate;

[0045] The hydrophilic organic solvents are selected from one or more of ethanol, tert-butyl alcohol, n-propanol and isopropanol;

[0046] The protein concentration of the GLP-1 analogue fermentation liquid is 9-13 mg / mL.

[0047] As a preferred method, the inorganic salts are dipotassium hydrogen phosphate.

[0048] As a preferred method of the above, the hydrophilic organic solvent is ethanol.

[0049] As a preferred method of the above, the amount of the inorganic salt added is 13% to 24% based on the total mass of the system.

[0050] As a preferred method of the above, the amount of the inorganic salt added is 13% to 24% based on the total mass of the system.

[0051] As a preferred method of the above, the amount of the inorganic salt added is 13% to 24% based on the total mass of the system.

[0052] As a preferred method of the above, the pH is adjusted to 9.5.

[0053] As a preferred method of the above, the method of the purification treatment is: after the enterokinase enzyme digestion GLP-1 analogue crude product is dissolved in Tris buffer and then loaded onto a reverse preparation liquid chromatography column, and the peak product at the 21 min peak position is collected, wherein: the mobile phase of the reverse preparation liquid chromatography separation is: A (0.1% (volume fraction) TFA in water), B (0.1% TFA (volume fraction) acetonitrile); the sample loading solution is 10% (volume fraction) acetonitrile solution; the chromatography 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; and then the next batch is recycled.

[0054] Since GLP-1 analogues coexist with other proteins and impurities in the fermentation broth, the traditional separation and purification method has problems such as complex operation, low yield and low purity, which restricts the further promotion of the genetic engineering production of GLP-1 analogues in clinical application.

[0055] The above method is aimed at GLP-1 analogue fermentation broth, and by adjusting the ratio between the hydrophilic organic solvent and the soluble inorganic salt, two aqueous layers are formed, thereby realizing efficient enrichment of GLP-1 analogues, providing a new technical approach for the separation and purification of GLP-1 analogues. Further, the crude product after efficient enrichment can be purified by reverse preparation liquid chromatography to obtain GLP-1 analogues with high purity. The above method fills the technical gap in the field of separation and purification of GLP-1 analogues in fermentation broth.

[0056] The application will be further described below with reference to the examples. Those skilled in the art will be able to implement the application based on the description. In addition, the examples of the application described below are generally only examples of part of the application, not all examples. Therefore, all other examples obtained by those skilled in the art based on the examples in the application without creative labor shall fall within the scope of protection of the application.

[0057] The GLP-1 analogue standard used in the examples of the application is from Lu'nan Pharmaceutical Group, with batch number F027-1-RS-221101; the GLP-1 analogue is a semaglutide precursor, and the amino acid sequence of the semaglutide precursor is shown in SEQ ID NO. 1: EGTFTSDVSSYLEGQAAKEFIAWLVRGRG. The GLP-1 analogue fermentation broth (not extracted) used in the examples of the application is from Lu'nan Pharmaceutical Group, and its liquid chromatogram is shown in Figure 13 . The determination conditions of the liquid chromatogram are as follows: the chromatographic column is Sepax Bio-C8 (5 μm, 4.6*250 mm); 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 as follows: 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 recycled.

[0058] The pH conditions in the experiments of the examples of the application are adjusted using 1 mol / L hydrochloric acid solution or sodium hydroxide solution; and the protein concentration of the fermentation broth in the experiments of the examples of the application is adjusted using deionized water, and the protein concentration of the original fermentation broth is 13 mg / mL.

[0059] Example 1: Preliminary screening of the aqueous two-phase extraction system

[0060] In this example, the preliminary screening of the aqueous two-phase extraction system is carried out, and the following steps are taken:

[0061] Step 1: Take 60% (w / w) of the mixed and uniform GLP-1 analogue fermentation broth in the total system (100 g) in a centrifugal tube.

[0062] Step 2: Take soluble inorganic salt anhydrous sodium citrate, anhydrous ammonium sulfate, anhydrous dipotassium hydrogen phosphate, anhydrous sodium bicarbonate, anhydrous sodium carbonate, anhydrous sodium phosphate salt buffer respectively, at the same time, take hydrophilic low molecular organic solvent anhydrous ethanol, isopropyl alcohol, tert-butyl alcohol, n-propanol respectively. Among them, the soluble inorganic salt accounts for 20% (w / w) of the total system mass percentage, and the hydrophilic low molecular organic solvent accounts for 20% (w / w) of the total system mass percentage.

[0063] Step 3: Under ice water bath, the soluble inorganic salt described in step 2 is combined and mixed with the hydrophilic low molecular organic solvent respectively, and after each combination is fully mixed and uniformly mixed with the GLP-1 analogue fermentation broth, 4500g centrifugation is carried out at 4°C for 20min to accelerate the formation of aqueous two-phase, and the phase formation results are recorded, see Table 1. Among the 11 systems with better phase formation effect, five systems can extract GLP-1 analogue into the upper phase. .

[0064] Step 4: Among the 11 systems with better phase formation effect, five systems can extract GLP-1 analogue into the upper phase, and the GLP-1 analogue recovery rate of the upper phase after new type aqueous two-phase extraction is analyzed by liquid phase, and the results are shown in Figure 1 . Among them, the GLP-1 analogue recovery rate determination method is: dilute the GLP-1 analogue solution after new type aqueous two-phase extraction and the original fermentation broth to the same concentration, and determine the concentration of GLP-1 analogue in the original fermentation broth and the GLP-1 analogue solution after new type aqueous two-phase extraction by reverse phase liquid chromatography. Among them, the parameters of reverse phase liquid chromatography are: the chromatographic column is Sepax Bio-C8 (5μm, 4.6*250mm); the mobile phase is A (containing 0.1% (volume fraction) TFA in water) and B (containing 0.1% (volume fraction) TFA in acetonitrile); 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; then the next cycle.

[0065] The recovery rate of the isopropanol-ammonium sulfate system was 54%, the recovery rate of the isopropanol-dipotassium hydrogen phosphate system was 45%, the recovery rate of the isopropanol-sodium citrate system was 43%, the recovery rate of the ethanol-ammonium sulfate system was 41%, and the recovery rate of the ethanol-dipotassium hydrogen phosphate system was the highest, reaching 48%. Thus, in the preliminary screening of the aqueous two-phase extraction system, the isopropanol-ammonium sulfate system had the highest recovery rate, the ethanol-dipotassium hydrogen phosphate system had the second highest recovery rate, and the recovery rate of the isopropanol-dipotassium hydrogen phosphate system was similar to that of the ethanol-dipotassium hydrogen phosphate system.

[0066] Example 2 Final screening of the aqueous two-phase extraction system

[0067] In this example, the final screening of the aqueous two-phase extraction system was performed according to the following steps (in the extraction system, the pH was 9.5, the extraction temperature was 0°C (under ice water bath conditions), and the protein concentration in the fermentation broth was adjusted to 11 mg / mL):

[0068] Step 1: The aqueous two-phase extraction system was used as the total system (100 g), and 60% (w / w) of the mixed and uniform GLP-1 analogue fermentation broth was taken in a centrifuge tube.

[0069] Step 2: The soluble inorganic salt anhydrous ammonium sulfate and the soluble inorganic salt anhydrous dipotassium hydrogen phosphate were taken, and the hydrophilic low molecular organic solvent anhydrous ethanol and isopropanol were taken at the same time. The soluble inorganic salt accounted for 20% (w / w) of the total system, and the hydrophilic low molecular organic solvent accounted for 20% (w / w) of the total system.

[0070] Step 3: Under ice water bath conditions, the soluble inorganic salt and the hydrophilic low molecular organic solvent were combined and mixed, and each combination was fully mixed with the GLP-1 analogue fermentation broth. After centrifugation at 4°C and 4500g for 20 min, the aqueous two-phase system was formed.

[0071] Step 4: The recovery rate of GLP-1 analogue in the upper phase after aqueous two-phase extraction was analyzed by liquid phase analysis, and the method was as described in step 4 of Example 1. The results are shown in Table 2. Figure 2 .

[0072] Step 5: The upper phase after new aqueous two-phase extraction was taken, and the purification factor (PF) was calculated. The results are shown in Table 3. Figure 2 .

[0073] From the recovery rate and purification factor determination results, it can be seen that the recovery rate of the isopropanol-ammonium sulfate system is 18%, the recovery rate of the isopropanol-dipotassium hydrogen phosphate system is 31%, the recovery rate of the ethanol-ammonium sulfate system is 31%, and the recovery rate of the ethanol-dipotassium hydrogen phosphate system is 48%; and the purification factor of the ethanol-dipotassium hydrogen phosphate system is 3.41, which is better than that of other systems, so the best aqueous two-phase extraction system for GLP-1 analog is the ethanol-dipotassium hydrogen phosphate system.

[0074] Example 3 Optimization screening of anhydrous dipotassium hydrogen phosphate content and anhydrous ethanol content

[0075] In this example, the ethanol-dipotassium hydrogen phosphate system is used as the aqueous two-phase extraction system to optimize the screening of the anhydrous dipotassium hydrogen phosphate content and the anhydrous ethanol content, which is carried out according to 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):

[0076] Step 1: Take 15%, 18%, 21%, 24%, 27%, and 30% of the total system mass percentage of anhydrous dipotassium hydrogen phosphate, respectively, and combine them with 14%, 18%, 22%, 26%, 30%, and 34% of the total system mass percentage of anhydrous ethanol, respectively, to form the aqueous two-phase extraction system.

[0077] Step 2: Take 100 g of the total system mass, and mix the mixed GLP-1 analog fermentation broth with the different content of anhydrous ethanol and anhydrous dipotassium hydrogen phosphate obtained in step 1 to form the aqueous two-phase extraction system.

[0078] Step 3: Under ice water bath, mix the different content of anhydrous ethanol and anhydrous dipotassium hydrogen phosphate to form the aqueous two-phase extraction system with the GLP-1 analog fermentation broth in it, and centrifuge at 4°C for 20 min at 4500g to accelerate the formation of the aqueous two-phase system. 36 groups of experiments are conducted, each repeated three times, and the test results are averaged.

[0079] Step 4: Take the upper phase after extraction of the aqueous two-phase extraction system, and determine the impurity removal effect. The GLP-1 analog recovery rate determination results are shown in Table 2, Figure 3 , and the determination method is shown in step 4 of Example 1. Among them, the highest recovery rate of GLP-1 analog is 99%, which is the 15% dipotassium hydrogen phosphate-18% ethanol system.

[0080] Step 5: Take the upper phase after extraction of the aqueous two-phase extraction system, and calculate the purification factor (PF), the results are shown in Table 3, Figure 4 . Among them, the highest recovery rate corresponds to a purification factor of 2.6.

[0081] Step 6: Take the upper phase after extraction in the aqueous two-phase extraction system and determine the pigment removal effect: Take the extracted GLP-1 analog solution and the original GLP-1 analog fermentation broth and dilute them to the same concentration. Use water as a blank and measure the absorbance at 350nm, 420nm and 610nm. The results show that the pigment removal rate of the 15% dipotassium hydrogen phosphate-18% ethanol system is 88%.

[0082] Step 7: Take the upper phase after extraction in the aqueous two-phase extraction system and determine the cell removal effect: Take the extracted GLP-1 analog solution and the supernatant of the original GLP-1 analog fermentation broth, and use the supernatant of the fermentation broth as a blank. Measure the absorbance at 600 nm. The results show that the cell removal rate of the 15% dipotassium hydrogen phosphate-18% ethanol system is 99%.

[0083] Example 4: Final determination of anhydrous dipotassium hydrogen phosphate content and anhydrous ethanol content

[0084] In this embodiment, the ethanol-dipotassium hydrogen phosphate system was used as the aqueous two-phase extraction system. The optimal content ratio of this system was determined by following these steps (the pH of the extraction system was 9.5, the extraction temperature was 0℃ (ice-water bath conditions), and the protein concentration in the fermentation broth was adjusted to 11 mg / mL):

[0085] Step 1: Using the aqueous two-phase extraction system as the total system, with a total system mass of 100g, take anhydrous dipotassium hydrogen phosphate at a mass percentage of 13%, 15%, and 17% of the total system, and combine it with anhydrous ethanol at a mass percentage of 16%, 18%, 20%, 22%, 24%, and 26% of the total system, respectively, to form an aqueous two-phase extraction system.

[0086] Step 2: The total mass of the system is controlled to be 100g. Take the well-mixed GLP-1 analog fermentation broth and mix it with the aqueous two-phase extraction system composed of different contents of anhydrous ethanol and anhydrous dipotassium hydrogen phosphate obtained in Step 1.

[0087] Step 3: Under ice-water bath conditions, the aqueous two-phase extraction system consisting of different concentrations of anhydrous ethanol and anhydrous dipotassium hydrogen phosphate was thoroughly mixed with the fermentation broth of the GLP-1 analogue. The mixture was then centrifuged at 4500g for 20 minutes at 4°C to accelerate the formation of the aqueous two-phase system. Eighteen pairs of crossover experiments were conducted, with each experiment repeated three times. The average value of the results was taken.

[0088] Step 4: Take the upper phase after extraction from the aqueous two-phase extraction system and determine the impurity removal effect. The results of the GLP-1 analogue recovery rate determination are shown in [link to relevant documentation]. Figure 5 The method is the same as step 4 in Example 1. This step shows that the highest recovery rate of the GLP-1 analog is 99% in a 15% dipotassium hydrogen phosphate-20% ethanol system.

[0089] Step 5: Take the upper phase after the aqueous two-phase extraction system extraction, calculate the purification factor (PF), and the results are shown in Table 4. Figure 6 The results show that the highest purification factor is 4.0, which is the 15% dipotassium hydrogen phosphate-16% ethanol system; the purification factor of the 15% dipotassium hydrogen phosphate-20% ethanol system is 2.9.

[0090] Step 6: Take the upper phase after the aqueous two-phase extraction system extraction, and determine the pigment removal effect: the method is shown in Step 6 of 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%.

[0091] Step 7: Take the upper phase after the aqueous two-phase extraction system extraction, and determine the cell removal effect, the method is shown in Step 7 of Example 3, and the results show that the cell 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 99%.

[0092] Example 5 Determination of the Optimal Temperature of the Aqueous Two-Phase Extraction System

[0093] In this example, the 15% dipotassium hydrogen phosphate-18% ethanol system is used as the aqueous two-phase extraction system to determine the optimal temperature for extraction, and the following steps are followed (in the extraction system, the pH is 9.5, and the protein concentration in the fermentation broth is adjusted to 11 mg / mL):

[0094] Step 1: Take 5 portions of GLP-1 analogue fermentation broth, each weighing 65 g.

[0095] Step 2: In an ice water bath, add 15 g of dipotassium hydrogen phosphate to each portion of GLP-1 analogue fermentation broth, and mix well using a vortex shaker. After the salt is completely dissolved, adjust the system temperature to 0°C, 25°C, 30°C, 35°C, and 40°C respectively, and then add 20 g of pre-cooled or pre-heated anhydrous ethanol, and mix well. Each group of experiments is repeated three times, and the test results are averaged.

[0096] Step 3: Centrifuge at 4500g for 20 min at 4°C to separate the phases.

[0097] Step 4: Take the upper phase after the aqueous two-phase extraction system extraction, and determine the GLP-1 analogue recovery rate and impurity removal effect, and the results are shown in Table 5. Figure 7 , and the method is shown in Step 4 of Example 1. The highest GLP-1 analogue recovery rate is 92%, which is obtained at 0°C.

[0098] Step 5: Take the upper phase after the aqueous two-phase extraction system extraction, calculate the purification factor (PF), and the results are shown in Table 4. Figure 8The results show that the highest purification factor is 3.4, which is obtained at 0℃.

[0099] Step 6: Take the upper phase after extraction by the aqueous two-phase extraction system, and determine the pigment removal effect. The method is shown in step 6 of Example 3. The results show that the pigment removal rate is 92% at 0℃.

[0100] Step 7: Take the upper phase after extraction by the aqueous two-phase extraction system, and determine the cell removal effect. The method is shown in step 7 of Example 3. The results show that the cell removal rate is 99% at 0℃.

[0101] Example 6 Determination of the Optimal pH of the Aqueous Two-Phase Extraction System

[0102] In this example, the 15% potassium phosphate dibasic-18% ethanol system is used as the aqueous two-phase extraction system to determine the optimal pH. The following steps are followed (in the extraction system, the extraction temperature is 0℃ (ice water bath condition), and the protein concentration in the fermentation broth is adjusted to 11 mg / mL):

[0103] Step 1: Take 5 portions of GLP-1 analogue fermentation broth, each 65g.

[0104] Step 2: In an ice water bath, add 15g of potassium phosphate dibasic to each portion of GLP-1 analogue fermentation broth, and mix well using a vortex shaker. After the salt is completely dissolved, add 20g of pre-cooled or pre-heated anhydrous ethanol, and mix well.

[0105] 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 is repeated three times, and the test results are averaged.

[0106] Step 4: Centrifuge at 4500g for 20min at 4℃ to separate the phases.

[0107] Step 5: Take the upper phase after extraction by the aqueous two-phase extraction system, and determine the GLP-1 analogue recovery rate and impurity removal effect. The results are shown in Figure 9 , and the method is shown in step 4 of Example 1. The highest GLP-1 analogue recovery rate is 97%, which is obtained at pH 9.5.

[0108] Step 6: Take the upper phase after extraction by the aqueous two-phase extraction system, and calculate the purification factor (PF). The results are shown in Figure 10 . The results show that the highest purification factor is 2.8, which is obtained at pH 9.5.

[0109] Step 7: Take the upper phase after extraction by the aqueous two-phase extraction system, and determine the pigment removal effect. The method is shown in step 6 of Example 3. The results show that the highest pigment removal rate is 88%, which is obtained at pH 9.5.

[0110] Step 8: Take the upper phase after the aqueous two-phase extraction system extraction, and determine the bacteria removal effect. The method is shown in step 7 of Example 3. The results show that the highest bacteria removal rate is 99%, which is obtained under the condition of pH 9.5.

[0111] Example 7 Determination of the optimal protein concentration of the aqueous two-phase extraction system

[0112] In this example, the 15% potassium phosphate dibasic-18% ethanol system is used as the aqueous two-phase extraction system to determine the optimal protein concentration. The following steps are taken (in the extraction system, the pH is 9.5, and the extraction temperature is 0°C (ice water bath condition)):

[0113] Step 1: Take 5 portions of GLP-1 analogue fermentation broth, each containing different protein concentrations, i.e. 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, and 13 mg / mL.

[0114] Step 2: In an ice water bath, add potassium phosphate dibasic 15 g to each portion of GLP-1 analogue fermentation broth, and mix well using a vortex shaker. After the salt is completely dissolved, add pre-cooled anhydrous ethanol 20 g, and mix well.

[0115] Step 3: Adjust the pH of the system to 9.5, and repeat each experiment three times.

[0116] Step 4: Centrifuge at 4500g for 20 min at 4°C to separate the phases.

[0117] Step 5: Take the upper phase after the aqueous two-phase extraction system extraction, and determine the GLP-1 analogue recovery rate and impurity removal effect. The results are shown in Figure 11 , and the method is shown in step 4 of Example 1. The results show that the highest GLP-1 analogue recovery rate is 98%, which is obtained under the condition of a protein concentration of 11 mg / mL.

[0118] Step 6: Take the upper phase after the aqueous two-phase extraction system extraction, and calculate the purification factor (PF). The results are shown in Figure 12 . The results show that the highest purification factor is 3.2, which is obtained under the condition of a protein concentration of 11 mg / mL.

[0119] Step 7: Take the upper phase after the aqueous two-phase extraction system extraction, and determine the pigment removal effect. The method is 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.

[0120] Step 8: Take the upper phase after the aqueous two-phase extraction system extraction, and determine the bacteria removal effect. The method is shown in step 7 of Example 3. The results show that the highest bacteria removal rate is 99%, which is obtained under the condition that the protein concentration is 11 mg / mL.

[0121] Example 8 Further purification of GLP-1 analogue

[0122] In this example, the aqueous two-phase extraction system of 15% potassium phosphate dibasic-18% ethanol system is used to further purify the GLP-1 analogue, and the following steps are taken:

[0123] Step 1: Take 201 g of GLP-1 analogue fermentation broth with a protein concentration of 11 mg / mL.

[0124] Step 2: Add potassium phosphate dibasic 45 g in an ice water bath, and mix well using a vortex oscillator. After the salt is completely dissolved, add 54 g of pre-cooled anhydrous ethanol, and mix well.

[0125] Step 3: Adjust the pH of the system to 9.5. Set up 3 groups of parallel experiments, and take the average value of the test results.

[0126] Step 4: Centrifuge at 4500g for 20 min at 4°C to separate the phases.

[0127] Step 5: Take the upper phase after the aqueous two-phase extraction system extraction, and test the liquid chromatogram. The results are shown in 14. Take the upper phase after the aqueous two-phase extraction system extraction, and determine the GLP-1 analogue recovery rate and impurity removal effect. The method is shown in step 4 of Example 1. The GLP-1 analogue recovery rate is 97%.

[0128] Step 6: Take the upper phase after the aqueous two-phase extraction system extraction, and calculate the purification factor (PF). The results show that the purification factor is 3.4.

[0129] Step 7: Take the upper phase after the aqueous two-phase extraction system extraction, and determine the pigment removal effect. The method is shown in step 6 of Example 3. The results show that the pigment removal rate is 88%.

[0130] Step 8: Take the upper phase after the aqueous two-phase extraction system extraction, and determine the bacteria removal effect. The method is shown in step 7 of Example 3. The results show that the bacteria removal rate is 99%.

[0131] Step 9: Take the upper phase after the aqueous two-phase extraction system extraction, and perform chromatographic analysis. The results are shown in Figure 14 ; and determine by 15% reduced SDS-PAGE electrophoresis, with a loading amount of 10 uL. The results are shown in Figure 15 . The electrophoresis results show that the impurity protein bands are significantly reduced. For example, Figure 14As shown, after chromatographic analysis, the impurity peaks are obviously reduced.

[0132] Step 10: After the upper phase of the sample was desalted, 15 mL of enterokinase buffer was added, and after mixing evenly, 3.5 mg of enterokinase was added, and the enzyme was cut for 16 h at 25℃.

[0133] Step 11: After the tag of the fusion protein was removed, the solution was adjusted to pH 4.2-4.5, precipitated at 10℃ for 30 min, centrifuged at 5000 r for 30 min, the supernatant was discarded, and the precipitate was dissolved by adding 20 mmol / L Tris buffer at pH 8.0 to obtain the crude product of GLP-1 analogue. The GLP-1 analogue after enzyme cutting was taken for liquid chromatogram test, and the results are shown in Figure 16 .

[0134] Step 12: Reverse phase preparative liquid chromatography was used to further remove impurities and improve purity. The GLP-1 analogue crude product was dissolved and loaded on a reverse phase preparative liquid chromatography column, and the target peak product was collected: the mobile phase was A (0.1% (volume fraction) TFA in water) and B (0.1% TFA (volume fraction) acetonitrile); the sample loading solution was 10% (volume fraction) acetonitrile solution; the chromatography column was Sepax Bio-C8 (10 μm, 21.2*250 mm); the elution conditions were as follows: 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 was recycled. The target peak collection time was selected as 21 min. As shown in Figure 17 , it is the reverse phase preparative liquid chromatogram of the GLP-1 analogue after enzyme cutting.

[0135] Step 13: The GLP-1 analogue sample collected after purification was taken for chromatographic analysis, and the results are shown in Figure 18 ; and 15% reduced SDS-PAGE electrophoresis was used for determination, and the loading amount was 10 uL, and the results are shown in Figure 15 . The electrophoresis results show that the GLP-1 analogue sample after purification is a single band. The product prepared by reverse phase preparative liquid chromatography has obviously reduced impurity peaks, and the purification effect is excellent. The chromatography column analysis shows that the purity is 99.3% (HPLC) Figure 18 .

[0136] The raw materials and equipment used in the present application are all common raw materials and equipment in the art unless otherwise specified; and the methods used in the present application are all conventional methods in the art unless otherwise specified.

[0137] The above is only the preferred embodiment of the present application, and does not limit the present application, and any simple modification, change and equivalent transformation of the above embodiment according to the technical essence of the present application still belongs to the protection scope of the technical scheme of the present application.

Claims

1. A method for separating and purifying GLP-1 analogs in fermentation broth using aqueous two-phase extraction coupled with reversed-phase column chromatography, characterized in that: The method comprises the following steps: At 0-30 DEG C, inorganic salt and hydrophilic organic solvent are added into GLP-1 analogue fermentation liquor, the system is uniformly suspended, the pH is adjusted to 8.5-10.5, the system is centrifuged to separate the phases, the upper phase is taken, subjected to enterokinase enzymolysis, and then subjected to reverse preparation liquid chromatography purification treatment; wherein: The inorganic salt is added in an amount of 10%-35% based on the total mass of the system, and the hydrophilic organic solvent is added in an amount of 15%-40% based on the total mass of the system. The inorganic salt and the hydrophilic organic solvent are selected from any one of the following combinations: isopropyl alcohol and ammonium sulfate, isopropyl alcohol and dipotassium hydrogen phosphate, isopropyl alcohol and sodium citrate, ethanol and ammonium sulfate, and ethanol and dipotassium hydrogen phosphate. The protein concentration of the GLP-1 analogue fermentation liquor is 9-13 mg / mL. The GLP-1 analogue is semaglutide precursor, and the amino acid sequence of the semaglutide precursor is: EGTFTSDVSSYLEGQAAKEFIAWLVRGRG.

2. The method for separating and purifying GLP-1 analogs in fermentation broth by aqueous two-phase extraction coupled with reversed-phase column chromatography according to claim 1, characterized in that: The inorganic salt is added in an amount of 13%-24% based on the total mass of the system.

3. The method for separating and purifying GLP-1 analogs in fermentation broth by aqueous two-phase extraction coupled with reversed-phase column chromatography according to claim 2, characterized in that: The inorganic salt is added in an amount of 13%-17% based on the total mass of the system.

4. The method of claim 1, wherein the method is for separating and purifying GLP-1 analogs from fermentation broth by aqueous two-phase extraction coupled with reversed-phase column chromatography. The hydrophilic organic solvent is added in an amount of 16%-26% based on the total mass of the system.

5. The method of claim 1, wherein the fermentation broth is purified by aqueous two-phase extraction coupled with reversed-phase column chromatography. The pH is adjusted to 9.5.

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