Post-treatment method of polypeptide lysate

Abstract

The invention discloses a post-treatment method of a polypeptide lysate. The method belongs to the field of polypeptide drug preparation. The method comprises the following steps: firstly, concentrating a polypeptide lysate, and then spraying pre-cooled isopropyl ether or methyl tert-butyl ether into the concentrated solution until a large amount of samples are separated out; and carrying out solid-liquid separation by using a filter bag type centrifuge, and carrying out on-line leaching by using isopropyl ether or methyl tert-butyl ether. The solid obtained through the method is large in particle size, solid-liquid separation is facilitated, the operation time is greatly shortened, and the method is extremely beneficial to large-scale industrial production. In addition, an on-line leaching mode is adopted, so that contact with people is avoided, and green industrial production is facilitated.

Description

Technical Field

[0001] The present invention belongs to the field of polypeptide drug preparation, and particularly relates to a post-treatment method for a polypeptide cleavage solution. Background Art

[0002] As an important bioactive substance, polypeptides are widely used in the fields of medicine, food, etc., and the current research enthusiasm is constantly increasing.

[0003] The wide application of polypeptides stems from their unique properties. Since Merrifield proposed the solid-phase polypeptide synthesis method in 1963, the synthesis technology of polypeptides has been continuously developed. At present, there are mainly two methods for polypeptide synthesis: solid-phase synthesis and liquid-phase synthesis. Among them, solid-phase synthesis is further divided into two methods: Fmoc and Boc. The Fmoc method is widely used because of its mild reaction conditions, few side reactions, high yield, and easy monitoring.

[0004] In the medical field, polypeptides have the effects of promoting cell proliferation and repair, enhancing immunity, regulating hormone secretion, treating inflammation, promoting muscle growth and recovery, etc. The global market scale of polypeptide drugs is constantly expanding. In 2019, the global sales of polypeptides (including insulin) exceeded $50 billion. Currently, there are about 80 approved polypeptide drugs globally. In recent years, with the continuous progress of peptide therapy and peptide synthesis technology, the research on polypeptide drugs has developed rapidly.

[0005] In the food field, polypeptides also play an important role. Polypeptides can be used as food additives such as sweeteners, flavor enhancers, emulsifiers, antioxidants, preservatives, etc., and have the advantages of high sweetness intensity, good thermal stability, and natural origin. In addition, the application of functional polypeptides in food is also becoming more and more extensive. For example, calcium absorption promoting peptide (CPP) can promote calcium absorption, lipid-lowering peptide can reduce blood lipid and adjust blood pressure, and sleep peptide can promote slow-wave sleep.

[0006] The synthesis methods of polypeptides mainly include liquid-phase synthesis and solid-phase synthesis. The commonly used protecting groups in liquid-phase synthesis are Boc and Cbz (Z), and the stepwise synthesis method and fragment synthesis method are mainly adopted. Compared with the solid-phase synthesis method, the liquid-phase synthesis method is more suitable for the synthesis of short peptides, with small amounts of reagents, solvents, etc., lower costs, in line with the principles of green chemistry, and more selective protecting groups, which is conducive to subsequent purification and large-scale production. The solid-phase synthesis method uses an insoluble resin as a carrier, fixes the C-terminal of the first amino acid on the resin, and gradually synthesizes the target polypeptide through condensation reactions.

[0007] At present, after the peptide resin obtained by conventional solid-phase synthesis is cleaved, the cleavage solution is added to isopropyl ether or methyl tert-butyl ether, and solid-liquid separation is carried out by centrifugation or suction filtration. Then the solid is homogenized and washed, repeated three times, and dried to obtain the crude product. The sample particles precipitated by this method are small, resulting in a long time required for solid-liquid separation. Inevitably, human contact occurs during the washing of the sample, and both isopropyl ether and methyl tert-butyl ether are flammable, so there are serious safety hazards in the operation process. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a post-treatment method for a polypeptide cleavage solution in view of the deficiencies of the prior art. This method increases the concentration of the sample in the cleavage solution, and a large amount of the sample precipitates and agglomerates by adding isopropyl ether or methyl tert-butyl ether, which is convenient for filtration, reduces the post-treatment time, and at the same time adopts an online rinsing method to avoid human contact, which is beneficial to green industrial production.

[0009] In order to achieve the above-mentioned invention object, the present invention provides the following technical solutions: The present invention is a post-treatment method for a polypeptide cleavage solution, which is characterized by including the following steps: (1) Concentrate the polypeptide cleavage solution; (2) Spray isopropyl ether or methyl tert-butyl ether pre-cooled to -5°C to -20°C into the rapidly stirred cleavage solution in step (1) until a large amount of the sample precipitates; (3) Pump the solid-liquid mixture precipitated in step (2) into a filter bag centrifuge for centrifugation and online rinsing, and collect the filter cake; (4) Dry the filter cake to obtain the polypeptide crude product.

[0010] For the post-treatment method of a polypeptide cleavage solution described in the present invention, a further preferred technical solution is: In step (1), the concentration temperature is 20 to 25°C.

[0011] In step (1), the volume after concentration is 70% to 90%.

[0012] In step (2), the sample precipitation temperature is -5 to 10°C.

[0013] In step (2), the volume ratio of isopropyl ether or methyl tert-butyl ether to the concentrated solution is 4 to 8:1.

[0014] In step (3), the method of solid-liquid separation is centrifugal filtration.

[0015] In step (3), the online rinsing volume of isopropyl ether or methyl tert-butyl ether is 1 to 4 times that of the concentrated solution.

[0016] In step (4), the sample is dried by vacuum drying.

[0017] Preferably, the specific steps of the method of the present invention are as follows: Take semaglutide peptide resin, prepare a cleavage solution according to the volume ratio of TFA:TIS:water:EDT = 90:5:3:2, add the resin to the cleavage solution, stir and react at room temperature, filter the resin, collect the filtrate into a concentration tank, wash the resin with TFA, and combine the filtrates; Turn on the stirring and jacket heat medium of the concentration tank, control the temperature in the concentration tank to be 20°C to 25°C, turn on the vacuum pump, concentrate, turn off the vacuum and heat medium, turn on the jacket refrigerant of the concentration tank, wait for the temperature to drop to -5°C to 10°C, and slowly spray methyl tert-butyl ether pre-cooled to -5°C to -20°C into the concentration tank with a pneumatic pump until a large amount of the sample precipitates, pump the solid-liquid mixture into a filter bag centrifuge for centrifugation, and perform online rinsing; Collect the solid, dry it in a vacuum drying oven to obtain semaglutide crude peptide dry powder.

[0018] Compared with the prior art, the present invention has the following beneficial effects: In the method of the present invention, solid-liquid separation is carried out by a filter bag centrifuge, and then isopropyl ether or methyl tert-butyl ether is used for online rinsing. The solid obtained by this method has a larger particle size, which is convenient for solid-liquid separation, and the filtration time is short, generally can be completed within 1 hour. Compared with 6-7 hours of the prior art, the operation time is greatly reduced, which is extremely beneficial for large-scale industrial production. In addition, the method of online rinsing is adopted, avoiding contact with people, which is beneficial for green industrial production. Description of the Drawings

[0019] Figure 1 Is the high-performance liquid chromatography spectrum of the sample obtained in Example 1; Figure 2 Is the high-performance liquid chromatography spectrum of the sample obtained in Comparative Example 1.

[0020] Specific Embodiment Cases The present invention discloses a post-treatment method for a polypeptide cleavage solution. The embodiments are only a part of the present invention, not all of it. The role of the embodiments is to illustrate and explain the technicality and characteristics of the present invention, so that those skilled in the art can better understand the present invention and be able to implement it, but it cannot limit the protection scope of the present invention. All equivalent changes or modifications made according to the essence of the present invention are included in the protection scope of the present invention.

[0021] Example 1: Refer to Figure 1 , the post-treatment method of semaglutide peptide resin cleavage solution Take 1000.0 g of semaglutide peptide resin, prepare 10000 mL of cleavage solution according to the volume ratio of TFA:TIS:water:EDT = 90:5:3:2. Add the resin into the cleavage solution and stir at room temperature for 2 h. Filter the resin, collect the filtrate into the concentration tank, wash the resin twice with 1000 mL of TFA, and combine the filtrates. Turn on the stirring of the concentration tank and the jacket heat medium, control the temperature in the concentration tank to be 23 °C, turn on the vacuum pump, concentrate to 4500 mL of liquid, with a concentration time of 1 hour. Turn off the vacuum and heat medium, turn on the jacket refrigerant of the concentration tank. Wait until the temperature drops to 0 °C, and slowly spray 45000 mL of pre-cooled methyl tert-butyl ether into the concentration tank using a pneumatic pump until a large amount of the sample precipitates. Pump the solid-liquid mixture into a filter bag centrifuge for centrifugation, and perform an online rinse for 30 min with a rinse volume of 25000 mL. Collect the solid, dry it in a vacuum drying oven for 12 h to obtain 523.2 g of semaglutide crude peptide dry powder.

[0022] Comparative Example 1: Refer to Figure 2 , Take 1000.0 g of semaglutide peptide resin, prepare 10000 mL of cleavage solution according to the volume ratio of TFA:TIS:water:EDT = 90:5:3:2. Add the resin into the cleavage solution and stir at room temperature for 2 h. Filter the resin, collect the filtrate, wash the resin twice with 1000 mL of TFA, and combine the filtrates. Turn on the refrigerant of the precipitation tank, pump 72000 mL of pre-cooled methyl tert-butyl ether into the precipitation tank, turn on the stirring, and slowly add the cleavage solution into the precipitation tank until a large amount of the sample precipitates. Add the solid-liquid mixture into a cup centrifuge for solid-liquid separation, and wash the solid by pulping 3 times, 10000 mL each time. Collect the solid, dry it in a vacuum drying oven for 12 h to obtain 510.3 g of semaglutide crude peptide dry powder.

[0023] It can be seen from the above comparison: From the data of the above examples and comparative examples, it can be seen that the post-treatment method of the polypeptide cleavage solution provided by the present invention greatly shortens the treatment time without reducing the purity compared with the conventional operation, and avoids the risk of contact between people and materials, which is beneficial to the production of green chemical industry.

[0024] Example 2: Post-treatment method of liraglutide cleavage solution Take 1000.0 g of liraglutide peptide resin, prepare 10000 mL of cleavage solution according to the volume ratio of TFA:TIS:water:EDT = 90:5:3:2. Add the resin into the cleavage solution and stir at room temperature for 2 h. Filter the resin, collect the filtrate into the concentration tank, wash the resin twice with 1000 mL of TFA, and combine the filtrates. Turn on the stirring of the concentration tank and the jacket heat medium, control the temperature in the concentration tank at 23 °C, turn on the vacuum pump, concentrate to 4500 mL of liquid, turn off the vacuum and heat medium, turn on the jacket refrigerant of the concentration tank. When the temperature drops to 0 °C, slowly spray 45000 mL of methyl tert-butyl ether pre-cooled in advance into the concentration tank with a pneumatic pump until a large amount of the sample precipitates. Pump the solid-liquid mixture into a filter bag centrifuge for centrifugation, and perform an online rinse for 30 min with a rinse volume of 25000 mL. Collect the solid, dry it in a vacuum drying oven for 12 h to obtain 481.3 g of crude liraglutide peptide dry powder with a purity of 83.5%.

Claims

1. A post-treatment method for a polypeptide lysate, characterized in that: The following steps are involved: (1) Concentrating the peptide lysate; (2) Spray isopropyl ether or methyl tert-butyl ether precooled to -5°C to -20°C into the rapidly stirred lysate of step (1) until a large amount of sample precipitates; (3) pumping the solid-liquid mixture precipitated in step (2) into a filter bag centrifuge for centrifugation, eluting online, and collecting the filter cake; (4) Dry the filter cake to obtain the crude polypeptide.

2. The post-treatment method of a polypeptide lysate according to claim 1, characterized in that: The concentration temperature is 20℃~25℃.

3. The post-treatment method of a polypeptide lysate according to claim 1, characterized in that: The volume after concentration is 70%~90%.

4. The post-treatment method of a polypeptide lysate according to claim 1, characterized in that: The sample precipitation temperature is -5℃~10℃.

5. The post-treatment method of a polypeptide lysate according to claim 1, characterized in that: The volume ratio of isopropyl ether or methyl tert-butyl ether to the concentrated solution is 4~8:

1.

6. The post-treatment method of a polypeptide lysate according to claim 1, characterized in that: The method for solid-liquid separation is centrifugal filtration.

7. The post-treatment method of a polypeptide lysate according to claim 1, characterized in that: The online elution volume of isopropyl ether or methyl tert-butyl ether is 1 to 4 times that of the concentrate.

8. The post-treatment method of a polypeptide lysate according to claim 1, characterized in that: The samples were dried by vacuum drying.

9. A post-treatment method for a polypeptide lysate according to any one of claims 1 to 8, characterized in that: The specific steps are as follows: take semaglutide peptide resin, prepare a cleavage solution according to the volume ratio of TFA:TIS:water:EDT=90:5:3:2, add the resin to the cleavage solution and stir at room temperature for reaction, filter the resin, collect the filtrate into a concentration tank, wash the resin with TFA, and combine the filtrate; Turn on the stirring and jacket heat medium of the concentration tank, control the temperature in the concentration tank to 20°C~25°C, turn on the vacuum pump, concentrate, turn off the vacuum and heat medium, turn on the jacket refrigerant of the concentration tank, wait until the temperature drops to -5°C~10°C, use a pneumatic pump to slowly spray methyl tert-butyl ether precooled to -5°C~-20°C into the concentration tank until a large amount of sample precipitates, pump the solid-liquid mixture into a filter bag centrifuge for centrifugation, and rinse online; collect the solid, dry it in a vacuum drying oven, and obtain the crude semaglutide peptide powder.

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