CN112321720A - Method for purifying exenatide human serum albumin fusion protein - Google Patents
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/57563—Vasoactive intestinal peptide [VIP]; Related peptides
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Abstract
The invention relates to a purification method of exenatide human serum albumin fusion protein, which is characterized in that yeast fermentation supernatant of the exenatide human serum albumin fusion protein which is expressed in a recombinant mode is subjected to ultrafiltration concentration, purification is carried out through an ion column, a hydrophobic column and an ion column in sequence, and finally desalting is carried out through a molecular sieve; the ion column is at least one strong anion column and/or one weak alkaline anion column, and a hydrophobic column is used between the two ion columns for one-time purification. According to the purification method of the exenatide human serum albumin fusion protein, an optimized purification process of fermentation, ultrafiltration concentration, strong anion glue adsorption separation, hydrophobic glue and ion exchange impurity removal, and endotoxin and salt removal by a molecular sieve is established according to the physicochemical characteristic characteristics of the exenatide human serum albumin fusion protein, so that the high-purity recombinant exenatide human serum albumin fusion protein is obtained, the purity is more than 98%, and the purity of the produced purified recombinant exenatide human serum albumin fusion protein meets the requirement of quality standard.
Description
Technical Field
The invention belongs to the technical field of biotechnology and pharmaceutical engineering, and particularly relates to a purification method of exenatide human serum albumin fusion protein.
Background
Exenatide (Exenatide) is a glucagon-like peptide-1 (GLP-1) analogue found in the saliva of exendin dulcis in south America and has a molecular weight of 4186.6 kDa. The exenatide N-terminal 9 amino acids are highly homologous with the human glucagon-like peptide-1, can be combined with a human GLP-1 receptor to play the same physiological function as the human GLP-1, but the bioactivity of the exenatide is higher and more stable than that of the human GLP-1, and the exenatide N-terminal 9 amino acids have the effects of protecting pancreatic islets, promoting insulin secretion, promoting sugar utilization, reducing hepatic glycogen decomposition, reducing blood sugar and the like, and the insulin secretion promoting effect is blood sugar dependent and can not cause hypoglycemic reaction, so the exenatide N-terminal 9 amino acids are called intelligent hypoglycemic agents and are widely used for treating type II diabetes. However, due to the small molecular weight of exenatide, the half-life period in vivo is only 60-90 minutes, the duration of drug effect in vivo is short, patients need to inject 2 times a day to achieve satisfactory treatment effect, the use is inconvenient, and the compliance and treatment value of clinical treatment are limited.
In order to solve the problem of short drug effect time of exenatide, the applicant of the invention connects the gene coding exenatide with the gene coding human serum albumin together to construct an engineering yeast Ex-HSA/CICIM Y0600 which externally secretes and expresses exenatide human serum albumin fusion protein in saccharomyces cerevisiae, and clinical tests show that the half-life period of the exenatide human serum albumin fusion protein in human body can reach 100 hours, the in vivo drug effect can be maintained for a week or longer, the diabetic can obtain good treatment effect by injecting the exenatide human serum albumin fusion protein once a week, and the exenatide human serum albumin fusion protein is expected to be an outstanding long-acting treatment drug for type II diabetes, is a 1.0 innovative drug with independent intellectual property rights in China, and brings good news to the patients with type II diabetes in China.
Because the exenatide human serum albumin fusion protein is a brand new protein, the structure and the physicochemical properties of the exenatide human serum albumin fusion protein have particularity, a set of suitable purification method needs to be established to prepare the high-purity exenatide human serum albumin fusion protein, and sufficient raw material medicaments are provided for the large-scale production of clinical preparations.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention aims to provide a method for purifying exenatide human serum albumin fusion protein.
In order to achieve the purpose and achieve the technical effect, the invention adopts the technical scheme that:
a purification method of exenatide human serum albumin fusion protein comprises the following steps: performing ultrafiltration and concentration on yeast fermentation supernatant of the recombinant expressed exenatide human serum albumin fusion protein, purifying by an ion column, a hydrophobic column and an ion column in sequence, and desalting by a molecular sieve; the ion column is at least one strong anion column and/or one weak alkaline anion column, and a hydrophobic column is used for one-time purification between the two ion columns.
Further, yeast fermentation supernatant of the recombinant expressed exenatide human serum albumin fusion protein is subjected to ultrafiltration concentration, then is purified by an SP Sepharose XL ion column, a Butyl Sepharose 4FF hydrophobic column and a DEAE Sepharose FF ion column in sequence, and then is desalted by a Sephacryl HR S-200 molecular sieve.
Further, the SP Sepharose XL ion column purification step comprises:
1) ultrafiltration and sample treatment
Carrying out ultrafiltration concentration on yeast fermentation supernatant of the recombinant expressed exenatide human serum albumin fusion protein to obtain a concentrated sample, adding diluent to enable the conductivity to be 2-5 s/cm, then adding sodium acetate to enable the final concentration to be 10-40mM, and adjusting the pH value to be 4.9 +/-0.5;
2) column packing and column processing
Loading 10-20 mL SP Sepharose XL resin into each liter of fermentation liquor, washing 3-5 column volumes with water, washing 2-5 column volumes with NaOH, and washing with water until the pH value is less than 9;
3) column balance
Balancing the column with an equilibrium buffer until the conductivity and the pH are consistent with those of the equilibrium buffer a;
4) sample loading
Loading the treated fermentation liquor into a balanced SP Sepharose XL ion column;
5) washing machine
Washing the column with an equilibrium buffer a for 3-5 column volumes;
6) elution is carried out
Eluting the target protein by using the elution buffer a, and collecting an elution peak.
Further, the equilibration buffer a is: 5mM sodium caprylate, 5mM EDTA, 20mM NaAC, pH 4.0-5.5, conductivity less than 5 ms/cm; the elution buffer a is: 5mM sodium caprylate, 5mM EDTA, 20mM NaAC, 400mM NaCl, pH 4.0-5.5.
Further, the Butyl Sepharose 4FF hydrophobic column purification step comprises:
1) column packing and column processing
Filling Butyl Sepharose 4FF hydrophobic column according to 10-20mg protein sample adsorbed by resin per ml, removing ethanol, washing 2-5 column volumes with NaOH, and removing alkali;
2) sample processing
Adding NaCl into an exenatide human serum albumin fusion protein sample obtained after SP Sepharose XL ion column purification to a final concentration of 2-3M, adjusting the pH to 4.0-7.0, and adjusting the conductivity to be more than 150 ms/cm;
3) sample loading
Loading the sample processed in the step 2) on a column, wherein the linear flow velocity is less than 150 cm/h;
4) washing machine
After the sample loading is finished, washing 3-5 column volumes by using an equilibrium buffer solution b;
5) elution is carried out
Eluting the target protein by using an elution buffer b, and collecting an elution peak.
Further, the equilibration buffer b is: 20mM PB with pH value of 6.5-7.5 and 5mM sodium caprylate, and the conductance is less than 5 ms/cm; the elution buffer b is 20mM PB, 5mM sodium caprylate and 200mM sodium chloride, and has a pH value of 6.5-6.8.
Further, the DEAE Sepharose FF ion column purification step comprises:
1) column packing and column processing
Loading a protein sample with 30-50 mg per ml of resin for adsorption into a column, removing ethanol, cleaning for 30-90min by using NaOH, and then removing alkali;
2) sample processing
Desalting target elution peak collected in Butyl Sepharose 4FF hydrophobic column chromatography by ultrafiltration, adjusting buffer solution concentration of sample to 10-40mM PB, pH to 6.0-8.0, and conductance less than 5 ms/cm;
3) sample loading
Loading a processed DEAE Sepharose FF sample, wherein the linear flow rate is 300-600 cm/h;
4) washing machine
Washing 3-5 column volumes with an equilibration buffer c;
5) elution is carried out
Eluting the target protein by using an elution buffer c, and collecting an elution peak.
Further, the equilibration buffer c is: 20mM PB with pH value of 6.5-7.5 and 5mM sodium caprylate, and the conductivity is less than 5 ms/cm; the elution buffer c is: 20mM PB, 5mM sodium caprylate, 200mM NaCl, pH 6.5-6.8.
Further, the Sephacryl HR S-200 molecular sieve desalting step comprises the following steps:
1) column packing and column processing
Removing ethanol, washing with NaOH for 30-90min, and removing alkali; balancing the column with an equilibration buffer solution d until the conductivity and pH of the effluent are consistent with those of the equilibration buffer solution d;
2) sample loading
Loading DEAE eluate at flow rate of 1-10mL/min with loading amount less than 5% of column volume;
3) washing machine
Elution was performed with equilibration buffer d and the main peak was collected.
Further, the equilibration buffer d is: 10mM PB, 5mM NaCl; pH 7.0. + -. 0.5.
Furthermore, the exenatide human serum albumin is derived from an exenatide human serum albumin fusion protein recombinant expression plasmid transformed saccharomyces cerevisiae recipient bacteria, and supernatant is collected through fermentation.
Compared with the prior art, the invention has the beneficial effects that:
the invention discloses a purification method of exenatide human serum albumin fusion protein, which comprises the following steps: performing ultrafiltration and concentration on yeast fermentation supernatant of the recombinant expressed exenatide human serum albumin fusion protein, purifying by an ion column, a hydrophobic column and an ion column in sequence, and desalting by a molecular sieve; the ion column is at least one strong anion column and/or one weak alkaline anion column, and the hydrophobic column is used for carrying out one-time chromatographic purification between the two ion columns. According to the purification method of the exenatide human serum albumin fusion protein, an optimized purification process of fermentation, ultrafiltration concentration, strong anion glue adsorption separation, hydrophobic glue and ion exchange impurity removal, and endotoxin and salt removal by a molecular sieve is established according to the physicochemical characteristic characteristics of the exenatide human serum albumin fusion protein, so that the high-purity recombinant exenatide human serum albumin fusion protein is obtained, the purity reaches more than 98%, and the purity of the produced purified recombinant exenatide human serum albumin fusion protein meets the requirement of quality standard.
Drawings
FIG. 1 is a flow diagram of the purification process of the present invention;
FIG. 2 is a diagram showing the analysis of the purity of exenatide human serum albumin fusion protein by HPLC molecular sieve method of the present invention.
Detailed Description
The following detailed description of the embodiments of the present invention is provided to enable those skilled in the art to more easily understand the advantages and features of the present invention, and to clearly and clearly define the scope of the present invention.
As shown in FIG. 1-2, a method for purifying exenatide human serum albumin fusion protein comprises the following steps: performing ultrafiltration and concentration on yeast fermentation supernatant of the recombinant expressed exenatide human serum albumin fusion protein, sequentially performing chromatography and purification on the yeast fermentation supernatant through an ion column, a hydrophobic column and an ion column, and finally desalting the yeast fermentation supernatant through a molecular sieve; the ion column is at least one strong anion column and/or one weak alkaline anion column, and a hydrophobic column is used between the two ion columns for one-time purification.
The ion column chromatography exchange medium in front of the hydrophobic column is SP Sepharose XL; hydrophobic column chromatography exchange medium is Butyl Sepharose 4 FF; the ion column chromatography exchange medium after the hydrophobic column is DEAE Sepharose FF; the molecular sieve medium is Sephacryl HR S200.
SP Sepharose XL ion column purification exenatide human serum albumin fusion protein:
1.1, exenatide human serum albumin source:
the exenatide human serum albumin fusion protein recombinant expression plasmid is transformed into saccharomyces cerevisiae recipient bacteria, and supernatant is collected through fermentation.
1.2 solution preparation
(1) And (3) ultrafiltration concentration of the diluent: 2mM sodium caprylate, 2mM EDTA; precooling to below 10 ℃ for later use;
(2) SP Sepharose XL ion column equilibration buffer a: 5mM sodium caprylate, 5mM EDTA, 20mM NaAC; the pH value is 4.9, and the electric conductivity is less than 5 ms/cm;
(3) elution buffer a was: 5mM sodium caprylate, 5mM EDTA, 20mM NaAC, 400mM NaCl; pH 4.9;
(4)2M NaCl solution
(5) 20% ethanol solution
(6)0.5M NaOH。
1.3, ultrafiltration
(1) Filtering with 1000kDa ultrafiltration membrane
Filtering the centrifuged fermentation liquor by a 1000kDa ultrafiltration membrane package according to the operating specification of an ultrafiltration machine for further clarification, and collecting filtrate;
(2) ultrafiltration concentration with 10kDa ultrafiltration membrane
And placing the sample introduction pipeline and the return pipeline in the fermentation liquor after 1000kDa filtration, placing the permeate in another container, starting a pump, refluxing for a period of time, adjusting a reflux valve, continuously adding the ultrafiltration concentrated diluent when the inlet pressure is less than 0.7MPa and the concentration is about 1/8-10 of the total volume, and performing ultrafiltration concentration until the conductivity is 2-5 s/cm. The above operation always keeps the fermentation broth under cooling.
1.4 SP Sepharose XL ion column purification step
(1) Column packing and column processing
And (3) packing 10-20 mL of SP Sepharose XL resin into each liter of fermentation liquor, and operating according to the column packing procedure after newly packing the column. Washing 3-5 column volumes with water for injection, washing 2 column volumes with 0.5M NaOH, and washing the column with water for injection until the pH is less than 9;
(2) column balance
Equilibrating the column with equilibration buffer a until the conductivity and pH are consistent with equilibration buffer a;
(3) sample loading processing
Adding sodium acetate into the treated ultrafiltrate to make the final concentration of the ultrafiltrate 20mM, and adjusting the pH value to 4.9 +/-0.1 by using acetic acid;
(4) sample loading
Loading the treated fermentation liquor into a column, and cleaning the column by using an equilibrium buffer solution a for 3-5 column volumes;
(5) elution is carried out
Eluting the target protein by using the elution buffer a, and collecting an elution peak.
Purification of exenatide human serum albumin fusion protein by Butyl Sepharose 4FF hydrophobic column chromatography
2.1 solution preparation
(1)0.5M NaOH
(2) And (3) an equilibration buffer b: 20mM PB (pH 7.2), 5mM sodium caprylate; the conductivity is less than 5 ms/cm;
(3) elution buffer b: 20mM PB, 5mM sodium caprylate, 200mM sodium chloride; the pH value is 6.5-6.8;
(4)2M NaCl
(5) 20% ethanol.
2.2, Butyl Sepharose 4FF hydrophobic column chromatography purification step:
(1) column packing and column processing
A Butyl Sepharose 4FF hydrophobic column was loaded with 1mL of a resin adsorbing 10-20mg of protein sample. Washing with water for injection to remove ethanol, washing with 0.5M NaOH for 2 column volumes, and washing with water for injection to remove alkali;
(2) sample processing
Adding NaCl into an SP Sepharose XL sample to ensure that the final concentration of the SP Sepharose XL sample is 2.5M, adjusting the pH value to 5.5, and ensuring that the conductivity is more than 150 ms/cm;
(3) sample loading
Elution of the treated SP Sepharose XL sample, UV280nmDetecting absorption, wherein the linear flow velocity is less than 150 cm/h;
(4) column balance
After the sample loading is finished, washing the column by 3-5 column volumes by using an equilibrium buffer solution b;
(5) elution is carried out
Eluting the target protein by using an elution buffer b, and collecting an elution peak.
Purification of exenatide human serum albumin fusion protein by DEAE Sepharose FF ion column chromatography
3.1 solution preparation
(1)0.5M NaOH
(2) And (3) an equilibration buffer c: 20mM PB (pH 7.2), 5mM sodium caprylate; the conductivity is less than 5 ms/cm;
(3) the elution buffer c was: 20mM PB (pH 7.2), 5mM sodium caprylate, 200mM sodium chloride; the pH value is 6.5-6.8.
3.2, DEAE Sepharose FF ion column chromatography purification step:
(1) column packing and column processing
Loading a protein sample with 30-50 mg per ml of resin for adsorption into a column, washing with injection water to remove ethanol, washing with 0.5M NaOH for 60min, and washing with injection water to remove alkali;
(2) sample processing
Desalting a target elution peak collected in Butyl Sepharose 4FF hydrophobic column chromatography by ultrafiltration (10 KD); the buffer system of the sample was adjusted to 20mM PB, pH 7.2;
(3) sample loading
Loading a processed DEAE Sepharose FF purified sample, wherein the linear flow rate is 300-600 cm/h;
(4) washing machine
After the sample loading is finished, washing the column by 3-5 column volumes by using an equilibrium buffer solution c;
(5) elution is carried out
Eluting the target protein by using an elution buffer c, and collecting an elution peak.
Sephacryl HR S-200 molecular sieve chromatography desalting
4.1 solution preparation
(1)0.5M NaOH
(2) And (3) an equilibration buffer d: 10mM PB, 5mM NaCl; pH 7.0. + -. 0.5
(3)2M NaCl
(4) 20% ethanol solution
4.2, Sephacryl HR S-200 molecular sieve chromatography desalting step:
(1) column packing and column processing
Column volume 1500mm (50 x 1000mm), flow rate less than 8mL/min, use water for injection to wash ethanol, then 0.5M NaOH washes for 60min, then use water for injection to wash and remove alkali;
(2) column balance
Balancing the column with an equilibrium buffer d until the conductivity and the pH are consistent with those of the equilibrium buffer d;
(3) sample loading
Eluting with a DEAE Sepharose FF ion column, and then loading the sample, wherein the loading amount is less than 5% (about 30-55 mL) of the column volume, and the flow rate is 5 mL/min;
(4) elution is carried out
Eluting with an equilibrium buffer solution d, and collecting a main peak; and (3) repeatedly loading, wherein the balance time before each loading is more than 4h, combining all collected peaks of the batch, and performing quality detection according to the quality standard, wherein the quality detection result shows that the purified exenatide human serum albumin fusion protein meets the requirement of the quality standard, and the product purity reaches 99.43%, as shown in figure 2.
The parts of the invention not specifically described can be made of existing products or existing technologies, and are not described herein.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes performed by the present specification and drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (10)
1. A method for purifying exenatide human serum albumin fusion protein is characterized by comprising the following steps: performing ultrafiltration and concentration on yeast fermentation supernatant of the recombinant expressed exenatide human serum albumin fusion protein, purifying by an ion column, a hydrophobic column and an ion column in sequence, and desalting by a molecular sieve; the ion column is at least one strong anion column and/or one weak alkaline anion column, and a hydrophobic column is used for one-time purification between the two ion columns.
2. The method of claim 1, wherein the yeast fermentation supernatant of the recombinant exenatide human serum albumin fusion protein is concentrated by ultrafiltration, purified by passing through an SP Sepharose XL ion column, a Butyl Sepharose 4FF hydrophobic column, and a DEAE Sepharose FF ion column in sequence, and then desalted by passing through a Sephacryl HR S-200 molecular sieve.
3. The method of claim 2, wherein the SP Sepharose XL ion column purification step comprises:
1) ultrafiltration and sample treatment
Carrying out ultrafiltration concentration on yeast fermentation supernatant of the recombinant expressed exenatide human serum albumin fusion protein to obtain a concentrated sample, adding diluent to enable the conductivity to be 2-5 s/cm, then adding sodium acetate to enable the final concentration to be 10-40mM, and adjusting the pH value to be 4.9 +/-0.5;
2) column packing and column processing
Loading 10-20 mL SP Sepharose XL resin into each liter of fermentation liquor, washing 3-5 column volumes with water, washing 2-5 column volumes with NaOH, and washing with water until the pH value is less than 9;
3) column balance
Balancing the column with an equilibrium buffer until the conductivity and the pH are consistent with those of the equilibrium buffer a;
4) sample loading
Loading the treated fermentation liquor into a balanced SP Sepharose XL ion column;
5) washing machine
Washing the column with an equilibrium buffer a for 3-5 column volumes;
6) elution is carried out
Eluting the target protein by using the elution buffer a, and collecting an elution peak.
4. The method of claim 3, wherein the equilibration buffer a is: 5mM sodium caprylate, 5mM EDTA, 20mM NaAC, pH 4.0-5.5, conductivity less than 5 ms/cm; the elution buffer a is: 5mM sodium caprylate, 5mM EDTA, 20mM NaAC, 400mM NaCl, pH 4.0-5.5.
5. The method of claim 2, wherein the Butyl Sepharose 4FF hydrophobic column purification step comprises:
1) column packing and column processing
Filling Butyl Sepharose 4FF hydrophobic column according to 10-20mg protein sample adsorbed by resin per ml, removing ethanol, washing 2-5 column volumes with NaOH, and removing alkali;
2) sample processing
Adding NaCl into an exenatide human serum albumin fusion protein sample obtained after SP Sepharose XL ion column purification to a final concentration of 2-3M, adjusting the pH to 4.0-7.0, and adjusting the conductivity to be more than 150 ms/cm;
3) sample loading
Loading the sample processed in the step 2) on a column, wherein the linear flow velocity is less than 150 cm/h;
4) washing machine
After the sample loading is finished, washing 3-5 column volumes by using an equilibrium buffer solution b;
5) elution is carried out
Eluting the target protein by using an elution buffer b, and collecting an elution peak.
6. The method of claim 5, wherein the equilibration buffer b is: 20mM PB with pH value of 6.5-7.5 and 5mM sodium caprylate, and the conductance is less than 5 ms/cm; the elution buffer b is 20mM PB, 5mM sodium caprylate and 200mM sodium chloride, and has a pH value of 6.5-6.8.
7. The method of claim 2, wherein the DEAE Sepharose FF column purification step comprises:
1) column packing and column processing
Loading a protein sample with 30-50 mg per ml of resin for adsorption into a column, removing ethanol, cleaning for 30-90min by using NaOH, and then removing alkali;
2) sample processing
Desalting target elution peak collected in Butyl Sepharose 4FF hydrophobic column chromatography by ultrafiltration, adjusting buffer solution concentration of sample to 10-40mM PB, pH to 6.0-8.0, and conductance less than 5 ms/cm;
3) sample loading
Loading a processed DEAE Sepharose FF sample, wherein the linear flow rate is 300-600 cm/h;
4) washing machine
Washing 3-5 column volumes with an equilibration buffer c;
5) elution is carried out
Eluting the target protein by using an elution buffer c, and collecting an elution peak.
8. The method of claim 7, wherein the equilibration buffer c is: 20mM PB with pH value of 6.5-7.5 and 5mM sodium caprylate, and the conductivity is less than 5 ms/cm; the elution buffer c is: 20mM PB, 5mM sodium caprylate, 200mM NaCl, pH 6.5-6.8.
9. The method of claim 2, wherein the Sephacryl HR S-200 molecular sieve desalting step comprises:
1) column packing and column processing
Removing ethanol, washing with NaOH for 30-90min, and removing alkali; balancing the column with an equilibration buffer solution d until the conductivity and pH of the effluent are consistent with those of the equilibration buffer solution d;
2) sample loading
Loading DEAE eluate at flow rate of 1-10mL/min with loading amount less than 5% of column volume;
3) washing machine
Elution was performed with equilibration buffer d and the main peak was collected.
10. The method of claim 9, wherein the equilibration buffer d is: 10mM PB, 5mM NaCl; pH 7.0. + -. 0.5.
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