Preparation method of Icodec insulin analogue precursor
By constructing engineered bacteria and using enzyme cleavage and reverse phase chromatography purification, a high-purity Icodec insulin analog precursor was prepared, which solved the problem of daily injection of existing long-acting insulin preparations, and achieved a significant extension of the insulin action time and simplification of the process.
Patent Information
- Application Number
- CN202411594672.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-10
- Publication Date
- 2025-03-21
AI Technical Summary
The existing long-acting insulin preparations require daily injection, which cannot meet the actual needs of diabetic patients. The existing technical means have limitations in extending the time of insulin action.
By constructing engineered bacteria, expressing and purifying the Icodec insulin analog precursor, and using enzyme cleavage and reverse phase chromatography purification, high-purity Icodec insulin precursor was prepared, which simplified the process flow and improved the recovery rate.
The efficient preparation of Icodec insulin precursors is achieved, with a purity of more than 99.0%, which simplifies the process, improves the recovery rate, and significantly extends the action time of insulin.
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Figure CN119662693A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology and relates to an Icodec insulin analog precursor and a preparation method thereof. Background Art
[0002] The 2023 World Health Statistics Report pointed out that diabetes caused 2 million deaths worldwide in 2019 alone. Diabetes has become one of the top 10 DALYs (DALYs is a summary indicator of the total number of years lost due to premature death and disability, representing the loss of healthy life time, such as some non-fatal causes such as back and neck pain will lead to an increase in DALYs.).
[0003] The 10th edition of the International Diabetes Forum Diabetes Atlas reports that 537 million adults worldwide have diabetes, and this number is expected to increase to 783 million by 2045, confirming that diabetes is a major global challenge to the health of individuals, families, and society. In December 2023, the International Diabetes Federation (IDF) official website released the 2023 Diabetes and Kidney Disease Report, which pointed out that diabetes is one of the main causes of CKD, with about 30% to 40% of diabetic patients developing CKD, of which type 2 diabetes is the largest factor causing the burden of diabetes-related CKD.
[0004] Type 2 diabetes is mainly treated by subcutaneous injection of insulin or glucagon-like peptide (GLP-1) receptor agonists. In recent decades, various insulin preparations with different durations of action, such as rapid-acting insulin preparations, intermediate-acting insulin preparations and long-acting insulin preparations, have been widely used in the treatment of type I and type II diabetes. Among them, long-acting and ultra-long-acting insulins simulate physiological basal insulin secretion better than intermediate-acting insulin. Existing long-acting insulins are daily preparations that require patients to inject daily, which cannot meet the actual needs of diabetic patients. Existing technical means mainly use macromolecular fusion technology (including connecting macromolecular PEG and IgG Fc domain) or linking fatty acid side bonds to further extend the duration of insulin action, thereby significantly reducing the number of injections and solving the key obstacles of daily preparations.
[0005] Icodec insulin continues the technology of adding fatty acid side chains used in degludec insulin and detemir insulin, removes the threonine at position B30 of human insulin, and connects the 20-carbon fatty acid side chain to the lysine at position B29 through a linker [2x oligoethylene glycol (OEG) yL-Glu]. In addition, Icodec insulin has three amino acid substitutions, including tyrosine at position A14 replaced by glutamic acid (A14E), tyrosine at position B16 replaced by histidine (B16H), and phenylalanine at position B25 replaced by histidine (B25H). The 20-carbon fatty acid side chain can bind firmly and reversibly to albumin, slowing down receptor-mediated clearance. The replacement of three amino acids can improve molecular stability, reduce enzyme degradation, and significantly prolong the duration of action.
[0006] Insulin precursor refers to the single-chain polypeptide that can be converted into human insulin by one or more subsequent chemical and / or enzymatic steps. Insulin analog precursor refers to the insulin precursor molecule that A and / or B chain have one or more sudden changes, substitutions, deletions and / or additions relative to human insulin molecule. In order to obtain more ultra-long-acting human insulin analog precursor and higher recovery rate, simplicity of technology, therefore, this ultra-long-acting human insulin analog precursor and preparation method are proposed. Summary of the invention
[0007] The purpose of the present invention is to address the above-mentioned problems in the prior art and to propose a method for preparing an Icodec insulin analog precursor.
[0008] The purpose of the present invention can be achieved by the following technical solutions:
[0009] A method for preparing an Icodec insulin analog precursor, characterized in that it comprises the following steps:
[0010] (1) Construction and cultivation of engineered bacteria:
[0011] The base sequence of the fusion protein containing Icodec insulin precursor is inserted into an expression vector, and a recombinant expression vector is synthesized and constructed. The expression vector is recombined into yeast or Escherichia coli to obtain an engineered bacterium; the engineered bacterium is fermented and induced to express to obtain a fermentation broth or bacterial cell containing the Icodec insulin precursor fusion protein.
[0012] (2) Enzyme digestion:
[0013] The fermentation broth of yeast engineered bacteria is purified by chromatography and then subjected to enzyme digestion; the bacterial cells obtained by fermentation of Escherichia coli engineered bacteria need to be broken, renatured and then subjected to enzyme digestion. The method is to adjust the pH to 8.5-9.5, add Lys-C enzyme or trypsin according to the mass ratio of Lys-C enzyme or trypsin: Icodec insulin precursor fusion protein of 1:5000 to 10000, perform enzyme digestion reaction, and obtain enzyme digestion reaction solution;
[0014] (3) Reverse phase chromatography purification:
[0015] The enzyme cleavage reaction solution is purified by reverse phase chromatography using C8 silica gel resin to obtain a reverse phase chromatography solution;
[0016] (3) Sediment collection:
[0017] The reverse phase chromatography solution was diluted and the pH was adjusted to 5.5-6.0, and the precipitate was collected by centrifugation.
[0018] Preferably, in step (1), the amino acid sequence of the fusion protein of Icodec insulin precursor is:
[0019] Leader-FVNQHLCGSHLVEALHLVCGERGFHYTPK-Linker-GIVEQCCT SICSLEQLENYCN;
[0020] Among them, Leader is a leader peptide with K at the C-terminus;
[0021] Leader is composed of 7-15 amino acids;
[0022] Linker is a connecting peptide with K at the C-terminus;
[0023] Linker is composed of 3-32 amino acids.
[0024] Preferably, in step (2), the fermentation broth of the yeast engineering bacteria is subjected to chromatography purification before enzyme digestion.
[0025] Preferably, the chromatography purification method is:
[0026] The fermentation broth is centrifuged to collect the supernatant, and the supernatant is purified by cation exchange chromatography and then the elution solution is collected.
[0027] Preferably, in step (2), the cells of the engineered Escherichia coli obtained by fermentation need to be broken, renatured and then subjected to enzyme digestion treatment.
[0028] Preferably, the method of crushing and renaturing:
[0029] The inclusion bodies are collected by mechanical disruption, dissolved in Tris+DTT buffer solution, and then diluted and renatured with a reconstitution solution containing cysteine or cystine.
[0030] Compared with the prior art, the preparation method of Icodec insulin precursor in the present application has fewer chromatography processes, simpler processes and higher recovery rates. The purity of Icodec insulin precursor obtained after purification of yeast fermentation broth is above 99.0%; the purity of Icodec insulin precursor obtained after purification of Escherichia coli fermentation cells is above 98.5%. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] Figure 1 This is the HPLC analysis spectrum of Icodec insulin precursor obtained after purification from yeast fermentation broth;
[0032] Figure 2 This is the HPLC analysis chart of Icodec insulin precursor obtained after purification of Escherichia coli fermentation cells;
[0033] Figure 3 This is the LC-MS analysis spectrum of Icodec insulin precursor obtained after purification from yeast fermentation broth;
[0034] Figure 4 This is the HPLC analysis chart of Icodec insulin precursor obtained after purification of Escherichia coli fermentation cells. DETAILED DESCRIPTION
[0035] The following are specific embodiments of the present invention and the accompanying drawings to further describe the technical solution of the present invention, but the present invention is not limited to these embodiments.
[0036] Embodiment 1
[0037] The method for preparing Icodec insulin precursor by purifying yeast fermentation broth comprises the following steps:
[0038] Construction and cultivation of engineered bacteria
[0039] First, the base sequence containing the Icodec insulin precursor fusion protein was designed and inserted into the expression vector PPIC9K. The recombinant expression vector was synthesized and constructed by GenScript, and then the expression vector was recombined into Pichia pastoris GS115 to obtain an engineered bacterium. After fermentation and induction of expression, a fermentation broth containing the Icodec insulin precursor fusion protein was obtained.
[0040] The amino acid sequence of the fusion protein of this example is shown in SEQ ID NO.1, specifically:
[0041] Leader-FVNQHLCGSHLVEALHLVCGERGFHYTPK-Linker-GIVEQCCT SICSLEQLENYCN;
[0042] Among them, the Leader is EAEFAELQRK and the Linker is NAK.
[0043] Cation exchange chromatography:
[0044] The fermentation broth was diluted and centrifuged, filtered and then subjected to cation exchange chromatography. The chromatographic medium was Agaros ix MC90-MMC, the column filling height was 25 cm, the loading capacity was 28 g / L medium, and the flow rate was not higher than 350 cm / h; buffer A: pH 3.5-4.0, 50 mM sodium acetate + acetic acid buffer; buffer B: pH 3.5-4.0, 50 mM sodium acetate + acetic acid + 0.5 M sodium chloride buffer; the elution gradient was 0-100% B linear elution 15-20CV, and the elution peak was collected.
[0045] Enzyme digestion:
[0046] The pH value of the elution peak collected by cation exchange chromatography was adjusted to between 8.5 and 9.5, and Lys-C enzyme (or trypsin) was added at a mass ratio of 1:(5000-10000) to Icodec insulin precursor fusion protein, and the enzyme digestion was carried out at 25°C for 5-10 hours.
[0047] Reverse phase chromatography purification:
[0048] After the digested sample is filtered, it is purified by C8 silica gel resin. The filling height of the chromatographic column is 25cm, the loading capacity is 12g / L medium, and the flow rate is not higher than 350cm / h; buffer A is 50mmol / L Tris + 2% ammonium sulfate solution, pH 7.5-8.5; buffer B is 90% acetonitrile solution; 10% buffer A is used for balance during the process, and 10% acetonitrile solution is added during the loading process. The elution gradient is 10-60% B linear elution 20-30CV, and the elution peak is collected.
[0049] Precipitate collection:
[0050] Add 2 volumes of purified water to the sample obtained by reverse phase chromatography, adjust the pH value to 5.5-6.0 to precipitate the sample, stir the precipitated sample evenly and collect it by centrifugation at a temperature of 2-8°C, discard the supernatant and collect the precipitate. Wash the precipitate twice with 2 times the volume of purified water, set the centrifugal force ≥7500g, the centrifugation time ≥20min, the temperature 2-8°C, discard the supernatant and collect the precipitate. Spread the precipitate evenly on a freeze-drying plate and freeze-dry it to collect Icodec insulin precursor. Figure 3 As shown, the molecular weight of Icodec insulin precursor was determined to be 5632.5549 by LC-MS.
[0051] like Figure 1 As shown, the process was carried out for two batches of 5 liter fermentations and the results are shown in the table below.
[0052]
[0053] Embodiment 2
[0054] The method for preparing Icodec insulin precursor by purifying Escherichia coli fermentation cells comprises the following steps:
[0055] Construction and cultivation of engineered bacteria:
[0056] First, the fusion protein base sequence containing Icodec insulin precursor was designed and inserted into an expression vector. The recombinant expression vector was synthesized and constructed by Shanghai Bioengineering, and then the expression vector was inserted into Escherichia coli BL21 to obtain an engineered bacterium.
[0057] The amino acid sequence of the fusion protein is shown in SEQ ID NO.2, specifically:
[0058] Leader-FVNQHLCGSHLVEALHLVCGERGFHYTPK-Linker-GIVEQCCT SICSLEQLENYCN:
[0059] Among them, the leader is MHAEFAELQRK; the linker is EAEDLQVGQVELGGGPGAGSLQPLALEGSLQK.
[0060] Bacteria fragmentation:
[0061] The E. coli cells collected by centrifugation were suspended in TE buffer, and the suspension was broken by a homogenizer 2-3 times, with the breaking pressure controlled at 750-850 bar, and the inclusion bodies were collected after centrifugation. The inclusion bodies were washed twice with 20 mmol / LPB buffer containing EDTA and Tween 80, and then collected.
[0062] Variation:
[0063] After washing, the inclusion bodies were added with 0.05 mol / L DTT in Tris buffer at a ratio of 1:10 to the weight of the inclusion bodies, and the pH was 10.0-12.0. After low temperature dissolution for 5-10 hours, the centrifugal force was set to ≥7500g, the centrifugal time was ≥30min, and the temperature was 2-8°C. The supernatant was collected. According to the protein concentration of 0.15-0.2 mg / ml, it was added to 50 mmol / L glycine-sodium hydroxide, pH 10.0 refolding buffer containing 1 mol / L urea, 5 mmol / L cysteine, and 1 mmol / L cystine, and refolded at low temperature for 6-14 hours.
[0064] Enzyme digestion:
[0065] The renatured sample is concentrated by ultrafiltration and its pH is adjusted to between 8.5 and 9.5. Lys-C enzyme (or trypsin) is added at a mass ratio of 1:(5000-10000) to Icodec insulin precursor fusion protein at 25°C for 5-16 hours.
[0066] Reverse phase chromatography purification:
[0067] The sample after enzyme digestion was adjusted to pH 7.5-8.5, filtered and purified using C8 silica gel resin. The filling height of the chromatographic column was 25 cm, the loading capacity was 9 g / L medium, and the flow rate was not higher than 350 cm / h; buffer A was 50 mmol / L Tris + 2% ammonium sulfate solution, pH 7.5-8.5; buffer B was 90% acetonitrile solution; 10% buffer A was used for balance during the process, and 10% acetonitrile solution was added during the loading process. The elution gradient was 10-60% B linear elution for 20-30CV, and the elution peak was collected.
[0068] Precipitate collection:
[0069] Add 2 volumes of purified water to the sample obtained by reverse phase chromatography, adjust the pH value to 5.5-6.0 to precipitate the sample, stir the precipitated sample evenly and collect it by centrifugation, the centrifugation temperature is 2-8°C, and the supernatant of the centrifugation is collected. Wash the precipitate twice with 2 times the volume of purified water of the precipitate, set the centrifugal force ≥7500g, the centrifugation time ≥20min, the temperature is 2-8°C, discard the supernatant of the centrifugation, and collect the precipitate. Spread the precipitate evenly on a freeze-drying plate and freeze-dry it to collect Icodec insulin precursor. Figure 4 As shown, the molecular weight of Icodec insulin precursor was determined to be 5632.5577 by LC-MS.
[0070] like Figure 2 As shown, the process was carried out in one batch of 5 L fermentation and one batch of 30 L fermentation, and the results are shown in the table below.
[0071]
[0072]
[0073] The specific embodiments described herein are merely examples of the spirit of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in similar ways, but they will not deviate from the spirit of the present invention or exceed the scope defined by the appended claims.
Claims
1. A method for preparing an Icodec insulin analog precursor, characterized in that: The following steps are involved: (1) Construction and cultivation of engineered bacteria: The base sequence of the fusion protein containing Icodec insulin precursor is inserted into an expression vector, and a recombinant expression vector is synthesized and constructed. The expression vector is recombined into yeast or Escherichia coli to obtain an engineered bacterium; the engineered bacterium is fermented and induced to express to obtain a fermentation broth or bacterial cell containing the Icodec insulin precursor fusion protein. (2) Enzyme digestion: The fermentation broth of yeast engineered bacteria is purified by chromatography and then subjected to enzyme digestion; the bacterial cells obtained by fermentation of Escherichia coli engineered bacteria need to be broken, renatured and then subjected to enzyme digestion. The method is to adjust the pH to 8.5-9.5, add Lys-C enzyme or trypsin according to the mass ratio of Lys-C enzyme or trypsin: Icodec insulin precursor fusion protein of 1:5000 to 10000, perform enzyme digestion reaction, and obtain enzyme digestion reaction solution; (3) Reverse phase chromatography purification: The enzyme cleavage reaction solution is purified by reverse phase chromatography using C8 silica gel resin to obtain a reverse phase chromatography solution; (4) Collection of precipitation: The reverse phase chromatography solution was diluted and the pH was adjusted to 5.5-6.0, and the precipitate was collected by centrifugation.
2. The method for preparing an Icodec insulin analog precursor according to claim 1, characterized in that: The amino acid sequence of the fusion protein of Icodec insulin precursor is: Leader-FVNQHLCGSHLVEALHLVCGERGFHYTPK-Linker-GIVEQCCT SICSLEQLENYCN; Among them, Leader is a leader peptide with K at the C-terminus; Leader is composed of 7-15 amino acids; Linker is a connecting peptide with K at the C-terminus; Linker is composed of 3-32 amino acids.
3. The method for preparing an Icodec insulin analog precursor according to claim 1, characterized in that: In step (2), the yeast engineering bacteria fermentation broth needs to be purified by chromatography before enzyme digestion.
4. The method for preparing an Icodec insulin analog precursor according to claim 3, characterized in that: The method of chromatography purification treatment: The fermentation broth is centrifuged to collect the supernatant, and the supernatant is purified by cation exchange chromatography and then the elution solution is collected.
5. The method for preparing an Icodec insulin analog precursor according to claim 1, characterized in that: In step (2), the Escherichia coli engineered bacteria obtained by fermentation need to be broken and renatured before enzyme digestion.
6. The method for preparing an Icodec insulin analog precursor according to claim 5, characterized in that: The method of crushing and renaturing: The inclusion bodies are collected by mechanical disruption, dissolved in Tris+DTT buffer solution, and then diluted and renatured with a reconstitution solution containing cysteine or cystine.