Methods of making exenatide variants and pegylated conjugates thereof

Abstract

The present application belongs to the technical field of protein drugs, and relates to a preparation method of an exenatide variant and a polyethylene glycol conjugate thereof. Specifically, Cys 39 The preparation method of Exendin-4 adopts three-step reversed-phase column chromatography for purification, while the preparation method of mPEG-ppMAL-Cys 39 The preparation method of Exendin-4 adopts three-step reversed-phase column chromatography for purification, while the preparation method of mPEG-ppMAL-Cys 39 The preparation method of Exendin-4 adopts three-step reversed-phase column chromatography for purification, while the preparation method of mPEG-ppMAL-Cys When the polypeptide is purified, three-step reversed-phase column chromatography is used, and the acidic substance used in the second step is optimized, so that the purity and yield of the product are obviously improved. When the conjugate is purified, ion exchange chromatography and three-step reversed-phase column chromatography are used in sequence, so that the purity of the product can reach more than 99.5%, and the yield is high. The combined use of the two preparation methods can meet the needs of industrial production.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of protein drugs, and relates to a preparation method of an exenatide variant and a preparation method of a pegylated exenatide variant. BACKGROUND

[0002] With the development of the economic society, the prolongation of the average life span of the population and the change of the lifestyle, diabetes has become a major health care problem in the world. The incidence of diabetes is rising sharply in both developed countries and developing countries. Diabetes is a metabolic disease characterized by high blood sugar due to defects in pancreatic secretion or insulin action. If the blood sugar of a diabetic patient is not well controlled for a long time, organ and tissue damage can occur, becoming the "source of all diseases", accompanied by various organ damage, especially eye, heart, blood vessels, kidney and nerve damage or organ dysfunction or failure, which can cause severe disability or death. According to the different pathogenesis, diabetes is mainly divided into type I diabetes, type II diabetes, gestational diabetes and other types of diabetes. Type II diabetes patients are the main body of diabetes, accounting for about 90%. The characteristics of type II diabetes are insulin secretion or action disorders and β-cell dysfunction, which leads to metabolic disorders of fat, carbohydrates and proteins, resulting in chronic hyperglycemia and eventually leading to various blood vessel and organ complications.

[0003] Diabetes is a progressive disease, and when blood sugar control cannot meet the standard after life intervention, drug intervention treatment is needed. Since 1920, when humans used animal insulin to treat diabetes, in the past 100 years, nine major categories of diabetes treatment drugs have been discovered and invented worldwide: biguanides, sulfonylureas, thiazolidinediones (TZDs), glinides, alpha-glucosidase inhibitors, DPP-4 inhibitors, SGLT-2 inhibitors, insulin, and GLP-1. Among them, GLP-1 was discovered in 1985, which is a product expressed by the proglucagon gene in the human body after eating and mainly secreted by L cells in the intestinal mucosa. It can stimulate pancreatic beta cells to secrete insulin and has an important role in stabilizing blood sugar levels. However, GLP-1 is easily degraded by DPP4 in the body, with a half-life of less than 2 minutes, and almost cannot become an effective anti-diabetic drug.

[0004] Exenatide (or Exendin-4) is a polypeptide found in the salivary secretions of the Gila monster, which has a high homology (53%) with GLP-1 (7-36). Studies have shown that Exendin-4 can also bind to GLP-1 receptor and exhibit similar pharmacological agonistic effects as GLP-1, such as increasing insulin synthesis and glucose-dependent insulin secretion; stimulating β-cell proliferation and regeneration, and inhibiting β-cell apoptosis, thereby increasing the number of β-cells; inhibiting glucagon secretion; inhibiting hepatic glucose production without causing severe hypoglycemia; inhibiting postprandial gastrointestinal motility and secretory function; reducing appetite and food intake; and protecting nerve cells. The effects of Exendin-4 in promoting insulin secretion and inhibiting postprandial glucagon secretion are glucose-dependent, which are superior to the currently used sulfonylurea hypoglycemic drugs, and are less likely to cause hypoglycemia, can greatly reduce the frequency of blood glucose monitoring, and can reduce body weight.

[0005] Polymer modification technology is a powerful modification technology developed in the 1970s, among which the PEGylation technology is the most representative. This technology is to chemically combine polyethylene glycol (PEG) with protein drugs to modify the surface of the protein. Through the modification of PEG, on the one hand, the molecular weight of the protein is increased, and the excretion rate of the protein in the kidney is reduced; on the other hand, the space steric hindrance effect is generated on the surface of the modified protein molecule by the coupled PEG chain, which reduces the hydrolysis of the protein by proteolytic enzymes in the blood, thereby effectively prolonging the residence time of the protein in the circulatory system, leading to an increase in the plasma cycle of the drug and systemic drug exposure and improving the therapeutic effect.

[0006] Compared with Exenatide or its variants, the PEGylated conjugates obtained by specific modification have obvious advantages in terms of duration of action, bioavailability and therapeutic effect. Clinical studies have shown that PEGylated Exenatide or its variants have excellent efficacy and safety for patients with type II diabetes. However, there is currently no synthesis and purification process for PEGylated Exenatide or its variants suitable for large-scale production. SUMMARY

[0007] Problems to be solved by the invention

[0008] In view of the fact that there is currently no synthesis and purification process for PEGylated Exenatide or its variants suitable for large-scale production, the present application develops a method for preparing a PEGylated conjugate of a specific Exenatide variant, and the present application also develops a method for preparing a specific Exenatide variant used as a raw material accordingly.

[0009] Solution to the problem

[0010] In one aspect, the present application provides an exenatide variant Cys 39 - a method for preparing Exendin-4, comprising the following steps: purifying Cys 39 - a crude Exendin-4.

[0011] Optionally, the Cys 39 - the crude Exendin-4 is obtained by a solid phase synthesis method.

[0012] Optionally, the chromatographic column used in each of the three steps of the three-step reverse phase column chromatography is independently selected from one or more of butylsilane-bonded silica gel chromatographic column, octylsilane-bonded silica gel chromatographic column and octadecylsilane-bonded silica gel chromatographic column, preferably octadecylsilane-bonded silica gel chromatographic column.

[0013] Optionally, the first step of the three-step reverse phase column chromatography uses a binary mobile phase system, wherein mobile phase A is an acid in water and mobile phase B is an acid in an organic solvent; the acid in mobile phase A and mobile phase B is independently selected from one or more of formic acid, acetic acid, trifluoroacetic acid, phosphoric acid, dipotassium hydrogen phosphate and monopotassium hydrogen phosphate, preferably trifluoroacetic acid; the organic solvent is selected from one or more of methanol, ethanol and acetonitrile, preferably acetonitrile. Further, the volume percentage of the acid in mobile phase A and mobile phase B used in the first step of the reverse phase column chromatography is independently 0.01%-1%, preferably 0.02-0.5%, more preferably 0.05-0.2%, even more preferably 0.1%.

[0014] Optionally, the first step of the reverse phase column chromatography comprises a gradient elution process using mobile phase B with a volume percentage of 0%-95%, preferably 1%-90%, more preferably 5%-85%, even more preferably 8%-80%. Further, the time of the gradient elution process is within 120 min, preferably within 110 min, more preferably within 100 min, even more preferably within 90 min.

[0015] Optionally, the first step of the reverse phase column chromatography further comprises an isocratic elution process after the gradient elution process, with the volume percentage of mobile phase B being the same as the end point of the gradient, preferably further comprising a column equilibration process before the gradient elution process and / or after the isocratic elution process, with the volume percentage of mobile phase B being the same as the start point of the gradient.

[0016] Optionally, the first step of the three-step reverse phase column chromatography is performed by first equilibrating the column with mobile phase B at 8% v / v, then performing a gradient elution with mobile phase B at 8%-80% v / v over 90 min, and finally performing an isocratic elution with mobile phase B at 80% v / v.

[0017] Optionally, the second step of the three-step reverse phase column chromatography is performed using a binary mobile phase system, wherein mobile phase A is an acidic solution in water and mobile phase B is an organic solvent; the acidic solution in mobile phase A is selected from one or more of phosphoric acid, dipotassium hydrogen phosphate, monopotassium phosphate, disodium hydrogen phosphate, and monosodium phosphate, preferably monopotassium phosphate and phosphoric acid; and the organic solvent is selected from one or more of methanol, ethanol, and acetonitrile, preferably acetonitrile. Further, the second step of the three-step reverse phase column chromatography is performed using a mobile phase A having a pH value of 1.0-6.0, preferably 2.0-5.0, more preferably 2.5-3.5, and even more preferably 3.0.

[0018] Optionally, the second step of the three-step reverse phase column chromatography comprises a gradient elution process using mobile phase B at 0%-85% v / v, preferably 1%-75% v / v, more preferably 2%-70% v / v, and even more preferably 5%-65% v / v. Further, the gradient elution process is performed within 100 min, preferably within 90 min, more preferably within 80 min, and even more preferably within 70 min.

[0019] Optionally, the second step of the three-step reverse phase column chromatography further comprises an isocratic elution process after the gradient elution process, wherein mobile phase B is at the same percentage as the end of the gradient, and preferably further comprises a column equilibration process before the gradient elution process and / or after the isocratic elution process, wherein mobile phase B is at the same percentage as the start of the gradient.

[0020] Optionally, the second step of the three-step reverse phase column chromatography is performed by first equilibrating the column with mobile phase B at 5% v / v, then performing a gradient elution with mobile phase B at 5%-65% v / v over 70 min, and finally performing an isocratic elution with mobile phase B at 65% v / v.

[0021] Optionally, the third step of the three-step reversed phase column chromatography uses a binary mobile phase system, wherein mobile phase A is an acidic substance in water and mobile phase B is an acidic substance in an organic solvent; the acidic substance in mobile phase A and mobile phase B is each independently selected from one or more of formic acid, acetic acid, trifluoroacetic acid, phosphoric acid, dipotassium hydrogen phosphate and monopotassium dihydrogen phosphate, preferably trifluoroacetic acid; the organic solvent is selected from one or more of methanol, ethanol and acetonitrile, preferably acetonitrile. Further, the volume percentage of the acidic substance in mobile phase A and mobile phase B used in the third step of the reversed phase column chromatography is each independently 0.005% to 0.5%, preferably 0.01 to 0.2%, more preferably 0.02 to 0.1%, even more preferably 0.02%.

[0022] Optionally, the third step of the reversed phase column chromatography comprises a gradient elution process using mobile phase B with a volume percentage of 0% to 95%, preferably 1% to 90%, more preferably 2% to 85%, even more preferably 5% to 80%. Further, the gradient elution process has a time of 100 min or less, preferably 90 min or less, more preferably 80 min or less, even more preferably 75 min.

[0023] Optionally, the third step of the reversed phase column chromatography further comprises an isocratic elution process after the gradient elution process, wherein the volume percentage of mobile phase B is the same as the end point of the gradient, preferably further comprises a column equilibration process before the gradient elution process and / or after the isocratic elution process, wherein the volume percentage of mobile phase B is the same as the start point of the gradient.

[0024] Optionally, the third step of the reversed phase column chromatography is performed as follows: first, a column equilibration process using mobile phase B with a volume percentage of 5%, second, a gradient elution process using mobile phase B with a volume percentage of 5% to 80% within 75 min, third, an isocratic elution process using mobile phase B with a volume percentage of 80%, and fourth, a column re-equilibration process using mobile phase B with a volume percentage decreasing to 5%.

[0025] In another aspect, the present application provides a method for preparing a pegylated exendin-4 variant mPEG-ppMAL-Cys 39 -Exendin-4, comprising the following steps:

[0026] Cys 39 -Exendin-4, comprising the following steps: 39 -Exendin-4, and then conjugating and purifying it with mPEG-ppMAL.

[0027] Optionally, the mPEG-ppMAL has a molecular weight of 5-100 kDa, preferably 10-50 kDa, more preferably 15-40 kDa, even more preferably 20-29 kDa, and even more preferably 23 kDa.

[0028] In still another aspect, the present application provides a pegylated exendin-4 variant mPEG-ppMAL-Cys 39 a method for preparing Exendin-4, comprising the steps of:

[0029] The purified Cys 39 -Exendin-4 is conjugated with mPEG-ppMAL, and then the mPEG-ppMAL-Cys 39 -Exendin-4 is purified by ion exchange chromatography and three-step reversed-phase column chromatography in sequence.

[0030] Optionally, the mPEG-ppMAL has a molecular weight of 5-100 kDa, preferably 10-50 kDa, more preferably 15-40 kDa, even more preferably 20-29 kDa, and even more preferably 23 kDa.

[0031] Optionally, the purified Cys 39 -Exendin-4 is prepared by the exendin-4 variant Cys 39 -Exendin-4 of the present application.

[0032] Optionally, the ion exchange chromatography is cation exchange chromatography, and the stationary phase used is MacroCap SP.

[0033] Optionally, the mobile phase system used in the ion exchange chromatography comprises mobile phase A, mobile phase B, mobile phase C and mobile phase D; mobile phase A is a solution of acetic acid and sodium acetate in water; the pH value of mobile phase A is 2.0-6.0, preferably 3.0-5.0, more preferably 4.0; the concentration of sodium acetate in mobile phase A is 5-50 mmol / L, preferably 10-30 mmol / L, more preferably 20 mmol / L; mobile phase B is a solution of acetic acid, sodium acetate and sodium chloride in water; the pH value of mobile phase B is 2.0-6.0, preferably 3.0-5.0, more preferably 4.0; the concentration of sodium acetate in mobile phase B is 5-50 mmol / L, preferably 10-30 mmol / L, more preferably 20 mmol / L; the concentration of sodium chloride in mobile phase B is 0.1-5 mol / L, preferably 0.2-4 mol / L, more preferably 0.5-2 mol / L, even more preferably 1 mol / L; mobile phase C is a mixture of mobile phase A and mobile phase B, the volume ratio of mobile phase A to mobile phase B is 0.5:1-10:1, preferably 1:1-5:1, more preferably 2:1-4:1, even more preferably 4:1; mobile phase D is a solution of sodium hydroxide in water, the concentration of sodium hydroxide in mobile phase D is 0.02-2 mol / L, preferably 0.05-1 mol / L, more preferably 0.1-0.5 mol / L, even more preferably 0.2 mol / L.

[0034] Optionally, the process of the ion exchange chromatography is as follows: first, flush the AKTA system with mobile phase D, mobile phase B and mobile phase A in sequence, second, dilute the pre-prepared pegylated exenatide variant mPEG-ppMAL-Cys 39 -Exendin-4 crude solution, and finally flush the AKTA system with mobile phase A and mobile phase C in sequence, and collect the components.

[0035] Optionally, the chromatographic column used in each step of the three-step reversed-phase column chromatography is independently selected from one or more of butylsilane-bonded silica gel chromatographic column (C4 column), octylsilane-bonded silica gel chromatographic column (C8 column) and octadecylsilane-bonded silica gel chromatographic column (C18 column), preferably octadecylsilane-bonded silica gel chromatographic column (C18 column).

[0036] Optionally, the first and third steps of the three-step reversed-phase column chromatography use the same binary mobile phase system and elution process, wherein mobile phase A is an acidic substance in water and mobile phase B is an acidic substance in an organic solvent; the acidic substance in mobile phase A and mobile phase B is each independently selected from one or more of formic acid, acetic acid, trifluoroacetic acid, phosphoric acid, dipotassium hydrogen phosphate, and monopotassium hydrogen phosphate, preferably trifluoroacetic acid; the organic solvent is selected from one or more of methanol, ethanol, and acetonitrile, preferably acetonitrile. Further, the volume percentage of the acidic substance in mobile phase A and mobile phase B used in the first and third steps of the reversed-phase column chromatography is each independently 0.01-1%, preferably 0.02-0.5%, more preferably 0.05-0.2%, even more preferably 0.1%.

[0037] Optionally, the first and third steps of the reversed-phase column chromatography comprise a gradient elution process using mobile phase B at a volume percentage of 0-95%, preferably 2-90%, more preferably 5-85%, even more preferably 9-80%. Further, the gradient elution process has a time of 100 min or less, preferably 90 min or less, more preferably 80 min or less, even more preferably 70 min or less.

[0038] Optionally, the second step of the three-step reversed-phase column chromatography uses a binary mobile phase system, wherein mobile phase A is an acidic substance in water and mobile phase B is an organic solvent; the acidic substance in mobile phase A is selected from one or more of phosphoric acid, dipotassium hydrogen phosphate, monopotassium hydrogen phosphate, disodium hydrogen phosphate, and sodium dihydrogen phosphate, preferably monopotassium hydrogen phosphate and phosphoric acid; the organic solvent is selected from one or more of methanol, ethanol, and acetonitrile, preferably acetonitrile. Further, the pH value of mobile phase A used in the second step of the reversed-phase column chromatography is 1.0-6.0, preferably 2.0-5.0, more preferably 2.5-3.5, even more preferably 3.0.

[0039] Optionally, the second step of the reversed-phase column chromatography comprises a gradient elution process using mobile phase B at a volume percentage of 0-85%, preferably 2-75%, more preferably 5-70%, even more preferably 8-65%. Further, the gradient elution process has a time of 100 min or less, preferably 90 min or less, more preferably 80 min or less, even more preferably 70 min or less.

[0040] Optionally, the preparation method further comprises an ultrafiltration process performed after the three-step reversed-phase column chromatography.

[0041] Optionally, the ultrafiltration process is: firstly, subjecting the sample obtained after the three-step reversed-phase column chromatography to ultrafiltration; secondly, diluting with water; thirdly, adjusting the pH value to 4.0-5.0 using a pH adjuster; and finally, subjecting to ultrafiltration again; the pH adjuster is an alkaline substance, preferably one or more of hydroxides, carbonates and bicarbonates of alkali metals, more preferably one or more of sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium hydroxide, potassium carbonate and potassium bicarbonate, and even more preferably sodium bicarbonate.

[0042] Effects of the invention

[0043] To make up for the blank of the mass production process of pegylated exendin-4 or its variants, the present application correspondingly develops a Cys 39 -Exendin-4 polypeptide and mPEG-ppMAL-Cys 39 -Exendin-4 conjugate preparation method. In the process of purifying Cys 39 -Exendin-4 polypeptide, the polypeptide preparation method of the present application creatively uses three-step reversed-phase column chromatography, and further optimizes the acidic substance used in the second reversed-phase column chromatography, so that the purity (up to more than 98%) and yield of the target product are significantly improved. In the process of purifying mPEG-ppMAL-Cys 39 -Exendin-4 conjugate, the conjugate preparation method of the present application creatively uses ion exchange chromatography and three-step reversed-phase column chromatography in sequence, so that the purity of the target product can reach more than 99.5%, and the yield is high. The combination of the polypeptide and conjugate preparation methods in the present application can basically meet the needs of industrial production. DETAILED DESCRIPTION

[0044] Definitions of terms

[0045] Unless otherwise specified, the term "chromatography" (or "chromatography") used in the context of the present application means a separation and analysis method which utilizes the differences in the physicochemical properties of each component, and produces different speeds of migration between the mobile phase (eluent) and the stationary phase (filler) due to adsorption, distribution or affinity, so as to achieve the purpose of separation, and then achieves the purpose of analysis by means of analysis. Chromatography is the most powerful means of separating and analyzing mixtures, with high sensitivity, high selectivity, high efficiency, fast analysis speed and wide application range.

[0046] The term "stationary phase" as used in the context of the present application means a phase that is stationary and produces a retention effect on the sample during the chromatographic separation. The choice of stationary phase plays an important role, sometimes even a decisive role, in the separation of the sample. Different types of chromatography employ different stationary phases. In gas-solid chromatography, the stationary phase is a solid adsorbent with adsorptive activity, such as polysiloxane, polyethylene glycol, alumina, etc. In gas-liquid chromatography, the stationary phase is a stationary liquid coated on the surface of a support. In liquid chromatography, the stationary phase is a variety of silica gel with different types of bonding, such as butylsilane bonded silica gel BS in C4 column, octylsilane bonded silica gel OS in C8 column, octadecylsilane bonded silica gel ODS in C18 column, etc. In ion exchange chromatography, the stationary phase is a variety of ion exchangers. In size exclusion chromatography, the stationary phase is a variety of gels with different types, etc.

[0047] The term "mobile phase" as used in the context of the present application means another phase that is in equilibrium with the stationary phase and carries the sample forward during the chromatographic separation. The choice of mobile phase also plays an important role in the separation of the sample. Different types of chromatography usually employ different mobile phases. In gas chromatography, the mobile phase (or "carrier gas") is usually high-purity helium. In liquid chromatography, the mobile phase is relatively complex and flexible. It can be a monophasic mobile phase system (such as water or its buffered salt solution, methanol, acetonitrile, etc.) or a biphasic mobile phase system (such as acetonitrile-trifluoroacetic acid aqueous solution, trifluoroacetic acid acetonitrile solution-trifluoroacetic acid aqueous solution, acetonitrile-phosphate buffer, acetonitrile-acetate buffer, etc.).

[0048] When a biphasic mobile phase system is used, gradient elution (or gradient elution or programmed elution) is usually employed. The term "gradient elution" as used in the context of the present application means that the concentration or volume percentage ratio of the two mobile phases is continuously changed according to a certain program within one analysis cycle. Gradient elution can separate components with large differences in properties in a complex sample under their respective optimal separation conditions.

[0049] Chromatography can be classified from different perspectives:

[0050] 1. According to the molecular aggregation state of the mobile phase and the stationary phase: In chromatography, the mobile phase can be a gas, a liquid and a supercritical fluid, corresponding to gas chromatography (GC; according to the different stationary phases, it can be further divided into gas-solid chromatography GSC and gas-liquid chromatography GLC), liquid chromatography (LC; according to the different stationary phases, it can be further divided into liquid-solid chromatography LSC and liquid-liquid chromatography LLC) and supercritical fluid chromatography (SFC).

[0051] 2. According to operation form: can be divided into column chromatography (CC; according to the different specifications of chromatographic column, it can also be divided into packed column chromatography PC, micro packed column chromatography MPC and capillary column chromatography CC; in liquid column chromatography, according to the difference of separation capacity, it can also be divided into classic liquid column chromatography LCC (i.e. the narrow sense of column chromatography), high performance liquid chromatography HPLC and ultra high performance liquid chromatography UPLC, etc.), flat plate chromatography (PC; according to the difference of stationary phase carrier, it can also be divided into paper chromatography PC using filter paper, thin layer chromatography TLC using glass plate or aluminum foil plate and thin film chromatography TFC using polymer film), capillary electrophoresis (CE) and the like.

[0052] Unless otherwise specified, the term "high performance liquid chromatography" used in the context of the present application means that, relative to classic liquid column chromatography, the mobile phase is delivered at high pressure, high efficiency stationary phase is used, online detector and instrumented liquid chromatography are adopted, which has the characteristics of high separation efficiency, fast analysis speed and wide application range, etc., so it is also called high speed liquid chromatography (HSLC), high pressure liquid chromatography (HPLC), high resolution liquid chromatography (HRLC) and the like.

[0053] 3. According to separation mechanism: can be divided into partition chromatography (PC), adsorption chromatography (AC), ion exchange chromatography (IEC), size exclusion chromatography (SEC) and affinity chromatography (AC) and the like.

[0054] Unless otherwise specified, the term "ion exchange chromatography" used in the context of the present application means a method for realizing separation by utilizing the difference of ion exchange capacity of separated components, and its stationary phase is ion exchange resin, which can also be divided into cation exchange resin and anion exchange resin according to the difference of charge sign of exchangeable ions, corresponding to cation exchange chromatography (CEC) and anion exchange chromatography (AEC) respectively.

[0055] 4. According to the polarity of stationary phase and mobile phase: can be divided into normal phase chromatography (NP-C) and reversed phase chromatography (RP-C), wherein the polarity of stationary phase is greater than that of mobile phase, it is normal phase chromatography; the polarity of mobile phase is greater than that of stationary phase, it is reversed phase chromatography; accordingly, high performance liquid chromatography (HPLC) can also be divided into normal phase high performance liquid chromatography (NP-HPLC) and reversed phase high performance liquid chromatography (RP-HPLC).

[0056] The term "ultrafiltration" as used in the context of the present application means a membrane separation technique driven by pressure for the purpose of separating large molecules from small molecules. Under certain pressure, small molecule solutes and solvent can pass through a separation membrane of certain pore size, while large molecule solutes cannot pass through, thus achieving a certain degree of purification of the large molecule substances. Ultrafiltration technique can be used to separate proteins, enzymes, nucleic acids, polysaccharides, polypeptides, antibiotics, viruses, etc. There is no phase transfer during the entire separation process, no need to add any strong chemicals, can be operated at low temperature, fast filtration rate, easy to perform sterile processing, avoiding the loss of activity and denaturation of biologically active substances.

[0057] The term "solid phase synthesis" as used in the context of the present application means a synthesis method in which the reactants are attached to a solid phase carrier that is insoluble in the reaction solvent, and is mainly used for the synthesis of polypeptides and other substances, i.e. "solid phase polypeptide synthesis" (SPPS). First, an amino acid with an amino group blocked by a protecting group is covalently attached to a solid phase carrier, and then the protecting group of the amino group is removed, thus completing the attachment of the first amino acid to the solid phase carrier. Then, the carboxyl group of the second amino acid with the amino group blocked is activated by a condensing agent, and then forms a peptide bond with the amino group of the first amino acid attached to the solid phase carrier, obtaining a dipeptide with a protecting group on the solid phase carrier. The above-mentioned peptide bond formation reaction is repeated to grow the peptide chain from the C-terminal to the N-terminal until the desired length of the peptide chain is reached. Finally, the amino protecting group of the N-terminal amino acid is removed, and a cleavage solution is used to strip the C-terminal of the polypeptide from the solid phase carrier, thus obtaining the target polypeptide. The main advantage of solid phase polypeptide synthesis is that the initial reactants and products are all attached to a solid phase carrier, so all reactions can be carried out in one reaction vessel, facilitating automated operation, and the use of excess reactants can obtain high yield of products, and the products are relatively easy to separate. Currently, solid phase polypeptide synthesis can be carried out on a program-controlled automated polypeptide synthesizer.

[0058] The term "exenatide variant" as used in the context of the present application means a Cys 39 - Exendin-4 (or Cys 39 - exenatide), which is a variant obtained by mutating the 39th serine (Ser) to cysteine (Cys) in the amino acid sequence of exenatide. Specifically, Cys 39The structure of -Exendin-4 is: H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Cys-NH2, as shown in SEQ ID NO:1.

[0059] Unless otherwise stated, the term "polyethylene glycol-modified exenatide variant" as used in the context of this invention refers to mPEG-ppMAL-Cys 39 -Exendin-4 (or mPEG-ppMAL-Cys) 39 -exenatide), which is Cys 39 The conjugate of exenatide with α-methoxy-polyethylene glycol-ω-[3-(3-maleimino-1-oxopropyl)amino]propyl ether (mPEG-ppMAL) can be simply referred to as the "exenatide variant-polyethylene glycol conjugate". Specifically, mPEG-ppMAL-Cys 39 The structure of -Exendin-4 is:

[0060]

[0061] The molecular weight of the polyethylene glycol modifier in the above-mentioned polyethylene glycolated exenatide variant can be 5-100 kDa, preferably 10-50 kDa, more preferably 15-40 kDa, such as 15 kDa, 16 kDa, 17 kDa, 18 kDa, 19 kDa, 20 kDa, 21 kDa, 22 kDa, 23 kDa, 24 kDa, 25 kDa, 26 kDa, 27 kDa, 28 kDa, 29 kDa, 30 kDa, 31 kDa, 32 kDa, 33 kDa, 34 kDa, 35 kDa, 36 kDa, 37 kDa, 38 kDa, 39 kDa, 40 kDa, even more preferably 20-29 kDa, and even more preferably 23 kDa.

[0062] Since polymers are composed of molecules with different degrees of polymerization within a certain distribution range, the molecular weight of a polymer is generally expressed as the average molecular weight. Specifically, it can be the number-average molecular weight or the weight-average molecular weight. Although the number-average molecular weight and the weight-average molecular weight may deviate when the degree of polymerization of polymers varies greatly, they tend to be equal for polymers with a narrow distribution range. For the polyethylene glycol conjugates of the present invention, when referring to their molecular weight, either the weight-average molecular weight or the number-average molecular weight can be used, with the number-average molecular weight being preferred.

[0063] Unless otherwise specified, the term "conjugate" used in the context of the present application means a compound formed by covalently linking a macromolecular compound such as a protein, a polypeptide, a saccharide, a lipid, a nucleic acid, etc. with a small molecule such as a polyethylene glycol modifier and / or another macromolecular compound; accordingly, the process of forming a covalent bond between different fragments included in a conjugate is "conjugation". Unless otherwise specified, the term "polyethylene glycol modifier" (or "PEG modifier") used in the context of the present application means a polyethylene glycol derivative (e.g., mPEG-NH2, mPEG-OTs, mPEG-ppMAL, etc.) having a functional group that can react with a molecule to be modified (e.g., a polypeptide or a protein), and is used for structural modification of a polypeptide or a protein drug to increase the in vivo half-life, reduce immunogenicity, and increase water solubility, etc.

[0064] Exenatide variant (Cys) 39 Preparation method of -Exendin-4)

[0065] In the context of the above-described exenatide variant, the present application provides a Cys 39 -Exendin-4 can be prepared by the following steps: purifying a Cys 39 -Exendin-4 crude product by a three-step reversed-phase column chromatography.

[0066] In an embodiment of the present application, the Cys 39 -Exendin-4 crude product can be obtained by a solid-phase synthesis method.

[0067] In an embodiment of the present application, the Cys 39 -Exendin-4 crude product can be obtained by a solid-phase polypeptide synthesis method.

[0068] The specific process of the above-described solid-phase synthesis method or solid-phase polypeptide synthesis method can be determined by a person skilled in the art according to actual needs, for example, a synthesis method using Fmoc-protected amino acid sequential connection with Fmoc-protected amino resin as a carrier.

[0069] The purification step in the above-described polypeptide preparation method can include a three-step reversed-phase column chromatography, and the column used in each step can be independently selected from each other, and can be completely the same, not completely the same, or completely different, for example, independently selected from one or more of C4 column, C8 column and C18 column.

[0070] In an embodiment of the present application, the purification step in the above-described polypeptide preparation method can include a three-step reversed-phase column chromatography (e.g., RP-HPLC, particularly preparative RP-HPLC), and the column used in each step can be a C18 column.

[0071] The first reversed-phase column chromatography of the three-step reversed-phase column chromatography can use a binary mobile phase system, wherein mobile phase A can be a solution of an acidic substance in water, and mobile phase B can be a solution of an acidic substance in an organic solvent; the acidic substance in mobile phase A and mobile phase B can each be independently selected, and can be the same as or different from each other, for example, each is independently selected from one or more of formic acid, acetic acid, trifluoroacetic acid, phosphoric acid, dipotassium hydrogen phosphate and monopotassium dihydrogen phosphate, preferably trifluoroacetic acid; the organic solvent can be a commonly used organic solvent in the art, for example, selected from one or more of methanol, ethanol and acetonitrile, preferably acetonitrile.

[0072] The volume percentage of the acidic substance in mobile phase A and mobile phase B used in the first reversed-phase column chromatography of the three-step reversed-phase column chromatography can be the same or different, for example, each is independently 0.01%-1%, preferably 0.02-0.5%, more preferably 0.05-0.2%, even more preferably 0.1%.

[0073] In an embodiment of the present application, the first reversed-phase column chromatography of the three-step reversed-phase column chromatography can use a binary mobile phase system, wherein mobile phase A can be a solution of trifluoroacetic acid in water, and mobile phase B can be a solution of trifluoroacetic acid in acetonitrile; the volume percentage of trifluoroacetic acid in mobile phase A and mobile phase B can each be 0.1%.

[0074] The first reversed-phase column chromatography of the three-step reversed-phase column chromatography can include a gradient elution process using mobile phase B with a volume percentage of 0%-95%, preferably 1%-90%, more preferably 5%-85%, even more preferably 8%-80%; the time of the gradient elution process can be within 120 min, preferably within 110 min, more preferably within 100 min, even more preferably within 90 min.

[0075] The first reversed-phase column chromatography of the three-step reversed-phase column chromatography can further include an isocratic elution process after the gradient elution process, in which the volume percentage of mobile phase B is the same as the gradient end point, and preferably can further include a column equilibration process before the gradient elution process and / or after the isocratic elution process, in which the volume percentage of mobile phase B is the same as the gradient start point.

[0076] In an embodiment of the present application, the process of the first reversed-phase column chromatography of the three-step reversed-phase column chromatography can be: first, column equilibration using mobile phase B with a volume percentage of 8%, second, gradient elution using mobile phase B with a volume percentage of 8%-80% within 90 min, third, isocratic elution using mobile phase B with a volume percentage of 80%, and finally, column re-equilibration using mobile phase B with a volume percentage reduced to 8%.

[0077] The second reversed-phase column chromatography of the three-step reversed-phase column chromatography can use a binary mobile phase system, wherein mobile phase A can be an acidic substance in water, and mobile phase B can be an organic solvent; the acidic substance in mobile phase A can be selected from one or more of phosphoric acid, dipotassium hydrogen phosphate, monopotassium phosphate, disodium hydrogen phosphate and monosodium phosphate, preferably monopotassium phosphate and phosphoric acid; the organic solvent can be a commonly used organic solvent in the art, for example, selected from one or more of methanol, ethanol and acetonitrile, preferably acetonitrile.

[0078] The pH value of mobile phase A used in the second reversed-phase column chromatography of the three-step reversed-phase column chromatography can be 1.0-6.0, preferably 2.0-5.0, more preferably 2.5-3.5, and even more preferably 3.0.

[0079] When the phosphate salt is contained in mobile phase A, the concentration of the phosphate salt can be 5-100 mmol / L, preferably 10-80 mmol / L, more preferably 15-70 mmol / L, and even more preferably 20-50 mmol / L.

[0080] In an embodiment of the present application, the second reversed-phase column chromatography of the three-step reversed-phase column chromatography can use a binary mobile phase system, wherein mobile phase A can be an aqueous solution of monopotassium phosphate and phosphoric acid, the concentration of monopotassium phosphate can be 35 mmol / L, the pH value of mobile phase A can be 3.0, and mobile phase B can be acetonitrile.

[0081] The second reversed-phase column chromatography of the three-step reversed-phase column chromatography can include a gradient elution process using mobile phase B with a volume percentage of 0%-85%, preferably 1%-75%, more preferably 2%-70%, and even more preferably 5%-65%; the time of the gradient elution process can be within 100 min, preferably within 90 min, more preferably within 80 min, and even more preferably within 70 min.

[0082] The second reversed-phase column chromatography of the three-step reversed-phase column chromatography can further include an isocratic elution process after the gradient elution process, in which the volume percentage of mobile phase B is the same as that at the end of the gradient, and preferably can further include a column equilibration process before the gradient elution process and / or after the isocratic elution process, in which the volume percentage of mobile phase B is the same as that at the start of the gradient.

[0083] In an embodiment of the present application, the second reversed-phase column chromatography in the three-step reversed-phase column chromatography can be performed by first equilibrating the column with mobile phase B at 5% (v / v), then performing a gradient elution with mobile phase B at 5%-65% (v / v) over 70 min, followed by an isocratic elution with mobile phase B at 65% (v / v), and finally re-equilibrating the column with mobile phase B at 5% (v / v).

[0084] The third reversed-phase column chromatography in the three-step reversed-phase column chromatography can use a binary mobile phase system, wherein mobile phase A can be an acidic solution in water and mobile phase B can be an acidic solution in an organic solvent; the acidic solution in water and the organic solvent can each be independently selected and can be the same or different from each other, for example, each independently selected from one or more of formic acid, acetic acid, trifluoroacetic acid, phosphoric acid, dipotassium hydrogen phosphate, and monopotassium dihydrogen phosphate, preferably trifluoroacetic acid; the organic solvent can be a commonly used organic solvent in the art, for example, selected from one or more of methanol, ethanol, and acetonitrile, preferably acetonitrile.

[0085] The acidic solution in mobile phase A and mobile phase B used in the third reversed-phase column chromatography in the three-step reversed-phase column chromatography can be the same or different, for example, each independently at 0.005%-0.5% (v / v), preferably 0.01-0.2% (v / v), more preferably 0.02-0.1% (v / v), and even more preferably 0.02% (v / v).

[0086] In an embodiment of the present application, the third reversed-phase column chromatography in the three-step reversed-phase column chromatography can use a binary mobile phase system, wherein mobile phase A can be a trifluoroacetic acid solution in water and mobile phase B can be a trifluoroacetic acid solution in acetonitrile; the trifluoroacetic acid in mobile phase A and mobile phase B can each be at 0.02% (v / v).

[0087] The third reversed-phase column chromatography in the three-step reversed-phase column chromatography can include a gradient elution process using mobile phase B at 0%-95% (v / v), preferably 1%-90% (v / v), more preferably 2%-85% (v / v), and even more preferably 5%-80% (v / v); the gradient elution process can be performed within 100 min, preferably within 90 min, more preferably within 80 min, and even more preferably within 75 min.

[0088] The third reversed-phase column chromatography in the three-step reversed-phase column chromatography can further include an isocratic elution process after the gradient elution process, wherein the volume percentage of mobile phase B is the same as the end point of the gradient, and preferably further includes a column equilibration process before the gradient elution process and / or after the isocratic elution process, wherein the volume percentage of mobile phase B is the same as the start point of the gradient.

[0089] In an embodiment of the present application, the third step of the above-mentioned three-step reverse phase column chromatography can be performed as follows: first, equilibration of the column using mobile phase B at 5% (v / v); second, gradient elution using mobile phase B at 5%-80% (v / v) over 75 min; third, isocratic elution using mobile phase B at 80% (v / v); and finally, re-equilibration of the column using mobile phase B at 5% (v / v).

[0090] Methods of making pegylated exenatide variants (mPEG-ppMAL-Cys 39 Exendin-4)

[0091] In an embodiment of the present application, the above-mentioned conjugate preparation method can be used to prepare a mPEG-ppMAL-Cys 39 In an embodiment of the present application, the above-mentioned conjugate preparation method can be used to prepare a mPEG-ppMAL-Cys 39 In an embodiment of the present application, the above-mentioned conjugate preparation method can be used to prepare a mPEG-ppMAL-Cys

[0092] In an embodiment of the present application, the mPEG-ppMAL used in the above-mentioned conjugate preparation method can have a molecular weight (e.g. number average molecular weight) of 5-100 kDa.

[0093] In an embodiment of the present application, the mPEG-ppMAL used in the above-mentioned conjugate preparation method can have a molecular weight (e.g. number average molecular weight) of 10-50 kDa.

[0094] In an embodiment of the present application, the mPEG-ppMAL used in the above-mentioned conjugate preparation method can have a molecular weight (e.g. number average molecular weight) of 15-40 kDa.

[0095] In an embodiment of the present application, the mPEG-ppMAL used in the above-mentioned conjugate preparation method can have a molecular weight (e.g. number average molecular weight) of 20-29 kDa.

[0096] In an embodiment of the present application, the mPEG-ppMAL used in the above-mentioned conjugate preparation method can have a molecular weight (e.g. number average molecular weight) of 23 kDa.

[0097] The conjugation step in the above-mentioned conjugate preparation method can be performed by any method known to one skilled in the art, for example, by addition reaction (e.g. Michael addition reaction).

[0098] The purification step in the above-mentioned conjugate preparation method can be performed by any method known to one skilled in the art, for example, by one or more of ion exchange chromatography (e.g. CEC) and reverse phase column chromatography (e.g. RP-HPLC, particularly preparative RP-HPLC).

[0099] In another aspect, the present application provides another mPEG-ppMAL-Cys conjugate of pegylated exenatide variant, which can comprise the following structure: 39 In another aspect, the present application provides a method for preparing Exendin-4, which can comprise the following steps: first purifying Cys 39 Exendin-4 is conjugated with mPEG-ppMAL, and then the mPEG-ppMAL-Cys conjugate is purified by ion exchange chromatography and three-step reversed-phase column chromatography in sequence. 39 Exendin-4 is purified by ion exchange chromatography and three-step reversed-phase column chromatography in sequence.

[0100] In one embodiment of the present application, the molecular weight (e.g. number average molecular weight) of mPEG-ppMAL in the above conjugate preparation method can be 5-100 kDa.

[0101] In one embodiment of the present application, the molecular weight (e.g. number average molecular weight) of mPEG-ppMAL in the above conjugate preparation method can be 10-50 kDa.

[0102] In one embodiment of the present application, the molecular weight (e.g. number average molecular weight) of mPEG-ppMAL in the above conjugate preparation method can be 15-40 kDa.

[0103] In one embodiment of the present application, the molecular weight (e.g. number average molecular weight) of mPEG-ppMAL in the above conjugate preparation method can be 20-29 kDa.

[0104] In one embodiment of the present application, the molecular weight (e.g. number average molecular weight) of mPEG-ppMAL in the above conjugate preparation method can be 23 kDa.

[0105] The specific process of the conjugation step in the above conjugate preparation method can be determined by those skilled in the art according to actual needs, for example, Cys 39 Exenatide is obtained by Michael addition reaction of the thiol group (-SH) in the 39th Cys residue of Exenatide with mPEG-ppMAL.

[0106] In one embodiment of the present application, the purified Cys 39 Exendin-4 can be obtained by the polypeptide preparation method of the present application.

[0107] In one embodiment of the present application, the ion exchange chromatography in the above conjugate preparation method can be CEC, for example, using MacroCap SP as the stationary phase.

[0108] The mobile phase system used in the ion exchange chromatography in the above conjugate preparation method can comprise mobile phase A, mobile phase B, mobile phase C and mobile phase D.

[0109] The mobile phase A in the mobile phase system used in the ion exchange chromatography can be an aqueous solution of acetic acid and sodium acetate; the pH value of the mobile phase A can be 2.0-6.0, preferably 3.0-5.0, more preferably 4.0; the concentration of sodium acetate in the mobile phase A can be 5-50 mmol / L, preferably 10-30 mmol / L, more preferably 20 mmol / L.

[0110] In an embodiment of the present application, the mobile phase A in the mobile phase system used in the ion exchange chromatography can be an aqueous solution of acetic acid and sodium acetate; the pH value of the mobile phase A can be 4.0; the concentration of sodium acetate in the mobile phase A can be 20 mmol / L.

[0111] The mobile phase B in the mobile phase system used in the ion exchange chromatography can be an aqueous solution of acetic acid, sodium acetate and sodium chloride; the pH value of the mobile phase B can be 2.0-6.0, preferably 3.0-5.0, more preferably 4.0; the concentration of sodium acetate in the mobile phase B can be 5-50 mmol / L, preferably 10-30 mmol / L, more preferably 20 mmol / L; the concentration of sodium chloride in the mobile phase B can be 0.1-5 mol / L, preferably 0.2-4 mol / L, more preferably 0.5-2 mol / L, even more preferably 1 mol / L.

[0112] In an embodiment of the present application, the mobile phase B in the mobile phase system used in the ion exchange chromatography can be an aqueous solution of acetic acid, sodium acetate and sodium chloride; the pH value of the mobile phase B can be 4.0; the concentration of sodium acetate in the mobile phase B can be 20 mmol / L, and the concentration of sodium chloride can be 1 mol / L.

[0113] The mobile phase C in the mobile phase system used in the ion exchange chromatography can be a mixture of the mobile phase A and the mobile phase B, and the volume ratio of the mobile phase A to the mobile phase B can be 0.5:1-10:1, preferably 1:1-5:1, more preferably 2:1-4:1, even more preferably 4:1.

[0114] In an embodiment of the present application, the mobile phase C in the mobile phase system used in the ion exchange chromatography can be a mixture of the mobile phase A and the mobile phase B, and the volume ratio of the mobile phase A to the mobile phase B can be 4:1.

[0115] The mobile phase D in the mobile phase system used in the above ion exchange chromatography can be a solution of sodium hydroxide in water, and the concentration of sodium hydroxide in the mobile phase D can be 0.02-2 mol / L, preferably 0.05-1 mol / L, more preferably 0.1-0.5 mol / L, and even more preferably 0.2 mol / L.

[0116] In an embodiment of the present application, the mobile phase D in the mobile phase system used in the above ion exchange chromatography can be a solution of sodium hydroxide in water, and the concentration of sodium hydroxide in the mobile phase D can be 0.2 mol / L.

[0117] In an embodiment of the present application, the ion exchange chromatography in the above conjugate preparation method can be performed by first flushing the AKTA system with the mobile phase D, the mobile phase B and the mobile phase A in sequence, secondly diluting the pre-prepared mPEG-ppMAL-Cys 39 -Exendin-4 crude solution, and finally flushing the AKTA system with the mobile phase A and the mobile phase C in sequence, and collecting the fractions.

[0118] The purification step in the above conjugate preparation method can comprise three steps of reverse phase column chromatography, and the chromatographic column used in each step can be independently selected from one or more of a C4 column, a C8 column and a C18 column.

[0119] In an embodiment of the present application, the purification step in the above conjugate preparation method can comprise three steps of reverse phase column chromatography (such as RP-HPLC, in particular preparative RP-HPLC), and the chromatographic column used in each step can be a C18 column.

[0120] In an embodiment of the present application, the first and third steps of reverse phase column chromatography in the above three steps of reverse phase column chromatography can use the same binary mobile phase system and elution process, wherein the mobile phase A can be a solution of an acidic substance in water, and the mobile phase B can be a solution of an acidic substance in an organic solvent; the acidic substance in the mobile phase A and the mobile phase B can be independently selected from one or more of formic acid, acetic acid, trifluoroacetic acid, phosphoric acid, dipotassium hydrogen phosphate and monopotassium hydrogen phosphate, preferably trifluoroacetic acid; the organic solvent can be a commonly used organic solvent in the art, for example, selected from one or more of methanol, ethanol and acetonitrile, preferably acetonitrile.

[0121] The volume percentage of the acidic substance in the mobile phase A and the mobile phase B used in the first and third step of the three-step reversed-phase column chromatography method described above can be the same or different, for example, each independently is 0.01%-1%, preferably 0.02-0.5%, more preferably 0.05-0.2%, even more preferably 0.1%.

[0122] In an embodiment of the present application, the first and third step of the three-step reversed-phase column chromatography method described above can use a binary mobile phase system, wherein the mobile phase A can be an aqueous solution of trifluoroacetic acid, and the mobile phase B can be an acetonitrile solution of trifluoroacetic acid; the volume percentage of trifluoroacetic acid in the mobile phase A and the mobile phase B can be both 0.1%.

[0123] The first and third step of the three-step reversed-phase column chromatography method described above can include a gradient elution process using the mobile phase B with a volume percentage of 0%-95%, preferably 2%-90%, more preferably 5%-85%, even more preferably 9%-80%; the time of the gradient elution process can be within 100 min, preferably within 90 min, more preferably within 80 min, even more preferably within 70 min.

[0124] The first and third step of the three-step reversed-phase column chromatography method described above can further include an isocratic elution process after the gradient elution process, with the volume percentage of the mobile phase B being the same as the gradient end point, preferably further including a column equilibration process before the gradient elution process and / or after the isocratic elution process, with the volume percentage of the mobile phase B being the same as the gradient start point.

[0125] In an embodiment of the present application, the process of the first and third step of the three-step reversed-phase column chromatography method described above can be: first, column equilibration using the mobile phase B with a volume percentage of 9%, second, gradient elution using the mobile phase B with a volume percentage of 9%-80% within 70 min, third, isocratic elution using the mobile phase B with a volume percentage of 80%, and fourth, column re-equilibration using the mobile phase B with a volume percentage decreasing to 9%.

[0126] In an embodiment of the present application, the second step of the three-step reversed-phase column chromatography method described above can use a binary mobile phase system, wherein the mobile phase A can be a solution of an acidic substance in water, and the mobile phase B can be an organic solvent; the acidic substance in the mobile phase A can be selected from one or more of phosphoric acid, dipotassium hydrogen phosphate, monopotassium phosphate, disodium hydrogen phosphate, and monosodium phosphate, preferably monopotassium phosphate and phosphoric acid; the organic solvent can be a commonly used organic solvent in the art, for example, selected from one or more of methanol, ethanol, and acetonitrile, preferably acetonitrile.

[0127] The pH value of the mobile phase A used in the second reversed-phase column chromatography of the three-step reversed-phase column chromatography described above can be 1.0-6.0, preferably 2.0-5.0, more preferably 2.5-3.5, even more preferably 3.0.

[0128] When the mobile phase A contains a phosphate salt (e.g. potassium dihydrogen phosphate), the concentration of the phosphate salt can be 5-100 mmol / L, preferably 10-80 mmol / L, more preferably 15-70 mmol / L, even more preferably 20-50 mmol / L.

[0129] In an embodiment of the present application, the second reversed-phase column chromatography of the three-step reversed-phase column chromatography described above can use a binary mobile phase system, wherein the mobile phase A can be an aqueous solution of potassium dihydrogen phosphate and phosphoric acid, the concentration of potassium dihydrogen phosphate can be 35 mmol / L, the pH value of the mobile phase A can be 3.0, and the mobile phase B can be acetonitrile.

[0130] The second reversed-phase column chromatography of the three-step reversed-phase column chromatography described above can comprise a gradient elution process using the mobile phase B at a volume percentage of 0%-85%, preferably 2%-75%, more preferably 5%-70%, even more preferably 8%-65%; the time of the gradient elution process can be within 100 min, preferably within 90 min, more preferably within 80 min, even more preferably within 70 min.

[0131] The second reversed-phase column chromatography of the three-step reversed-phase column chromatography described above can further comprise an isocratic elution process after the gradient elution process, in which the volume percentage of the mobile phase B is the same as the gradient end point, preferably further comprising a column equilibration process before the gradient elution process and / or after the isocratic elution process, in which the volume percentage of the mobile phase B is the same as the gradient start point.

[0132] In an embodiment of the present application, the process of the second reversed-phase column chromatography of the three-step reversed-phase column chromatography described above can be: first, column equilibration using the mobile phase B at a volume percentage of 8%, second, gradient elution using the mobile phase B at a volume percentage of 8%-65% within 70 min, third, isocratic elution using the mobile phase B at a volume percentage of 65%, and finally, column re-equilibration using the mobile phase B at a volume percentage reduced to 8%.

[0133] The purification step in the conjugate preparation method described above can further comprise an ultrafiltration process after the three-step reversed-phase column chromatography.

[0134] The ultrafiltration process in the above conjugate preparation method can be: firstly, subjecting the sample obtained after purification by the three-step reversed-phase column chromatography to ultrafiltration, secondly, diluting with water, thirdly, adjusting the pH value to 4.0-5.0 using a pH adjuster, and finally, subjecting to ultrafiltration again; the pH adjuster is a basic substance, preferably one or more of hydroxides, carbonates and bicarbonates of alkali metals, more preferably one or more of sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium hydroxide, potassium carbonate and potassium bicarbonate, and even more preferably sodium bicarbonate.

[0135] In an embodiment of the present application, the ultrafiltration process in the above conjugate preparation method can be: firstly, subjecting the sample obtained after purification by the three-step reversed-phase column chromatography to ultrafiltration (e.g. 4 times of ultrafiltration), secondly, diluting with water (e.g. purified water) (e.g. 1-fold dilution), thirdly, adjusting the pH value to 4.0-5.0 using sodium bicarbonate (e.g. 0.1M aqueous solution), and finally, subjecting to ultrafiltration again (e.g. 3 times of ultrafiltration).

[0136] The technical solutions of the present application will be further illustrated below in combination with specific examples. Unless otherwise specified, the materials, reagents, instruments and the like used in the following examples can be obtained through conventional commercial means.

[0137] Unless otherwise specified, the terms represented by abbreviations in the present application have the following meanings.

[0138] Terms Abbreviations Dichloromethane DCM N,N-diisopropylethylamine DIPEA N,N-dimethylformamide DMF 9-fluorenylmethyloxycarbonyl Fmoc O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate HBTU Nitrogen [N2] Trifluoroacetic acid TFA Acetic acid HAc Sodium acetate NaAc Sodium chloride NaCl Hours h Minutes min Column volume CV

[0139] Example 1: Exenatide variant Cys 39 - Synthesis of Exendin-4

[0140] The exenatide variants in the present application can be obtained by using conventional solid-phase synthesis methods. For example, the following method is used for synthesis:

[0141] (1) Fmoc-Rink Linker MBHA resin is added to the reaction tank of the synthesizer, DCM is added, stirring is started, and swelling is performed for 30 min. Then, N2 is filled into the reaction tank until the end of the entire synthesis reaction;

[0142] (2) After the resin is swelled, the solution is drained, and piperidine / DMF is prepared as a deprotection agent according to the volume of the resin, which is added to the reaction tank and stirred with the resin to obtain a deprotected resin;

[0143] (3) Fmoc-protected amino acids and HBTU / DIPEA as a condensing agent are dissolved in DMF and added to the reaction tank for 1-5 h, and the completion of the condensation reaction is monitored by the ninhydrin method;

[0144] (4) Prepare piperidine / DMF as deprotection agent, add the deprotection agent into the reaction tank, and react for 10-30 min. Monitor whether the protecting group is completely removed by the ninhydrin method;

[0145] (5) According to the amino acid sequence of the target polypeptide, use the corresponding Fmoc-protected amino acid in turn, and repeat steps (3) and (4) until the last amino acid in the sequence is coupled;

[0146] (6) Add the pre-prepared TFA cleavage solution into the reaction tank, mix with the coupled polypeptide resin, and react for 3-5 hours. Filter, add ether to the filtrate, and then filter after standing. Collect the precipitate to obtain the Exendin-4 variant Cys 39 - Exendin-4 crude product.

[0147] Example Two: Purification of Exendin-4 variant Cys 39 - Exendin-4 crude product.

[0148] According to the method described in Example One, three batches of Exendin-4 variant Cys 39 - Exendin-4 crude product, each batch of crude product is divided into three parts, and each part is dissolved using 40% v / v acetic acid to obtain a crude solution.

[0149] Each of the three samples in each batch of crude product is purified using the following purification processes (1)-(3), respectively, and then the samples are collected, freeze-dried, and subjected to relevant tests:

[0150] Purification Process (1): Method a + Method c + Method d;

[0151] Purification Process (2): Method a + Method d;

[0152] Purification Process (3): Method a + Method b + Method d.

[0153] The specific conditions of methods a-d used in the above purification processes are as follows:

[0154] Method a: RP-HPLC

[0155] Chromatographic column packing: C18 SMB200-10;

[0156] Detection wavelength: 230 nm;

[0157] Mobile phase A: 0.1% v / v TFA-H2O;

[0158] Mobile phase B: 0.1% v / v TFA-CH3CN;

[0159] Elution conditions: Initial equilibration of the column with 8% v / v mobile phase B followed by a gradient elution with 8-80% v / v mobile phase B over 90 min, then isocratic elution using 80% v / v mobile phase B, and finally re-equilibration of the column using 8% v / v mobile phase B.

[0160] Method b: RP-HPLC

[0161] Column packing: C18 SMB200-10;

[0162] Detection wavelength: 230 nm;

[0163] Mobile phase A: 35 mmol / L KH2PO4.H2O adjusted to pH = 3.0 ± 0.2 with H3PO4;

[0164] Mobile phase B: CH3CN;

[0165] Elution conditions: Initial equilibration of the column with 5% v / v mobile phase B followed by a gradient elution with 5-65% v / v mobile phase B over 70 min, then isocratic elution using 65% v / v mobile phase B, and finally re-equilibration of the column using 5% v / v mobile phase B.

[0166] Method c: RP-HPLC

[0167] Column packing: C18 SMB200-10;

[0168] Detection wavelength: 230 nm;

[0169] Mobile phase A: 1% v / v HAc.H2O;

[0170] Mobile phase B: CH3CN;

[0171] Elution conditions: Initial equilibration of the column with 5% v / v mobile phase B followed by a gradient elution with 5-90% v / v mobile phase B over 90 min, then isocratic elution using 90% v / v mobile phase B, and finally re-equilibration of the column using 5% v / v mobile phase B.

[0172] Method d: RP-HPLC

[0173] Column packing: C18 SMB200-10;

[0174] Detection wavelength: 230 nm;

[0175] Mobile phase A: 0.02% v / v TFA.H2O;

[0176] Mobile phase B: 0.02% v / v TFA CH3CN;

[0177] Elution conditions: Equilibration of the column with 5% v / v mobile phase B initially, followed by a gradient elution with 5% to 80% v / v mobile phase B over 75 min, then isocratic elution using 80% v / v mobile phase B, and finally re-equilibration of the column using 5% v / v mobile phase B.

[0178] The overall test results for each batch and each purification process are as follows:

[0179]

[0180] From the above test results, it can be seen that under the conditions of purification process (3), the yield and purity of the three batches of products are both high.

[0181] Subsequently, the content of each type of component contained in the products of each batch and each purification process was also detected, and the test results are as follows:

[0182]

[0183] From the comparison results of the sample quality of the above three different purification processes, it can be seen that the water content of purification processes (1), (2) and (3) is roughly equivalent, the peptide content of purification process (3) is the highest, and the TFA content of purification process (3) is the lowest. The results of reverse phase high performance liquid chromatography show that the purity of the 3 small batches of samples obtained by purification process (3) is the highest, which can reach more than 98.0%, and the contents of Cys39 deamidation and Met14 oxidation impurities, Asn28 deamidation impurities, and dimer impurities as known impurities are at a relatively low level, and the maximum unknown single impurity is not more than 0.3%; the ion chromatography detection results show that the D-His impurity content is also low. The purities of the samples obtained by the other two purification processes are significantly lower under the conditions of reverse phase high performance liquid chromatography and ion chromatography.

[0184] Example Three: PEGylated Exendin-4 variant mPEG-ppMAL-Cys 39 - Synthesis of Exendin-4

[0185] Phosphate buffer solution with pH = 6.5 was prepared in a reaction bottle, N2 was replaced for 30-50 min, and then Exendin-4 variant Cys 39Exendin-4 (obtained by the purification method in Example 2) and mPEG-ppMAL (molecular weight 23±2.3 kDa) were added into a reaction bottle containing phosphate buffer at a molar ratio of 1:2, the reaction temperature was controlled at 22±2°C, the reaction was stirred for 60 min, and the reaction progress was detected by sampling. After the reaction was completed, the reaction solution was diluted with a sodium acetate / acetic acid solution at pH=4.0, and the pH value of the mixture was adjusted to 4.0 by adding an appropriate amount of glacial acetic acid, to obtain the PEGylated exendin-4 variant mPEG-ppMAL-Cys 39 Exendin-4 crude solution.

[0186] Example 4: PEGylated exendin-4 variant mPEG-ppMAL-Cys 39 Purification of Exendin-4

[0187] Three batches of PEGylated exendin-4 variant mPEG-ppMAL-Cys were prepared according to the method described in Example 3 39 Exendin-4 crude solution, which was divided into four parts for each batch.

[0188] Each of the four samples in the crude solution of each batch was purified using the following purification processes (one) to (four), respectively, and relevant tests were performed:

[0189] Process (one): Method A→Method B→Method A→Method C;

[0190] Process (two): Method D→Method C;

[0191] Process (three): Method A→Method B→Method A→Method D→Method C;

[0192] Process (four): Method D→Method A→Method B→Method A→Method C.

[0193] In the above-mentioned purification processes, both processes (three) and (four) need to undergo four steps of purification, the difference being that process (three) first undergoes three steps of reverse phase chromatography purification, and then one step of ion exchange purification, while process (four) first undergoes one step of ion exchange purification, and then three steps of reverse phase chromatography purification.

[0194] The specific conditions of methods A-D used in the above-mentioned purification processes are as follows:

[0195] Method A: RP-HPLC

[0196] Chromatographic column packing: C18 SMB200-10;

[0197] Detection wavelength: 230 nm;

[0198] Mobile phase A: 0.1% v / v TFA in H2O;

[0199] Mobile phase B: 0.1% v / v TFA in CH3CN;

[0200] Elution condition: Equilibration of column with 9% v / v mobile phase B initially, followed by gradient elution with 9%-80% v / v mobile phase B within 70 min, then isocratic elution with 80% v / v mobile phase B maintained, and finally re-equilibration of column with 9% v / v mobile phase B.

[0201] Method B: RP-HPLC

[0202] Column packing: C18 SMB200-10;

[0203] Detection wavelength: 230 nm;

[0204] Mobile phase A: 35 mmol / L KH2PO4 in H2O, adjusted to pH = 3.0 ± 0.2 with H3PO4;

[0205] Mobile phase B: CH3CN;

[0206] Elution condition: Equilibration of column with 8% v / v mobile phase B initially, followed by gradient elution with 8%-65% v / v mobile phase B within 70 min, then isocratic elution with 65% v / v mobile phase B maintained, and finally re-equilibration of column with 8% v / v mobile phase B.

[0207] Method C: Ultrafiltration

[0208] After ultrafiltration for 4 times, the sample was diluted about 1-fold with purified water, and then adjusted to pH 4.0-5.0 with 0.1 mol / L NaHCO3, and then ultrafiltrated for 3 times. If the ultrafiltration process is slow due to too high product concentration, purified water can be added for dilution. The final product concentration is about 20-40 mg / mL.

[0209] Method D: Ion exchange chromatography

[0210] Column packing: MacroCap SP;

[0211] Detection wavelength: 230 nm;

[0212] Mobile phase A: 20 mmol / L NaAc, adjusted to pH = 4.0 with HAc;

[0213] Mobile phase B: 20 mmol / L NaAc + 1 mol / L NaCl, adjusted to pH = 4.0 with HAc;

[0214] Mobile phase C: Mobile phase A: Mobile phase B = 8:2 (V / V);

[0215] Mobile phase D: 0.2 mol / L NaOH;

[0216] Elution condition:

[0217] Step 1: flush the AKTA system with 1-3 CV of mobile phase D until the conductivity is substantially constant for more than 5 min and the baseline is substantially stable;

[0218] Step 2: flush the AKTA system with 1-3 CV of mobile phase B until the conductivity is substantially constant for more than 5 min and the baseline is substantially stable;

[0219] Step 3: flush the AKTA system with 1-3 CV of mobile phase A until the conductivity is substantially constant for more than 5 min and the baseline is substantially stable;

[0220] Step 4: dilute the sample with 2 times the volume of mobile phase A and load into the AKTA system;

[0221] Step 5: flush the AKTA system with 2-5 CV of mobile phase A until the conductivity is substantially constant for more than 15 min;

[0222] Step 6: flush the AKTA system with 2-4 CV of mobile phase C and collect the fractions.

[0223] The detection results of each batch and each purification process are as follows:

[0224]

[0225] From the above data, it can be seen that the product obtained by purification process (I) contains part of free PEG, and the purity of the product obtained by purification process (II) is low and cannot meet the requirements; whether in terms of purity or yield, the exenatide variant mPEG-ppMAL-Cys 39 -Exendin-4 produced by purification process (IV) is better than that by purification process (III); in addition, the overall purification time of purification process (III) is longer than that of purification process (IV), and the time spent on ultrafiltration is also longer, so purification process (IV) can better meet the production requirements. SEQUENCE LISTING <110> PEGBOGEN OHEMICALS (SUZHOU) CO., LTD. <120> Preparation method of exenatide variant and its polyethylene glycol conjugate <130> 6C40-2173485I-SU <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Exenatide variant <220> <221> AMIDATION <222> (39)..(39) <223> -NH2 <400> 1 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Cys 35

Claims

1. An exenatide variant Cys 39 The preparation method of -Exendin-4 includes the following steps: Cys was purified using a three-step reversed-phase column chromatography method. 39 -Exendin-4 crude product, including Cys 39 The structure of -Exendin-4 is: H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Cys-NH2, as shown in SEQ ID NO:1; in, The chromatographic column used in the three-step reversed-phase column chromatography method is an octadecylsilane-bonded silica column. The first step of the three-step reversed-phase column chromatography method uses a binary mobile phase system, wherein mobile phase A is a solution of an acidic substance in water, and mobile phase B is a solution of an acidic substance in an organic solvent; the acidic substance in mobile phase A and mobile phase B is trifluoroacetic acid; the organic solvent is acetonitrile; and the volume percentage of the acidic substance in mobile phase A and mobile phase B is independently 0.05%-0.2%. The second step of the three-step reversed-phase column chromatography method uses a binary mobile phase system, wherein mobile phase A is a solution of an acidic substance in water, and mobile phase B is an organic solvent; the acidic substance in mobile phase A is selected from one or more of phosphoric acid, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, and sodium dihydrogen phosphate; the pH value of mobile phase A is 2.5-3.5; and the organic solvent is acetonitrile. The third step of the three-step reversed-phase column chromatography method uses a binary mobile phase system, wherein mobile phase A is a solution of an acidic substance in water, and mobile phase B is a solution of an acidic substance in an organic solvent; the acidic substance in mobile phase A and mobile phase B is trifluoroacetic acid; the organic solvent is acetonitrile; and the volume percentage of the acidic substance in mobile phase A and mobile phase B is independently 0.02%-0.1%.

2. The preparation method according to claim 1, wherein, The Cys 39 -Exendin-4 crude product was obtained by solid-phase synthesis.

3. The preparation method according to claim 1 or 2, wherein, The first step of the reversed-phase column chromatography includes a gradient elution process using mobile phase B with a volume percentage of 8%-80%, and the gradient elution process takes no more than 90 minutes.

4. The preparation method according to claim 1 or 2, wherein, The second step, reversed-phase column chromatography, includes a gradient elution process using mobile phase B at a volume percentage of 5%-65%, with the gradient elution process lasting no more than 70 minutes.

5. The preparation method according to claim 1 or 2, wherein, The third step of the reversed-phase column chromatography method includes a gradient elution process using mobile phase B with a volume percentage of 5%-80%, and the gradient elution process takes no more than 75 minutes.

6. A polyethylene glycol-modified exenatide variant, mPEG-ppMAL-Cys 39 The preparation method of -Exendin-4 includes the following steps: Cys is first obtained by the preparation method according to any one of claims 1 to 5. 39 -Exendin-4 was then conjugated and purified with mPEG-ppMAL.

7. A polyethylene glycol-modified exenatide variant, mPEG-ppMAL-Cys 39 The preparation method of -Exendin-4 includes the following steps: First, purify Cys 39 -Exendin-4 was conjugated with mPEG-ppMAL, and then mPEG-ppMAL-Cys was purified sequentially by ion exchange chromatography and three-step reversed-phase column chromatography. 39 -Exendin-4 crude product, including mPEG-ppMAL-Cys 39 The structure of -Exendin-4 is: ； The ion exchange chromatography method is a cation exchange chromatography method, and the stationary phase used is MacroCap SP; The mobile phase system used in the ion exchange chromatography method includes mobile phase A, mobile phase B, mobile phase C, and mobile phase D; Mobile phase A is a solution of acetic acid and sodium acetate in water; the pH of mobile phase A is 3.0-5.0; the concentration of sodium acetate in mobile phase A is 10-30 mmol / L. Mobile phase B is a solution of acetic acid, sodium acetate, and sodium chloride in water; the pH of mobile phase B is 3.0-5.0; the concentration of sodium acetate in mobile phase B is 10-30 mmol / L; the concentration of sodium chloride in mobile phase B is 0.5-2 mol / L. Mobile phase C is a mixture of mobile phase A and mobile phase B, with a volume ratio of mobile phase A to mobile phase B of 2:1 to 4:

1. Mobile phase D is a sodium hydroxide solution in water, and the concentration of sodium hydroxide in mobile phase D is 0.1-0.5 mol / L; The ion exchange chromatography method includes: first, washing the AKTA system sequentially with mobile phase D, mobile phase B, and mobile phase A; then, diluting the pre-prepared mPEG-ppMAL-Cys solution with mobile phase A. 39 After loading the crude Exendin-4 solution, the AKTA system was washed sequentially with mobile phase A and mobile phase C, and the components were collected. The chromatographic column used in the three-step reversed-phase column chromatography method is an octadecylsilane-bonded silica column. The first and third steps of the three-step reversed-phase column chromatography method use the same binary mobile phase system and elution process. Mobile phase A is a solution of the acidic substance in water, and mobile phase B is a solution of the acidic substance in an organic solvent. The acidic substance in mobile phases A and B is trifluoroacetic acid; the organic solvent is acetonitrile; and the volume percentage of the acidic substance in mobile phases A and B is independently 0.05%–0.2%. The second step of the three-step reversed-phase column chromatography method uses a binary mobile phase system, wherein mobile phase A is a solution of an acidic substance in water, and mobile phase B is an organic solvent; the acidic substance in mobile phase A is selected from one or more of phosphoric acid, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, and sodium dihydrogen phosphate; the pH value of mobile phase A is 2.5-3.5; and the organic solvent is acetonitrile.

8. The preparation method according to claim 7, wherein, The purified Cys 39 -Exendin-4 is obtained by the preparation method according to any one of claims 1 to 5.

9. The preparation method according to claim 7 or 8, wherein, The first and third reversed-phase column chromatography steps include a gradient elution process using mobile phase B with a volume percentage of 9%-80%, and the gradient elution process takes no more than 70 minutes. The second step, reversed-phase column chromatography, includes a gradient elution process using mobile phase B at a volume percentage of 8%-65%, with the gradient elution process lasting no more than 70 minutes.

10. The preparation method according to claim 7 or 8, wherein, The preparation method also includes an ultrafiltration process performed after the three-step reverse column chromatography; The ultrafiltration process is as follows: first, the sample purified by three-step reversed-phase column chromatography is ultrafiltered; second, it is diluted with water; third, the pH value is adjusted to 4.0-5.0 using a pH adjuster; and finally, ultrafiltration is performed again. The pH adjuster is sodium bicarbonate.

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