Purification method of retaglutide
Through two-step reverse-phase high-performance liquid chromatography, using an acetic acid-sodium acetate or acetic acid-ammonium acetate system and a carbonate aqueous solution for segmented gradient elution, the problems of long purification cycle and difficult impurity separation of retaglutide were solved, achieving an efficient and low-cost purification effect, which is suitable for the large-scale production of retaglutide.
Patent Information
- Application Number
- CN202511001604.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-21
- Publication Date
- 2025-10-17
AI Technical Summary
The existing purification methods for retaglutide have the disadvantages of long purification cycles, high environmental costs, difficulty in controlling the maximum single impurity below 0.1%, low purity, and difficulty in separating impurities.
A two-step reversed-phase high-performance liquid chromatography method is used. First, segmented gradient elution is performed with acetic acid-sodium acetate or acetic acid-ammonium acetate aqueous solution as phase A and acetonitrile as phase B. Then, a secondary purification is performed with carbonate aqueous solution as phase A and acetonitrile as phase B. By combining gradient elution and segmented gradient elution, the impurities are controlled below 0.1% and the purity is increased to 99.5%.
The purification time is shortened, the amount of acetonitrile used is reduced, the dissolution cost is lowered, the purification efficiency and product purity are improved, the applicability is strong, and it is suitable for large-scale production, achieving the purpose of energy saving and environmental protection.
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Figure CN120795119A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of polypeptide drug purification, and particularly relates to a purification method of Retatrutide. BACKGROUND
[0002] Retatrutide is an active compound named Retatrutide published by Lilly in June 2023, and its CAS number is 2381089-83-2. It is a triple (GLP-1 / GIP / Gcg) agonist based on the GLP / GIP dual receptor agonist Tirzepatide (Tirzepatide) by hybridizing the Glucagon Gcg related structure, which helps to control blood sugar and achieve weight loss effect by activating the receptor agonist in our body. Retatrutide enhances the activity of the peptide chain to GIP and glucagon receptor by modifying the 13th non-natural amino acid aMeL (a-methyl-L-leucine) residue. The 17th is Lys, and a binary fatty acid side chain is introduced at this residue through an AEEA and y-glutamic acid (yGlu) linker to achieve a prolonged action time in the body, and the 20th uses a non-natural amino acid Aib to prevent the peptide chain from being degraded by dipeptidyl peptidase 4 (DDP4) at this position. Retatrutide may even exceed semaglutide and tirzepatide in terms of weight loss effect, and is expected by Lilly, and is being developed at a faster pace, and is expected to be launched in the market in the next few years.
[0003] The current published purification method of Retatrutide has a long purification cycle, high environmental protection cost and is not suitable for long-term development of environmental protection, and the maximum single impurity is difficult to control below 0.1%, which brings a series of heavy work for subsequent impurity research and quality product control. Due to the long peptide chain in the solid phase synthesis of Retatrutide, the side chain contains a long fatty chain, forming a large steric hindrance, thereby causing the generation of more missing peptide impurities, racemization peptides generated by long reaction time, and further causing the low purity of the crude peptide and the similar polarity of the impurities to the product, greatly increasing the separation difficulty of purification. SUMMARY
[0004] The present application aims to overcome at least one of the deficiencies of the prior art and provide a purification method of Retatrutide.
[0005] The technical solution adopted by the present application is:
[0006] In a first aspect, the present application provides a purification method of Retatrutide, comprising the following steps:
[0007] 1) Dissolve the Retatrutide crude product in purified water, adjust the pH to 7.5-9.0, filter and collect the filtrate for use;
[0008] 2) the filtrate obtained in step 1) is subjected to a first purification step using a reverse phase packing as stationary phase, acetic acid-sodium acetate or acetic acid-ammonium acetate aqueous solution as phase A and a polar organic solvent as phase B, performing a stepwise gradient elution, collecting the fractions with purity greater than 98% and single impurities less than 0.5%;
[0009] 3) the fractions obtained in step 2) are subjected to a second purification step using a reverse phase packing as stationary phase, carbonate aqueous solution as phase A and a polar organic solvent as phase B, performing a stepwise gradient elution, collecting the fractions with purity greater than 99.5% and single impurities less than 0.1%;
[0010] 4) the fractions obtained in step 3) are subjected to a sterilization and freeze-drying, obtaining the recombinant human relaxin.
[0011] In some examples, the stepwise gradient elution conditions of step 2) are:
[0012] First, the initial wash operation is performed with a linear gradient elution for 8-12 minutes with a volume percentage of mobile phase A of 75-70% and a volume percentage of mobile phase B of 25-30%;
[0013] Then, the elution of the target operation is performed with a linear gradient elution for 33-37 minutes with a volume percentage of mobile phase A of 68-67% and a volume percentage of mobile phase B of 32-33%, collecting the fractions with purity greater than 98% and single impurities less than 0.5%;
[0014] Immediately after the collection of the target peak, the volume percentage of mobile phase B is increased to more than 75% and eluted for 7-10 minutes, rinsing the impurities on the column.
[0015] In some examples, the stepwise gradient elution conditions of step 3) are:
[0016] First, the initial wash operation is performed with a linear gradient elution for 8-12 minutes with a volume percentage of mobile phase A of 73-71% and a volume percentage of mobile phase B of 27-29%;
[0017] Then, the elution of the target operation is performed with a linear gradient elution for 28-32 minutes with a volume percentage of mobile phase A of 71-69% and a volume percentage of mobile phase B of 29-31%, collecting the fractions with purity greater than 99.5% and single impurities less than 0.1%;
[0018] Immediately after the collection of the target peak, the volume percentage of mobile phase B is increased to more than 75% and eluted for 7-10 minutes, rinsing the impurities on the column.
[0019] In some examples, the volume percentage of acetic acid in the acetic acid-sodium acetate or acetic acid-ammonium acetate aqueous solution of mobile phase A in step 2) is between 0.1% and 5.0%.
[0020] In some examples, the concentration of sodium acetate or ammonium acetate in the aqueous acetic acid-sodium acetate or ammonium acetate solution of the mobile phase A in step 2) is 20-500 mMol / L.
[0021] In some examples, the concentration of the aqueous carbonate solution in step 3) is 20-500 mMol / L.
[0022] In some examples, the concentration of the aqueous carbonate solution in step 3) is preferably 50-200 mMol / L.
[0023] In some examples, the carbonate in step 3) is selected from ammonium bicarbonate or ammonium carbonate.
[0024] In some examples, the reversed-phase filler is selected from a tetraalkylsilane bonded silica gel filler, an octaalkylsilane bonded silica gel filler, or an octadecylsilane bonded silica gel filler.
[0025] In some examples, the polar organic solvent is selected from at least one of methanol, ethanol, acetonitrile, or isopropanol.
[0026] The beneficial effects of the present application are:
[0027] 1. The solid-phase synthesis of Retalutide contains a large amount of trifluoroacetic acid after cleavage. The present application uses water to dissolve the crude peptide, ammonia water or aqueous sodium hydroxide solution to adjust the pH to 7.5-9.0, the dissolution volume is small, and the crude peptide concentration is high, which can reach 20-50 mg / mL, reducing the diffusion effect of the sample on the column and improving the separation capacity.
[0028] 2. The purification adopts an octaalkylsilane bonded silica gel filler, the first purification adopts an acetic acid-sodium acetate or acetic acid-ammonium acetate system, the second purification adopts ammonium carbonate, which can control the impurities of Retalutide to below 0.1%, especially the missing peptide impurities with similar polarity and main peak, missing peptide Aib 2 and missing peptide Tyr 1 -Aib 2 and missing peptide Ile 12 , missing peptide Gly 30 and other impurities, which are prone to occur in the existing synthesis process of Retalutide and are common impurities of Retalutide. More difficultly, the acetic acid-sodium acetate or acetic acid-ammonium acetate system has a good effect on the separation of racemic impurities, and the purification of single or double racemic impurities of Ser 8 , Ser 11 and Ser 39 has achieved ideal effect.
[0029] 3. The method uses gradient elution precise control method combined with subsection gradient to reduce the duration of high concentration of acetonitrile, shorten the overall preparation time, plus elution and column flushing time, the preparation completion time < 60 min, reduce the use of a large amount of acetonitrile, save the cost of dissolution while improving the purification efficiency, shorten the purification process time, in the demand of large-scale production, achieve the purpose of energy saving and environmental protection benefits.
[0030] 4. The ammonium carbonate in the secondary purification can be volatilized in the subsequent rotary evaporation and freeze-drying, and converted into a salt-free form, which improves the product quality while reducing the desalting step and shortening the purification process time period.
[0031] 5. The first purification of the method is under acidic conditions, and the second purification is under alkaline conditions, which has strong applicability and is widely used. Even under the condition that the purity of the crude peptide obtained by existing solid-phase synthesis is poor, the method can still achieve a purity of more than 99.5%, a single impurity control of 0.1% or less, and a total purification yield of more than 80%. BRIEF DESCRIPTION OF DRAWINGS
[0032] Figure 1 The chromatogram of the retahaparide freeze-dried powder collected in Example 1. DETAILED DESCRIPTION
[0033] The following disclosure provides many different embodiments or examples for implementing different aspects of the present application.
[0034] Example 1
[0035] This example provides a method for purifying crude retahaparide, and the specific steps are as follows:
[0036] 1. Sample dissolution and membrane filtration:
[0037] Dissolve 24 g of solid retahaparide crude peptide obtained by solid-phase synthesis in 800 mL of water, adjust the pH to 7.5-9.0 with ammonia water, and dissolve the crude peptide to a concentration of 30 mg / mL. After ultrasonic dissolution, filter with a 0.45 or 0.22 um water filter membrane, and collect the filtrate for use.
[0038] 2. First purification:
[0039] Purification conditions:
[0040] Chromatographic column: octylsilane-bonded silica gel as stationary phase, column diameter and length 150 mm * 250 mm.
[0041] Mobile phase A: acetic acid-sodium acetate aqueous solution, with a volume ratio of acetic acid of 2% and a concentration of sodium acetate of 100 mMol / L;
[0042] Mobile phase B phase: acetonitrile;
[0043] Flow rate: 500 mL / min;
[0044] Detection wavelength: 210 nm and 254 nm.
[0045] Equilibrium baseline: Turn on the machine, open the software and set up the preparation method, with a flow rate of 500 mL / min, 20% volume ratio of B phase to the baseline equilibrium. Start the loading program as shown in Table 1 below.
[0046] Start the gradient elution method: Open the software and set up the preparation method, start the gradient elution program as shown in Table 2 below.
[0047] Table 1
[0048] Time (min) Flow rate (mL / min) C pump 0 400 100％ 2 400 100％
[0049] Table 2 (percentage is volume ratio)
[0050] Time (min) Flow rate (mL / min) Phase A Phase B 0 500 75％ 25％ 10 500 70％ 30％ 10.1 500 68％ 32％ 45 500 7％ 33％ 45.1 500 25％ 75％ 52.1 500 25％ 75％
[0051] Collect fractions: according to the response value of the peak, start collection, after loading, carry out step gradient elution, collect the target peak, collect components with purity greater than 98% and single impurities less than 0.5%, dilute the components with equal volume of water to one time as the second purification sample.
[0052] Flush the column: after the collection of the target peak is completed, directly flush the column with 75% volume ratio of acetonitrile and 25% volume ratio of A phase for 7 min.
[0053] 3. Second purification:
[0054] The purification conditions are:
[0055] Chromatographic column: octylsilane bonded silica gel filler as stationary phase, column diameter and length is 150 mm*250 mm;
[0056] Mobile phase A phase: 50 mmol / L aqueous ammonium carbonate solution;
[0057] Mobile phase B phase: 100% acetonitrile;
[0058] Flow rate: 500 mL / min;
[0059] Detection wavelength: 210 nm and 254 nm.
[0060] Equilibrium baseline: Turn on the machine, open the software and set up the preparation method, with a flow rate of 500 mL / min, 20% B phase to the baseline equilibrium. Start the loading program as shown in Table 3 below.
[0061] Start the gradient elution method: open the software and set up the preparation method, start the gradient elution program as shown in Table 4.
[0062] Table 3
[0063] Time (min) Flow rate (mL / min) C pump 0 400 100％ 8 400 100％
[0064] Table 4 (percentage is volume percentage)
[0065] Time (min) Flow rate (mL / min) Phase A Phase B 0 500 73％ 27％ 10 500 71％ 29％ 40 500 69％ 31％ 40.1 500 25％ 75％ 47.1 500 25％ 75％
[0066] Preparation collection: according to the response value of the peak, collect the sample, after the sample is loaded, perform a step gradient elution, collect the component with purity greater than 99.5% and single impurity less than 0.1% as the secondary purification fraction.
[0067] Wash the column: after the target peak is collected, directly wash the column with 75% volume concentration of acetonitrile and 25% volume concentration of phase A for 7 min.
[0068] 4. Collect the purified component:
[0069] After the two purifications are completed, the second fraction is concentrated under reduced pressure, the component is calculated according to the standard, 50 mMol / L of sodium hydroxide solution is added, filtration is performed to remove bacteria, the qualified fractions are mixed, and the mixture is concentrated under reduced pressure at a water temperature of 30°C and a vacuum degree of -0.09 Mbar. The sample is placed in a freeze-drying tray, and freeze-drying is performed at a desorption temperature of 35°C and a vacuum pressure of 0.1 Pa, to obtain reteplase with a purity greater than 99.5% and a maximum single impurity less than 0.1% (see Table 6). Figure 1 The total purification yield is 82%.
[0070] Example 2
[0071] In this example, the volume content of the mobile phase B used in the elution of the target in the first step of purification is optimized, and the rest of the operations are the same as in Example 1.
[0072] A step gradient elution method is used to elute with four different acetonitrile proportions, and the test results are shown in Table 5. In experiment 1, the elution proportion of acetonitrile is too high, and the impurities are not fully separated before being eluted, resulting in poor purity. In experiment 4, the proportion of acetonitrile in B phase is too low, making it difficult to elute, and after the concentration is increased, the purity after elution is 80%.
[0073] Table 5 (percentage of A phase and B phase is volume percentage)
[0074] Experiment name Phase A Phase B Detection purity Experiment 1 67％-66％ 33％-34％ 91％ Experiment 2 68％-67％ 32％-33％ 98％ Experiment 3 69％-68％ 31％-32％ 97％ Experiment 4 71％-70％ 29％-30％ 80％
[0075] Example 3
[0076] The concentration of acetic acid-sodium acetate aqueous solution used in the first step of purification in this example was optimized, and the rest of the operations were the same as in Example 1. The specific optimization details are shown in Table 6 below.
[0077] Table 6
[0078] Experiment number Acetic acid volume concentration Sodium acetate molar concentration Maximum single impurity Detection purity 5 0.1％ 20 mmol / L 1.2％ 93.4％ 6 1％ 20 mmol / L 0.8％ 95.2％ 7 1％ 50 mmol / L 0.7％ 97.2％ 8 1％ 100 mmol / L 0.4％ 98.5％ 9 2％ 200 mmol / L 0.5％ 98.3％ 10 3％ 100 mmol / L 0.5％ 98.2％
[0079] According to Experiment 5 and Experiment 6, when the volume concentration of acetic acid is less than 0.1% and the molar concentration of sodium acetate is less than 20 mMol / L, the purity and separation degree of the product are relatively poor.
[0080] According to Experiment 7, 8, 9 and 10, when the volume percentage of acetic acid concentration is 1%-3% and the molar concentration of sodium acetate is 50-500 mMol / L, the product not only has good buffering capacity in acetic acid, but also has appropriate adsorption on the column, and the sample loading capacity for purification is high and the purification effect is the best.
[0081] When the concentration of sodium acetate is less than 10 mMol / L, the buffering capacity of the product in the mobile phase decreases, and the adsorption capacity of the product on the column becomes weak, resulting in poor separation effect of impurities and decreased sample loading capacity of the product.
[0082] When the concentration of sodium acetate is higher than 500 mMol / L, a high proportion of sodium acetate is difficult to dissolve in acetonitrile during column flushing, which is easy to precipitate and cause column blockage.
[0083] Example 4
[0084] In this example, the acetic acid-sodium acetate aqueous solution used in the first step of purification was optimized, and acetic acid-ammonium acetate was used to replace sodium acetate for purification. The rest of the operations were the same as in Example 1. The specific optimization details are shown in Table 7 below.
[0085] Table 7
[0086] Experiment number Acetic acid volume concentration Ammonium acetate molar concentration Maximum single impurity Detection purity 11 0.1％ 20 mmol / L 1.3％ 93.8％ 12 1％ 20 mmol / L 0.8％ 95.5％ 13 1％ 50 mmol / L 0.7％ 98.4％ 14 1％ 100 mmol / L 0.3％ 98.6％ 15 2％ 200 mmol / L 0.4％ 98.7％ 16 3％ 100 mmol / L 0.4％ 98.4％
[0087] According to Experiment 11 and Experiment 12, when the volume concentration of acetic acid is less than 0.1% and the molar concentration of ammonium acetate is less than 20 mMol / L, the purity and separation degree of the product are relatively poor.
[0088] According to Experiment 13, 14, 15 and 16, when the volume percentage of acetic acid concentration is 1%-3% and the molar concentration of ammonium acetate is 50-500 mMol / L, the product not only has good buffering capacity in acetic acid-ammonium acetate, but also has appropriate adsorption on the column, and the sample loading capacity for purification is high and the purification effect is the best. The qualified fractions and the maximum single impurities after purification in Experiment 8, 9 and Experiment 14, 15 meet the requirements, and ammonium acetate can achieve the same or similar purification effect as sodium acetate.
[0089] ammonium acetate
[0090] When the concentration of ammonium acetate is lower than 10 mMol / L, the buffering capacity of the product in the mobile phase decreases, the adsorption capacity of the product on the column becomes weak, which results in the poor separation effect of impurities and the decrease of the sample loading capacity of the product;
[0091] When the concentration of ammonium acetate is higher than 500 mMol / L, a high proportion of ammonium acetate is difficult to dissolve in acetonitrile and is easy to precipitate during the column washing, which causes the column to be blocked.
[0092] Example 5
[0093] In this example, the concentration of ammonium carbonate used in the first step of purification is optimized, and the rest of the operations are the same as those in Example 1. The specific optimization details are shown in Table 8.
[0094] The mobile phase A is ammonium carbonate with different concentrations, and the experimental results are shown in Table 7. In experiments 11 and 12, when the concentration of ammonium carbonate is too low, the buffering capacity of the product in the mobile phase decreases, the adsorption capacity of the product on the column becomes weak, which results in the poor separation effect. In experiment 15, when the concentration of ammonium carbonate is higher than 200 mMol / L, a high proportion of ammonium carbonate is difficult to dissolve in acetonitrile and is easy to precipitate during the column washing, which causes the column to be blocked, and the maximum single impurity is also larger. According to the results of experiments 13 and 14, when the concentration of ammonium carbonate is 50-100 mMol / L, the product has good buffering capacity in ammonium carbonate, high sample loading capacity, and good separation effect with impurities.
[0095] Table 8
[0096] Experiment name Ammonium carbonate molar concentration Maximum single impurity Detection purity Experiment 17 5 mMol 0.35％ 98.8％ Experiment 18 25 mMol 0.18％ 99.2％ Experiment 19 50 mMol 0.07％ 99.7％ Experiment 20 100 mMol 0.08％ 99.4％ Experiment 21 200 mMol 0.07％ 99.4％
[0097] The above are only preferred embodiments of the present application, and it should be pointed out that the above preferred embodiments should not be regarded as a limitation of the present application, and the protection scope of the present application should be limited by the scope defined by the claims. For ordinary skilled persons in the art, several improvements and refinements can be made without departing from the spirit and scope of the present application, and these improvements and refinements should also be regarded as the protection scope of the present application.
Claims
1. A method for purifying retaglutide, characterized in that: The following steps are involved: 1) Dissolve the crude retaglutide in purified water, adjust the pH to 7.5-9.0, and filter the filtrate for later use; 2) The filtrate obtained in step 1) was subjected to the first step of purification, using reverse phase filler as the stationary phase and acetic acid buffer as phase A, The polar organic solvent is phase B, and segmented gradient elution is performed to collect components with a purity greater than 98% and a single impurity less than 0.5%; 3) subjecting the fraction obtained in step 2) to a second purification step by performing stepwise gradient elution using a reverse phase filler as the stationary phase, a carbonate aqueous solution as phase A, and a polar organic solvent as phase B to collect fractions with a purity greater than 99.5% and a single impurity content less than 0.1%; 4) sterilizing and freeze-drying the components obtained in step 3) to obtain the retaglutide.
2. The purification method according to claim 1, wherein The step 2) of the step gradient elution conditions is as follows: first, the initial washing operation is: the volume percentage of mobile phase A is 75% to 70%, the volume percentage of mobile phase B is 25% to 30%, and a linear gradient elution is performed for 8-12 minutes; Then, the elution target operation is: the volume percentage of mobile phase A is 68% to 67%, the volume percentage of mobile phase B is 32% to 33%, and a linear gradient elution is performed for 33-37 minutes; Collect components with a purity greater than 98% and a single impurity less than 0.5%; After collecting the target peak, immediately increase the volume percentage of mobile phase B to above 75% and elute for 7-10 minutes to wash away the impurities on the column.
3. The purification method according to claim 1, wherein The step 3) of the step gradient elution conditions is as follows: first, the initial washing operation is: the volume percentage of mobile phase A is 73% to 71%, the volume percentage of mobile phase B is 27% to 29%, and a linear gradient elution is performed for 8-12 minutes; Then, the elution target operation is: the volume percentage of mobile phase A is 71% to 69%, the volume percentage of mobile phase B is 29% to 31%, and a linear gradient elution is performed for 28-32 minutes to collect components with a purity greater than 99.5% and a single impurity less than 0.1%; After collecting the target peak, immediately increase the volume percentage of mobile phase B to above 75% and elute for 7-10 minutes to wash away the impurities on the column.
4. The method according to claim 1, wherein In the step 2), the acetic acid buffer is an acetic acid aqueous solution-acetate aqueous solution, and the acetate is sodium acetate or ammonium acetate.
5. The method according to claim 1 or 4, characterized in that In the step 2), the volume percentage of acetic acid in the acetic acid aqueous solution in the mobile phase A is 0.1%-5.0%.
6. The method according to claim 1, characterized in that The concentration of acetate in the acetate aqueous solution of the mobile phase A in step 2) is 20-500 mMol / L.
7. The method according to claim 1, characterized in that The concentration of the carbonate aqueous solution in step 3) is 20-500 mMol / L.
8. The method according to claim 6, characterized in that The concentration of the carbonate aqueous solution in step 3) is 50-200 mMol / L.
9. The method according to claim 1, characterized in that In step 3), the carbonate is selected from ammonium bicarbonate or ammonium carbonate.
10. The method according to claim 1, characterized in that The polar organic solvent is selected from at least one of methanol, ethanol, acetonitrile or isopropanol.