Method for preparing lutetium ( 177lu) oxodotreotide and method for purifying precursor compound thereof

Abstract

Provided is a method for purifying a lutetium (177Lu) oxodotreotide precursor compound, comprising the following steps: (1) dissolving a crude product of a lutetium (177Lu) oxodotreotide precursor compound in a mixed solvent of an organic solvent and water, adjusting a pH value to 2.00-4.00, then adjusting the pH value to 7.08-7.91, and filtering; (2) purifying the filtrate obtained in step (1) by means of high-performance liquid chromatography, wherein octadecylsilyl silica gel is used as a chromatographic column packing material, an aqueous solution of KH2PO4 and K2HPO4 with a total concentration of 9-11 mM and a molar ratio of 1:9 is used as mobile phase A, and acetonitrile is used as mobile phase B, and performing gradient elution. The described method significantly increases the purity of lutetium (177Lu) oxodotreotide precursor compounds, and reduces individual impurity content in products.

Description

A type of lutetium 177 Preparation method of Lu-oxyoctreotide and purification method of its precursor compound Technical Field

[0001] This application belongs to the field of pharmaceutical technology. Specifically, this application relates to a lutetium [ 177 Preparation method of Lu]oxyoctreotide and purification method of its precursor compound. Background Technology

[0002] In 2018, the U.S. FDA approved lutetium from Novartis subsidiary Advanced Accelerator Applications of France. 177 Lutathera, an oxooctreotide, has been marketed for the treatment of SSTR-positive gastrointestinal pancreatic neuroendocrine tumors (GEP-NETs) that have progressed after standard therapy. 177 Lu]Octrete, the first approved radiopharmaceutical for radioligand therapy (RLT), reaches tumor cells by specifically binding to the growth hormone-inhibiting receptor on the surface of tumor cells and releasing radionuclides to generate radiation that kills tumor cells. Recent clinical trial results also indicate that its clinical application scope may be further expanded.

[0003] lute 177 Lu]Oxyoctreotide is obtained through radionuclide [ 177 Lu was prepared by chelating with a precursor compound (MS011) of Formula I as a chelating agent, which is composed of a polypeptide linked to the chelating agent DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) consisting of eight amino acids. The octardate ligand formed by the four nitrogen atoms on the dodecane heterocycle plus the four acetic acid groups attached to the nitrogen atoms provides electrons, thereby chelating with the centrally located lutetium [ 177 Lu atoms form stable chelates.

[0004] The industrial production of radionuclide-conjugated drugs places high demands on the yield and product purity of precursor compound preparation methods, as impurities can severely affect the subsequent radionuclide labeling process. Existing lutetium [ 177 The preparation methods for Lu]oxooctreotide precursor compounds generally suffer from low yields and low product purity, which hinders the realization of lutetium [ 177 The efficient and large-scale production of lutetium oxyoctreotide raw material poses a significant challenge to meet clinical needs. Therefore, improving lutetium oxyoctreotide... 177The purification process of Lu]oxooctreotide precursor compounds has become one of the urgent technical problems to be solved in the pharmaceutical field. Summary of the Invention

[0005] In view of this, the purpose of this application is to provide a lutetium [ 177 Purification method of Lu]O-octreotide precursor compound, a lutetium [ 177 A method for preparing lutetium-oxyoctreotide, the purification method of which can significantly improve the efficiency of lutetium-oxyoctreotide. 177 To improve the purity of the Lu]oxooctreotide precursor compound and reduce impurity content, for lutetium [ 177 This laid the foundation for the large-scale industrial production and clinical application of Lu]oxyoctreotide.

[0006] The above-mentioned objective of this application is achieved through the following technical solution:

[0007] A type of lutetium shown in Formula I [ 177 A purification method for the Lu]oxooctreotide precursor compound includes the following steps:

[0008] (1) The lutetium shown in Formula I [ 177 The crude Lu]oxooctreotide precursor compound was dissolved in a mixed solvent of organic solvent and water. The pH of the resulting solution was adjusted to 2.00–4.00, and then further adjusted to 7.08–7.91. The solution was then filtered.

[0009] (2) Purify the filtrate obtained in step (1) by high performance liquid chromatography (HPLC), wherein the HPLC conditions include: using octadecylsilane-bonded silica gel as the column packing material; using an aqueous solution of KH₂PO₄ and K₂HPO₄ with a total concentration of 9–11 mM, preferably 10 mM, in a molar ratio of 1:9 as mobile phase A, and acetonitrile as mobile phase B, and eluting according to the following gradient:

[0010] From 0 to 5 minutes, the volume ratio of mobile phase A to mobile phase B is 95:5;

[0011] For 5 to 95 minutes, the volume ratio of mobile phase A to mobile phase B is 87 to 89:13 to 11, preferably 88:12;

[0012] After 95 minutes, the volume ratio of mobile phase A to mobile phase B is 67-69:33-31, preferably 68:32;

[0013] According to some embodiments of this application, in step (1), the mixed solvent of the organic solvent and water is a mixed solvent of acetonitrile, DMSO and water; preferably, the volume ratio of acetonitrile, DMSO and water is 1:2:7; preferably, the lutetium [represented by Formula I] in the obtained solution... 177The concentration of the Lu]oxooctreotide precursor compound is 25–35 g / L, preferably 30 g / L.

[0014] According to some embodiments of this application, in step (1), the pH of the obtained solution is adjusted to 2.00-4.00 at 20-30°C, stirred for 1.5-5 hours, then adjusted to 7.10-7.90, stirred for 2-18 hours, and an equal weight of 10% acetonitrile aqueous solution is added and filtered; preferably, 5% ammonia solution is used to adjust the pH; preferably, a 0.45μm nylon filter membrane is used for filtration.

[0015] According to some embodiments of this application, in step (2), the chromatographic column packing is UniSil 10-100 C18 packing.

[0016] According to some embodiments of this application, in step (2), the sample loading of the chromatographic column is 16-18g.

[0017] According to some embodiments of this application, in step (2), the pH of the mobile phase A is 7.77 to 7.97, preferably 7.87.

[0018] According to some embodiments of this application, in step (2), the detection wavelength of the high performance liquid chromatography is 230 nm.

[0019] According to some embodiments of this application, in step (2), the elution flow rate of the mobile phase is 880 mL / min.

[0020] According to some embodiments of this application, in step (2), a fraction with a purity ≥99.0% and a single impurity ≤0.10% by weight is collected. For a fraction with a purity ≥95% and ≤99.0% and a single impurity >0.10% by weight, the high-performance liquid chromatography (HPLC) secondary purification is repeated to obtain a fraction with a purity ≥99.0% and a single impurity ≤0.10% by weight. Preferably, the fraction with a purity ≥95% and ≤99.0% and a single impurity >0.10% by weight is diluted with an equal weight of water before the secondary purification. Preferably, the sample loading of the chromatographic column for the secondary purification is ≤11.6g.

[0021] According to some embodiments of this application, the purification method further includes the following steps:

[0022] (3) The fraction obtained in step (2) is concentrated by salt conversion using high performance liquid chromatography (HPLC), wherein the HPLC conditions include: using octadecylsilane-bonded silica gel as the column packing material; using a 0.1% (v / v) trifluoroacetic acid aqueous solution as mobile phase A; using acetonitrile as mobile phase B; and eluting according to the following gradient:

[0023] From 0 to 25 minutes, the volume ratio of mobile phase A to mobile phase B is 95:5;

[0024] For 25 to 75 minutes, the volume ratio of mobile phase A to mobile phase B is 80:20;

[0025] After 75 minutes, the volume ratio of mobile phase A to mobile phase B was 30:70.

[0026] According to some embodiments of this application, in step (3), the chromatographic column packing is UniSil 10-100 C18 packing.

[0027] According to some embodiments of this application, in step (3), the sample loading of the chromatographic column is ≤42.6g.

[0028] According to some embodiments of this application, in step (3), the detection wavelength of the high performance liquid chromatography is 230 nm.

[0029] According to some embodiments of this application, in step (3), the elution flow rate of the mobile phase is 880 mL / min.

[0030] According to some embodiments of this application, in step (3), the fraction obtained in step (2) is diluted with an equal weight of water before salt concentration.

[0031] According to some embodiments of this application, in step (3), a fraction with a purity ≥99.0% and a single impurity ≤0.10% by weight is collected.

[0032] According to some embodiments of this application, the purification method further includes the following steps:

[0033] (4) Freeze-dry the fraction obtained in step (3).

[0034] According to some embodiments of this application, in step (4), the freeze-drying includes the following steps:

[0035] (4-1) Pre-freezing: Cool to -45 to -55°C within 30 minutes, preferably -45°C, and hold for 210 minutes;

[0036] (4-2) Sublimation: Under a vacuum of 0.2 to 0.25 mbar, preferably 0.2 mbar, the temperature is raised to -10 to -15°C, preferably -10°C, within 30 minutes and held for 510 to 1600 minutes, preferably 1600 minutes; then the temperature is raised to 5°C within 30 minutes and held for 1470 to 1500 minutes, preferably 1500 minutes;

[0037] (4-3) Desorption drying: Under a vacuum of 0.01 mbar, the temperature is raised to 25°C within 30 minutes and maintained for 790 to 2400 minutes, preferably 790 to 800 minutes.

[0038] According to some embodiments of this application, in step (4), the thickness of the freeze-dried fraction is 8 to 12 mm.

[0039] According to some embodiments of this application, in step (4), the lutetium [represented by formula I] in the fraction before freeze-drying 177 The concentration of the Lu]oxooctreotide precursor compound is ≤37.6 mg / mL.

[0040] According to some embodiments of this application, the purification method further includes the following steps:

[0041] (5) The freeze-dried fraction obtained in step (4) is dispensed; preferably, the dispensing is carried out at a humidity of ≤30%.

[0042] In addition, this application also provides a lutetium [ 177 A method for preparing lutetium oxorlene according to Formula I of this application. 177 The purification method of the Lu]oxooctreotide precursor compound yielded lutetium of formula I. 177 Lu]Oxyoctreotide precursor compound and lutetium[ 177 Lu] chelation.

[0043] This application has at least the following beneficial effects:

[0044] This application provides an optimized lutetium [ 177 A purification method for the Lu]oxooctreotide precursor compound was developed, which further reduced the content of individual impurities in the crude precursor compound and improved the purity of the precursor compound product through HPLC purification, resulting in an individual impurity content ≤0.10% by weight and a product purity ≥99.0%, which is lutetium [ 177 This laid the foundation for the large-scale industrial production and clinical application of Lu]oxyoctreotide. Attached Figure Description

[0045] The embodiments of this application are described in detail below with reference to the accompanying drawings, wherein:

[0046] Figure 1 shows the lutetium prepared in Example 1. 177 NMR spectrum of MS011, a precursor compound of Lu-oxy-octreotide;

[0047] Figure 2 shows the lutetium prepared in Example 1. 177 LC-MS spectrum of Lu]oxooctreotide precursor compound MS011;

[0048] Figure 3 shows the lutetium prepared in Example 1.177 Infrared spectrum of MS011, a precursor compound of Lu-oxy-octreotide;

[0049] Figures 4-6 show the lutetium [in Example 2] 177 HPLC chromatogram of crude solution of Lu]oxooctreotide precursor compound MS011;

[0050] Figures 7-9 show the lutetium [in Example 2] 177 HPLC chromatogram of the first purified fraction (MP) of the Lu]oxooctreotide precursor compound MS011;

[0051] Figures 10-12 show the lutetium [in Example 2] 177 HPLC chromatogram of the primary purified recovery buffer (RP) of the Lu]oxooctreotide precursor compound MS011;

[0052] Figures 13-15 show the lutetium [in Example 2] 177 HPLC chromatogram of the recovered fraction of the Lu]oxooctreotide precursor compound MS011 after primary purification;

[0053] Figures 16-18 show the lutetium [in Example 2] 177 HPLC chromatogram of the major fraction (combined MP) of the Lu]oxooctreotide precursor compound MS011 after one-time purification;

[0054] Figures 19-21 show the lutetium [in Example 2] 177 HPLC chromatogram of the collected solution of Lu]oxyoctreotide precursor compound MS011 after salt concentration. Detailed Implementation

[0055] The present application will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present application and are not intended to limit the scope of the present application.

[0056] Unless otherwise specified, the experimental methods used in the following examples and comparative examples are conventional methods. Unless otherwise specified, the reagents used in the following examples and comparative examples are commercially available products.

[0057] Abbreviation Table

[0058] Example 1: Lutetium [ 177 Preparation of Lu]Oxyoctreotide precursor compounds

[0059] 1. Lutetium 177 Synthetic route of Lu]O-octreotide precursor compound (MS011)

[0060] Key materials: Wang Resin, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-D-Phe-OH, DOTA-(t Bu)3-CO2H

[0061] Reagents: PyBOP, HOBt, DIEA, Ac2O, DIC, TFA, DODT, Tis, water, piperidine, iodine, vitamin C

[0062] Solvents: DMF, MeOH, DCM, MTBE, ACN

[0063] 2. Synthesis process of MS011

[0064] 2.1 Coupling of the first amino acid Fmoc-Thr(tBu)-OH

[0065] Using blank Wang Resin (Xi'an Lanxiao Technology Co., Ltd., crosslinking degree of 1%, mesh size of 100-200) with a substitution degree of 1.0-1.2 mmol / g, under nitrogen protection, 300±1g of Wang Resin (1eq) and 3.0±0.1L of DMF (10vol, v / w) were added sequentially to the synthesis vessel R-2105. The mixture was stirred under nitrogen bubbling for 30-35 minutes, then stirring was stopped and the solvent in the vessel was emptied.

[0066] Add 2.1 ± 0.1 L DMF (7 vol, v / w) to the synthesis vessel R-2105, bubble and stir with nitrogen for 3–5 minutes, and then purge the solvent; repeat this step once more.

[0067] Under nitrogen protection, 562.8±1g Fmoc-Thr(tBu)-OH (4.0eq), 736.9±1g PyBOP (4.0eq), 191.3±1g HOBT (4.0eq), and 0.82±0.01Kg DMF were added sequentially to the activation vessel R-0304, stirred to dissolve, and then cooled to 5±5℃.

[0068] Control the internal temperature of activation tank R-0304 to 0~10℃, add 366.0±1g DIEA (8.0eq) dropwise to activation tank R-0304, and stir for 3~5 minutes after the addition is completed.

[0069] The solution in the activation tank R-0304 was added dropwise to the synthesis tank R-2105, and the activation tank R-0304 was rinsed with 0.20±0.01Kg DMF. The rinsing solution was then transferred into the synthesis tank R-2105.

[0070] The internal temperature of the synthesis tank R-2105 was controlled at 25-30℃, and the reaction was maintained at this temperature for 2 hours. After the reaction was completed, the solvent in the synthesis tank R-2105 was emptied.

[0071] Add 6.0±0.1L of DMF to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Add 2.1±0.1L of DMF (7vol, v / w) to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step once. Add 2.1±0.1L of DMF (7vol, v / w) to the synthesis tank R-2105 and spray. Wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step twice.

[0072] Under nitrogen protection, add 0.85±0.1Kg DMF, 722.8±1g Ac2O (20eq), and 91.5±1g DIEA (2.0eq) sequentially to the activation tank R-0304, and stir for 5-10 minutes.

[0073] Under nitrogen protection, the solution in the activation tank R-0304 is transferred to the synthesis tank R-2105. The internal temperature of the synthesis tank R-2105 is controlled at 20-30℃, and the reaction is maintained for 15-20 hours. After the heat preservation is completed, the solvent in the synthesis tank R-2105 is emptied.

[0074] Add 6.0±0.1L of DMF to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Add 2.1±0.1L of DMF (7vol, v / w) to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step once. Add 2.1±0.1L of DMF (7vol, v / w) to the synthesis tank R-2105 and spray. Wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step twice.

[0075] Add 2.78±0.1 kg DCM (7 vol, v / w) to synthesis vessel R-2105, bubble and stir under nitrogen for 3–5 minutes, and drain the solvent; add 1.55±0.1 kg MTBE (7 vol, v / w), bubble and stir under nitrogen for 3–5 minutes, and drain the solvent; add 2.78±0.1 kg DCM (7 vol, v / w), bubble and stir under nitrogen for 3–5 minutes, and drain the solvent; add 1.55±0.1 kg MTBE (7 vol, v / w), bubble and stir under nitrogen for 3–5 minutes, and drain the solvent. Repeat this step 3 times.

[0076] Remove Fmoc-Thr(tBu)-Wang Resin from synthesis tank R-2105 and transfer it to vacuum drying oven D-2101. Control the jacket temperature at 30-35℃ and the vacuum degree at ≤-0.08MPa. Dry for 10 hours, turning the material over every 3-4 hours. Weigh the material starting from the 10th hour, then weigh it every 2 hours until the first process control test (In-Process Control 01, IPC01) shows a loss on drying (LOD) ≤10%. Discharge the material, pack it into double-layer LDPE bags, weigh it, and label it. Take samples for intermediate control and test the degree of substitution (Substitution = 0.4-0.7 mmol / g). During the testing period, the material should be refrigerated at 2-8℃.

[0077] In the above operation steps 2.1, the material feeding amount is calculated based on the resin; the stirring speed under nitrogen bubbling is set to 100 rpm.

[0078] In the following operating steps 2.2 to 2.10, the material feed rate is calculated based on Fmoc-Thr(tBu)-Wang Resin; the stirring speed under nitrogen bubbling is set to 100 rpm.

[0079] 2.2 Fmoc-Cys(Trt)-OH Coupling

[0080] Under nitrogen protection, 320±10g Fmoc-Thr(tBu)-Wang Resin(1eq) and 3.2±0.1L DMF (10vol, v / w) were added sequentially to the synthesis vessel R-2105. The mixture was stirred under nitrogen bubbling for 30-35 minutes, then the stirring was stopped and the solvent in the vessel was emptied.

[0081] Add 2.24±0.1L DMF (7vol, v / w) to the synthesis vessel R-2105, bubble and stir with nitrogen for 3-5 minutes, and drain the solvent; repeat this step once more.

[0082] Under nitrogen protection, add 6.98±0.1Kg PIP and 30.56±0.1Kg DMF to buffer tank R-2107 in sequence, stir well, and set aside for use (total amount for 8 uses).

[0083] Release approximately 2.08 ± 0.1 kg (6.5 vol, w / w) from buffer tank R-2107 into PP tank. Control the internal temperature of synthesis tank R-2105 at 20–30 °C. Add the solution from PP tank into synthesis tank R-2105. Bubble and stir with nitrogen for 20 minutes. Empty the solvent from synthesis tank R-2105. Repeat this step once more.

[0084] Add 2.24±0.1L DMF (7vol, v / w) to the synthesis tank R-2105, bubble and stir with nitrogen for 3-5 minutes, and drain the solvent; repeat this step 5 times (take a sample for Kaiser test, the resin solution should be blue).

[0085] Under nitrogen protection, 307.5±1g Fmoc-Cys(Trt)-OH (3.0eq), 74.5±1g HOBT (3.15eq), and 0.76±0.1Kg DMF were added sequentially to the activation vessel R-0304, stirred to dissolve, and then cooled to 5±5℃.

[0086] Control the internal temperature of the activation vessel R-0304 to 0-10℃, add 69.6±1g DIC (3.15eq) dropwise to the activation vessel R-0304, and stir for 5-10 minutes after the addition is completed.

[0087] The solution in the activation tank R-0304 was added dropwise to the synthesis tank R-2105, and the activation tank R-0304 was rinsed with 0.20±0.1Kg DMF. The rinsing solution was then transferred to the synthesis tank R-2105. After the addition was completed, the temperature inside the synthesis tank R-2105 was controlled at 20-30℃, nitrogen was bubbled, and the mixture was stirred for 2-3 hours (a sample was taken for Kaiser analysis; the resin and solution were colorless and transparent; ninhydrin was used for testing every hour until a negative result was obtained). After the reaction was completed, the solvent in the synthesis tank R-2105 was emptied.

[0088] Add 6.0±0.1L of DMF to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Add 2.24±0.1L of DMF (7vol, v / w) to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step once. Add 2.24±0.1L of DMF (7vol, v / w) to the synthesis tank R-2105 and spray. Wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step twice.

[0089] 2.3 Fmoc-Thr(tBu)-OH Coupling

[0090] Release approximately 2.08 ± 0.1 kg (6.5 vol, w / w) from buffer tank R-2107 into PP tank. Control the internal temperature of synthesis tank R-2105 at 20–30 °C. Add the solution from PP tank into synthesis tank R-2105. Stir with nitrogen bubbling for 20 minutes. Empty the solvent from synthesis tank R-2105. Repeat this step once more.

[0091] Add 2.24±0.1L DMF (7vol, v / w) to the synthesis tank R-2105, bubble and stir with nitrogen for 3-5 minutes, and drain the solvent; repeat this step 5 times (take a sample for Kaiser test, the resin solution should be blue).

[0092] Under nitrogen protection, 208.7±1g Fmoc-Thr(tBu)-OH (3.0eq), 74.5±1g HOBT (3.15eq), and 0.56±0.1Kg DMF were added sequentially to the activation vessel R-0304, stirred to dissolve, and then cooled to 5±5℃.

[0093] Control the internal temperature of the activation vessel R-0304 to 0-10℃, add 69.6±1g DIC (3.15eq) dropwise to the activation vessel R-0304, and stir for 5-10 minutes after the addition is completed.

[0094] The solution in the activation tank R-0304 was added dropwise to the synthesis tank R-2105, and the activation tank R-0304 was rinsed with 0.20±0.1Kg DMF. The rinsing solution was then transferred to the synthesis tank R-2105. After the addition was completed, the temperature inside the synthesis tank R-2105 was controlled at 20-30℃, nitrogen was bubbled, and the mixture was stirred for 2-3 hours (a sample was taken for Kaiser analysis; the resin and solution were colorless and transparent; ninhydrin was used for testing every hour until a negative result was obtained). After the reaction was completed, the solvent in the synthesis tank R-2105 was emptied.

[0095] Add 6.0±0.1L of DMF to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Add 2.24±0.1L of DMF (7vol, v / w) to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step once. Add 2.24±0.1L of DMF (7vol, v / w) to the synthesis tank R-2105 and spray. Wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step twice.

[0096] 2.4 Fmoc-Lys(Boc)-OH Coupling

[0097] Release approximately 2.08 ± 0.1 kg (6.5 vol, w / w) from buffer tank R-2107 into PP tank. Control the internal temperature of synthesis tank R-2105 at 20–30 °C. Add the solution from PP tank into synthesis tank R-2105. Stir with nitrogen bubbling for 20 minutes. Empty the solvent from synthesis tank R-2105. Repeat this step once more.

[0098] Add 2.24±0.1L DMF (7vol, v / w) to the synthesis tank R-2105, bubble and stir with nitrogen for 3-5 minutes, and drain the solvent; repeat this step 5 times (take a sample for Kaiser test, the resin solution should be blue).

[0099] Under nitrogen protection, 246±1g Fmoc-Lys(Boc)-OH (3.0eq), 74.5±1g HOBT (3.15eq), and 0.56±0.1Kg DMF were added sequentially to the activation vessel R-0304, stirred to dissolve, and then cooled to 5±5℃.

[0100] Control the internal temperature of the activation vessel R-0304 to 0-10℃, add 69.6±1g DIC (3.15eq) dropwise to the activation vessel R-0304, and stir for 5-10 minutes after the addition is completed.

[0101] The solution in the activation tank R-0304 was added dropwise to the synthesis tank R-2105, and the activation tank R-0304 was rinsed with 0.20±0.1Kg DMF. The rinsing solution was then transferred to the synthesis tank R-2105. After the addition was completed, the temperature inside the synthesis tank R-2105 was controlled at 20-30℃, nitrogen was bubbled, and the mixture was stirred for 2-3 hours (a sample was taken for Kaiser analysis; the resin and solution were colorless and transparent; ninhydrin was used for testing every hour until a negative result was obtained). After the reaction was completed, the solvent in the synthesis tank R-2105 was emptied.

[0102] Add 6.0±0.1L of DMF to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3–5 minutes. Drain the solvent. Add 2.24±0.1L of DMF (7vol, v / w) to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3–5 minutes. Drain the solvent. Repeat this step once. Add 2.24±0.1L of DMF (7vol, v / w) to the synthesis tank R-2105 and spray. Wash with nitrogen bubbling and stirring for 3–5 minutes. Drain the solvent. Repeat this step twice.

[0103] 2.5 Fmoc-D-Trp(Boc)-OH Coupling

[0104] Release approximately 2.08 ± 0.1 kg (6.5 vol, w / w) from buffer tank R-2107 into PP tank. Control the internal temperature of synthesis tank R-2105 at 20–30 °C. Add the solution from PP tank into synthesis tank R-2105. Bubble and stir with nitrogen for 20 minutes. Empty the solvent from synthesis tank R-2105. Repeat this step once more.

[0105] Add 2.24±0.1L DMF (7vol, v / w) to the synthesis tank R-2105, bubble and stir with nitrogen for 3-5 minutes, and drain the solvent; repeat this step 5 times (take a sample for Kaiser test, the resin solution should be blue).

[0106] Under nitrogen protection, 276.5±1g Fmoc-D-Trp(Boc)-OH (3.0eq), 74.5±1g HOBT (3.15eq), and 0.62±0.1Kg DMF were added sequentially to the activation vessel R-0304, stirred to dissolve, and then cooled to 5±5℃.

[0107] Control the internal temperature of the activation vessel R-0304 to 0-10℃, add 69.6±1g DIC (3.15eq) dropwise to the activation vessel R-0304, and stir for 5-10 minutes after the addition is completed.

[0108] The solution in the activation tank R-0304 was added dropwise to the synthesis tank R-2105, and the activation tank R-0304 was rinsed with 0.20±0.1Kg DMF. The rinsing solution was then transferred to the synthesis tank R-2105. After the addition was completed, the temperature inside the synthesis tank R-2105 was controlled at 20-30℃, nitrogen was bubbled, and the mixture was stirred for 2-3 hours (a sample was taken for Kaiser analysis; the resin and solution were colorless and transparent; ninhydrin was used for testing every hour until a negative result was obtained). After the reaction was completed, the solvent in the synthesis tank R-2105 was emptied.

[0109] Add 6.0±0.1L of DMF to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Add 2.24±0.1L of DMF (7vol, v / w) to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step once. Add 2.24±0.1L of DMF (7vol, v / w) to the synthesis tank R-2105 and spray. Wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step twice.

[0110] 2.6 Fmoc-Tyr(tBu)-OH Coupling

[0111] Release approximately 2.08 ± 0.1 kg (6.5 vol, w / w) from buffer tank R-2107 into PP tank. Control the internal temperature of synthesis tank R-2105 at 20–30 °C. Add the solution from PP tank into synthesis tank R-2105. Bubble and stir with nitrogen for 20 minutes. Empty the solvent from synthesis tank R-2105. Repeat this step once more.

[0112] Add 2.24±0.1L DMF (7vol, v / w) to the synthesis tank R-2105, bubble and stir with nitrogen for 3-5 minutes, and drain the solvent; repeat this step 5 times (take a sample for Kaiser test, the resin solution should be blue).

[0113] Under nitrogen protection, 241.3±1g Fmoc-Tyr(tBu)-OH (3.0eq), 74.5±1g HOBT (3.15eq), and 0.62±0.1Kg DMF were added sequentially to the activation vessel R-0304, stirred to dissolve, and then cooled to 5±5℃.

[0114] Control the internal temperature of the activation vessel R-0304 to 0-10℃, add 69.6±1g DIC (3.15eq) dropwise to the activation vessel R-0304, and stir for 5-10 minutes after the addition is completed.

[0115] The solution in the activation tank R-0304 was added dropwise to the synthesis tank R-2105, and the activation tank R-0304 was rinsed with 0.20±0.1Kg DMF. The rinsing solution was then transferred to the synthesis tank R-2105. After the addition was completed, the temperature inside the synthesis tank R-2105 was controlled at 20-30℃, nitrogen was bubbled, and the mixture was stirred for 2-3 hours (a sample was taken for Kaiser analysis; the resin and solution were colorless and transparent; ninhydrin was used for testing every hour until a negative result was obtained). After the reaction was completed, the solvent in the synthesis tank R-2105 was emptied.

[0116] Add 6.0±0.1L of DMF to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Add 2.24±0.1L of DMF (7vol, v / w) to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step once. Add 2.24±0.1L of DMF (7vol, v / w) to the synthesis tank R-2105 and spray. Wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step twice.

[0117] 2.7 Fmoc-Cys(Trt)-OH Coupling

[0118] Release approximately 2.08 ± 0.1 kg (6.5 vol, w / w) from buffer tank R-2107 into PP tank. Control the internal temperature of synthesis tank R-2105 at 20–30 °C. Add the solution from PP tank into synthesis tank R-2105. Bubble and stir with nitrogen for 20 minutes. Empty the solvent from synthesis tank R-2105. Repeat this step once more.

[0119] Add 2.24±0.1L DMF (7vol, v / w) to the synthesis tank R-2105, bubble and stir with nitrogen for 3-5 minutes, and drain the solvent; repeat this step 5 times (take a sample for Kaiser test, the resin solution should be blue).

[0120] Under nitrogen protection, 307.5±1g Fmoc-Cys(Trt)-OH (3.0eq), 74.5±1g HOBT (3.15eq), and 0.62±0.1L DMF were added sequentially to the activation vessel R-0304, stirred to dissolve, and then cooled to 5±5℃.

[0121] Control the internal temperature of the activation vessel R-0304 to 0-10℃, add 69.6±1g DIC (3.15eq) dropwise to the activation vessel R-0304, and stir for 5-10 minutes after the addition is completed.

[0122] The solution in the activation tank R-0304 was added dropwise to the synthesis tank R-2105, and the activation tank R-0304 was rinsed with 0.20±0.1Kg DMF. The rinsing solution was then transferred to the synthesis tank R-2105. After the addition was completed, the temperature inside the synthesis tank R-2105 was controlled at 20-30℃, nitrogen was bubbled, and the mixture was stirred for 2-3 hours (a sample was taken for Kaiser analysis; the resin and solution were colorless and transparent; ninhydrin was used for testing every hour until a negative result was obtained). After the reaction was completed, the solvent in the synthesis tank R-2105 was emptied.

[0123] Add 6.0±0.1L of DMF to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Add 2.24±0.1L of DMF (7vol, v / w) to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step once. Add 2.24±0.1L of DMF (7vol, v / w) to the synthesis tank R-2105 and spray. Wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step twice.

[0124] 2.8 Fmoc-D-Phe-OH Coupling

[0125] Release approximately 2.08 ± 0.1 kg (6.5 vol, w / w) from buffer tank R-2107 into PP tank. Control the internal temperature of synthesis tank R-2105 at 20–30 °C. Add the solution from PP tank into synthesis tank R-2105. Bubble and stir with nitrogen for 20 minutes. Empty the solvent from synthesis tank R-2105. Repeat this step once more.

[0126] Add 2.24±0.1L DMF (7vol, v / w) to the synthesis tank R-2105, bubble and stir with nitrogen for 3-5 minutes, and drain the solvent; repeat this step 5 times (take a sample for Kaiser test, the resin solution should be blue).

[0127] Under nitrogen protection, 203.4±1g Fmoc-D-Phe-OH (3.0eq), 74.5±1g HOBT (3.15eq), and 0.62±0.1L DMF were added sequentially to the activation vessel R-0304, stirred to dissolve, and then cooled to 5±5℃.

[0128] Control the internal temperature of the activation vessel R-0304 to 0-10℃, add 69.6±1g DIC (3.15eq) dropwise to the activation vessel R-0304, and stir for 5-10 minutes after the addition is completed.

[0129] The solution in the activation tank R-0304 was added dropwise to the synthesis tank R-2105, and the activation tank R-0304 was rinsed with 0.20±0.1Kg DMF. The rinsing solution was then transferred to the synthesis tank R-2105. After the addition was completed, the temperature inside the synthesis tank R-2105 was controlled at 20-30℃, nitrogen was bubbled, and the mixture was stirred for 2-3 hours (a sample was taken for Kaiser analysis; the resin and solution were colorless and transparent; ninhydrin was used for testing every hour until a negative result was obtained). After the reaction was completed, the solvent in the synthesis tank R-2105 was emptied.

[0130] Add 6.0±0.1L of DMF to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Add 2.24±0.1L of DMF (7vol, v / w) to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step once. Add 2.24±0.1L of DMF (7vol, v / w) to the synthesis tank R-2105 and spray. Wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step twice.

[0131] 2.9 DOTA-(tBu)3-CO2H Coupling

[0132] Release approximately 2.08 ± 0.1 kg (6.5 vol, w / w) from buffer tank R-2107 into PP tank. Control the internal temperature of synthesis tank R-2105 at 20–30 °C. Add the solution from PP tank into synthesis tank R-2105. Bubble and stir with nitrogen for 20 minutes. Empty the solvent from synthesis tank R-2105. Repeat this step once more.

[0133] Add 2.24±0.1L DMF (7vol, v / w) to the synthesis tank R-2105, bubble and stir with nitrogen for 3-5 minutes, and drain the solvent; repeat this step 5 times (take a sample for Kaiser test, the resin solution should be blue).

[0134] Under nitrogen protection, 200.5±1g DOTA-(tBu)3-CO2H (2.0eq), 47.3±1g HOBT (2.0eq), 182.1±1g PyBOP (2.0eq), and 0.62±0.1Kg DMF were added sequentially to the activation vessel R-0304, stirred to dissolve, and then cooled to 5±5℃.

[0135] Control the internal temperature of activation tank R-0304 to 0~10℃, add 90.5±1g DIEA (4.0eq) dropwise to activation tank R-0304, and stir for 5~10 minutes after the addition is completed.

[0136] The solution in the activation tank R-0304 was added dropwise to the synthesis tank R-2105, and the activation tank R-0304 was rinsed with 0.20±0.1Kg DMF. The rinsing solution was then transferred to the synthesis tank R-2105. After the addition was completed, the temperature inside the synthesis tank R-2105 was controlled at 20-30℃, nitrogen was bubbled, and the mixture was stirred for 2-3 hours (a sample was taken for Kaiser analysis; the resin and solution were colorless and transparent; ninhydrin was used for testing every hour until a negative result was obtained). After the reaction was completed, the solvent in the synthesis tank R-2105 was emptied.

[0137] Add 6.0±0.1L of DMF to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Add 2.24±0.1L of DMF (7vol, v / w) to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step once. Add 2.24±0.1L of DMF (7vol, v / w) to the synthesis tank R-2105 and spray. Wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step twice.

[0138] 2.10 Solid-phase cyclization

[0139] Under nitrogen protection, 3.3±0.1Kg DMF and 177.7±1g iodine (4.0eq) were added sequentially to the activation vessel R-0304 and stirred until homogeneous.

[0140] The solution in the activation tank R-0304 was added dropwise to the synthesis tank R-2105. The temperature inside the synthesis tank R-2105 was controlled at 27-32℃, and the reaction was maintained at this temperature for 40±5 minutes. The solvent in the synthesis tank R-2105 was then drained.

[0141] Add 6.0±0.1L of DMF to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Add 2.24±0.1L of DMF (7vol, v / w) to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step once. Add 2.24±0.1L of DMF (7vol, v / w) to the synthesis tank R-2105 and spray. Wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step twice.

[0142] Under nitrogen protection, 184.9±1g vitamin C (3.0eq), 0.99±0.05Kg methanol, and 5.9±0.1Kg DMF were added sequentially to the activation vessel R-0304 and stirred until dissolved.

[0143] Control the internal temperature of synthesis tank R-2105 at 20-30℃, add half of the solution (about 2.24±0.05L) from activation tank R-0304 into synthesis tank R-2105, keep warm and stir for 10 minutes, and then drain the solvent from synthesis tank R-2105.

[0144] Control the internal temperature of synthesis tank R-2105 at 20-30℃, add the remaining solution (approximately 2.24±0.05L) in activation tank R-0304 to synthesis tank R-2105, keep warm and stir for 10 minutes, and then drain the solvent from synthesis tank R-2105.

[0145] Add 6.0±0.1L of DMF to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Add 2.24±0.1L of DMF (7vol, v / w) to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step once. Add 2.24±0.1L of DMF (7vol, v / w) to the synthesis tank R-2105 and spray. Wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step twice.

[0146] Add 2.98±0.2 kg DCM (7 vol, v / w) to synthesis vessel R-2105, bubble and agitate under nitrogen for 3–5 minutes, then drain the solvent. Add 1.66±0.1 kg MTBE (7 vol, v / w) to synthesis vessel R-2105, bubble and agitate under nitrogen for 3–5 minutes, then drain the solvent. Add 2.98±0.2 kg DCM (7 vol, v / w) to synthesis vessel R-2105, bubble and agitate under nitrogen for 3–5 minutes, then drain the solvent. Repeat this step once. Add 1.66±0.1 kg MTBE (7 vol, v / w) to synthesis vessel R-2105, bubble and agitate under nitrogen for 3–5 minutes, then drain the solvent. Repeat this step once.

[0147] Remove the peptide resin from synthesis tank R-2105 and transfer it to vacuum drying oven D-2101. Control the jacket temperature at 30-35℃ and the vacuum degree at ≤-0.08MPa. Dry for 10 hours, turning the material over every 3-4 hours. Starting from the 10th hour, weigh the material every 2 hours until LOD ≤10%. Discharge the material, pack it into double-layer LDPE bags, weigh it, and label it. Refrigerate the material at 2-8℃.

[0148] 2.11 Peptide Resin Cleavage

[0149] Under nitrogen protection, add 153±1Kg MTBE (300vol, v / w) to the settling tank, cool to -20~-10℃, start stirring, and rotate at 100 rpm.

[0150] Under nitrogen protection, 27.5±0.1Kg TFA, 930±1g DODT, 480±1g Tis, and 0.62±0.05Kg purified water (volume ratio of 90:4:3:3) were added sequentially to the pyrolysis vessel. The internal temperature was lowered to -10 to -5℃, and the rotation speed was 100 rpm.

[0151] Under nitrogen protection, 690±1g of peptide resin was added to the pyrolysis vessel, the internal temperature was controlled at -10 to 0℃, and the feed port was rinsed with a small amount of pyrolysis solution.

[0152] Within 20 minutes, the temperature inside the pyrolysis vessel is raised to 5-10°C and maintained at 5-10°C with stirring for 1 hour.

[0153] Within 30 minutes, the temperature inside the pyrolysis reactor is raised to 26–30°C and maintained at 26–30°C with stirring for 3 hours.

[0154] Filter the solution and transfer the filtrate to the settling tank R-2101 (control the temperature of the settling tank to not exceed 20℃). Wash the resin in the pyrolysis vessel with TFA three times, stirring for 3 to 5 minutes each time. Use 0.22±0.02Kg of TFA for each wash and transfer the washing solution to the settling tank together.

[0155] Stir in the settling tank for 2-4 hours, then let it stand for 15-20 hours to settle.

[0156] Transfer the material in the settling tank into a filter with a screen (3-in-1 M-2101) for filtration, and wash with MTBE, using 1.5±0.1Kg MTBE (2.94vol, v / w) each time; each wash lasts 10 to 30 minutes; repeat the washing operation 3 times.

[0157] The filter cake from the three-in-one process is dried under reduced pressure for 12 hours, with stirring every 1 to 2 hours. The jacket temperature is 25 to 35°C, and the vacuum degree is ≤-0.085MPa. After drying, the material from the three-in-one process is transferred to a tray drying oven D-2101 and dried under reduced pressure. The material is weighed every 2 hours until the LOD is ≤10%.

[0158] Remove from the oven; transfer the material into a double-layer LDPE bag and thermoplasticize; then pack it into an aluminum foil bag and thermoplasticize; add a desiccant between the LDPE and aluminum foil bags, weigh, label, and store at -20±5℃ to obtain MS011 crude product (MS011-A).

[0159] In the operation step 2.11, the material feed amount is calculated based on peptide resin; the stirring speed under nitrogen bubbling is set to 100 rpm.

[0160] The nuclear magnetic resonance spectrum, LC-MS spectrum and infrared spectrum of the synthesized product are shown in Figures 1-3.

[0161] Example 2: Lutetium [ 177 Purification of Lu]O-octreotide precursor compounds

[0162] 1. Single purification

[0163] A certain amount of crude MS011-A (prepared in Example 1) was added to the reaction vessel. A mixture of acetonitrile, DMSO and purified water (acetonitrile:DMSO:purified water = 0.79:2.20:7.01 (w / w / w) or acetonitrile:DMSO:purified water = 1:2:7 (v / v / v)) was added to the sample. The concentration of the crude product solution was about 30 g / L. The mixture was stirred until no solid particles were visible.

[0164] Control the temperature inside the reactor at 20–30°C. Adjust the pH to 2.00–4.00 using 5% ammonia water (pre-prepared) or 5% trifluoroacetic acid aqueous solution (pre-prepared). Stir for 1.5–5 hours, then continue to add 5% ammonia water dropwise to adjust the pH to 7.10–7.90. Stir for 2–18 hours. At 20–30°C, add approximately equal weight of 10% acetonitrile aqueous solution (acetonitrile:pure water = 0.79:9 (w / w)) to the reactor.

[0165] The material in the reactor was filtered through a 0.45μm nylon filter membrane, and the filtrate was stored in a container for later use. The relevant substances and their contents were then tested.

[0166] The prepared sample solution was purified using a single-step purification method. Information on the single-step purification method is shown in Table 1.

[0167] Table 1

[0168] During purification, samples of each fraction are taken for testing. The purity of the purified fractions is then determined.

[0169] All fractions that meet the MP standard (purity ≥ 99.0% and individual impurities ≤ 0.10% by weight) are combined and temporarily stored in a 2-8℃ warehouse for salt concentration.

[0170] Combine all fractions that meet RP standards (purity ≥ 95.0% but not MP requirements).

[0171] 2. Recovery and purification

[0172] After diluting with an equal weight of purified water to a distillation tank conforming to RP standards, the product was recovered and purified according to the recovery and purification method. Information on the recovery and purification method is shown in Table 2.

[0173] Table 2

[0174] During the recovery and purification process, samples of each fraction were taken by Kaiser to test the resin color.

[0175] All fractions that meet the MP standard (purity ≥ 99.0% and individual impurities ≤ 0.10% by weight) are combined and temporarily stored in a 2-8℃ warehouse for salt concentration; fractions that do not meet the MP standard are discarded.

[0176] 3. Salt Concentration

[0177] After diluting with an equal weight of purified water to a distillation tank conforming to MP standards, salt concentration was carried out according to the salt concentration method. Information on the salt concentration method is shown in Table 3.

[0178] Table 3

[0179] During the salt concentration process, samples were taken from each fraction for testing.

[0180] All fractions meeting FP standards (purity ≥ 99.0% and individual impurities ≤ 0.10% by weight) were combined and filtered through a 0.22 μm hydrophobic polytetrafluoroethylene (PTFE) filter. The filtrate was sampled and the resin color was tested. The concentration of the test result must not exceed 37.6 mg / ml. If the concentration exceeds 37.6 mg / ml, it must be diluted with 0.1% trifluoroacetic acid aqueous solution to below 37.6 mg / ml.

[0181] 4. Freeze-drying

[0182] All FP solutions were lyophilized according to the lyophilization procedure, with each tray containing a sample solution to a height of 10 ± 2 mm. The lyophilization procedure is shown in Table 4.

[0183] Table 4

[0184] After freeze-drying, re-press, unload and package (packaging must be carried out under humidity ≤30%), and notify QC to take samples.

[0185] After sampling, the samples are packaged according to the packaging instructions and then stored in the warehouse.

[0186] 5. Purification results

[0187] Table 5

[0188] IPC sample storage conditions: room temperature. The purity test method for the MS011 crude solution is as follows:

[0189] Example 3: Screening of Crude Product Dissolution Conditions

[0190] 1. Take 100 mg of the crude product (batch number: MS011-20240126-E0042-A05, prepared according to the method of Example 1), add 3.3 mL of mixed solvent (DMSO / H2O / ACN = 2 / 1 / 7, volume ratio) to dissolve, to a concentration of approximately 30 mg / mL, and investigate the stability under the conditions in Table 6 below:

[0191] Table 6

[0192] 2. Take 2g of crude product (batch number: MS011-20240126-E0057-02, prepared according to the method of Example 1), add 66mL of mixed solvent (DMSO / H2O / ACN = 2 / 1 / 7, volume ratio) to dissolve, with a concentration of approximately 30mg / ml, and investigate the stability under the conditions in Table 7 below:

[0193] Table 7

[0194] Experiments 1 and 2 show that when the crude peptide solution is stirred at 30±1℃ for 1.5 to 5 hours at pH 2.28 and 3.98, the purity of the crude product remains relatively stable with no significant change. Experiments 3 and 4 show that when the crude peptide solution is stirred at 20±1℃ for 1.5 to 5 hours at pH 1.98 and 4.00, the purity of the crude product remains relatively stable with no significant change. Therefore, the pH of the crude peptide solution in the first stage of purification is set at 2.00-4.00, and the stirring temperature at 20-30℃.

[0195] 3. Divide the solutions from Experiments 3 and 4 into two equal portions and examine their stability under the conditions shown in Table 8 below:

[0196] Table 8

[0197] Experiments 5, 6, 7, and 8 show that when the crude peptide solution with pH 2.00-4.00 is stirred at 20-30℃ for 5 hours, and then the pH is adjusted to 7.08-7.91, followed by stirring at 20-30℃ for 2-4 hours, the purity of the crude product remains relatively stable with no significant change. Continuing to stir at 20-30℃ for 18 hours slightly reduces the purity of the crude peptide, and the impurity (RRT 0.18) increases by about 1%. However, due to the significant difference in properties compared to the product, this does not affect subsequent purification. Therefore, when the crude peptide solution with pH 2.00-4.00 is stirred at 20-30℃ for 1.5-5 hours, the pH is adjusted to 7.08-7.91 (e.g., 7.10-7.90) using 5% ammonia, and then stirred at 20-30℃ for 2-18 hours, the crude product remains relatively stable.

[0198] Example 4: Screening of primary purification buffer (phase A)

[0199] 1. Types of primary purification buffer (phase A)

[0200] The crude product solution was purified using aqueous solutions of 0.1% TFA-H2O, 0.1% H3PO4-H2O, 0.1% H3PO4 + triethylamine, 10mM ammonium bicarbonate, and 10% KH2PO4 + 90% K2HPO4 (10mM KH2PO4 and 10mM K2HPO4 solutions were mixed uniformly at a volume ratio of 1:9) as phase A and acetonitrile as phase B. The purity and yield of the purified samples were compared. The experimental results are shown in Table 9 below.

[0201] Table 9

[0202] According to the results in the table above, the samples obtained from Experiments 4 and 5 have the highest purity, both reaching over 99%. The yield of Experiment 5 is 87.7%, which is higher than the yield of Experiment 4 (78.5%). Taking all factors into consideration, the first purification buffer (phase A) is determined to be 10mM 10% KH2PO4 + 90% K2HPO4 (pH 7.87).

[0203] 2. Concentration of the first purification buffer (phase A)

[0204] The crude product solution was purified using 10% KH₂PO₄ + 90% K₂HPO₄ (8 mM), 10% KH₂PO₄ + 90% K₂HPO₄ (10 mM), and 10% KH₂PO₄ + 90% K₂HPO₄ (12 mM) as phase A and acetonitrile as phase B, respectively. The purity and yield of the purified samples were compared. The experimental results are shown in Table 10 below:

[0205] Table 10

[0206] As can be seen from the table above, the purity of all three groups of experiments is above 99%, the maximum single impurity is ≤0.1% by weight, and the yield is highest when the buffer concentration is 10mM. Taking all factors into consideration, the buffer concentration is determined to be 10±1mM.

[0207] 3. Primary purification buffer (phase A) pH

[0208] The crude product solution was purified using three buffer solutions: 10 mM 10% KH₂PO₄ + 90% K₂HPO₄ (pH 7.60), 10 mM 10% KH₂PO₄ + 90% K₂HPO₄ (pH 7.87), and 10 mM 10% KH₂PO₄ + 90% K₂HPO₄ (pH 8.00) as phase A and acetonitrile as phase B. The purity and yield of the purified samples were compared. The experimental results are shown in Table 11 below.

[0209] Table 11

[0210] As can be seen from the results in the table above, the purity of the samples obtained from the three groups of experiments is above 99%, the maximum single impurity is ≤0.1% by weight, the yield is above 80% and there is no significant difference. Taking all factors into consideration, the pH of the buffer solution is determined to be 7.87±0.1.

[0211] Example 5: Screening of chromatographic packing materials

[0212] Using 10% KH₂PO₄ + 90% K₂HPO₄ (10 mM) as buffer, samples were analyzed using UniSil 10-100 C18, UniHybrid 10-120 C18, and UniSil 10-120 C4 packing materials, respectively. The purity and resolution of the main peaks of samples under different packing materials were compared. The results are shown in Table 12 below:

[0213] Table 12

[0214] The crude peptide has a large amount of impurities adjacent to the main peak that are difficult to remove. According to the results in the table above, the UniSil 10-100 C18 packing material has a resolution of 1.3 for the impurities, which is the largest among the three groups of experiments. The purity of the main peak is 74.8%, which is the smallest among the three groups of experiments. Considering all these factors, the UniSil 10-100 C18 packing material was selected as the chromatographic packing material.

[0215] Example 6: Screening of primary purification buffer (phase B)

[0216] The samples were purified using a buffer solution of 10% 10mM KH₂PO₄ + 90% 10mM K₂HPO₄, with methanol and acetonitrile added respectively. The purity and yield of the purified samples were investigated, and the experimental results are shown in Table 13 below:

[0217] Table 13

[0218] As can be seen from the results in the table above, the purity of the sample obtained in Experiment 2 was 99.4%, and the yield was 87.7%, both of which were higher than the purity and yield of the sample obtained in Experiment 1. Taking all factors into consideration, acetonitrile was determined to be the organic phase for the first purification.

[0219] Example 7: Optimization of the elution gradient for a single purification step

[0220] The crude product solution was purified using different elution gradients: 5% (0 min) - 10% (5 min) - 30% (95 min) based on the volume percentage of phase B, 5% (0 min) - 12% (5 min) - 32% (95 min), and 5% (0 min) - 14% (5 min) - 34% (95 min). The purity and yield of the purified samples were compared. The experimental results are shown in Table 14 below.

[0221] Table 14

[0222] The results in the table above show that the purity of the sample obtained in Experiment 2 was 99.4%, and the yield was 87.2%. The purity and maximum single impurity of the samples obtained in Experiments 1 and 3 also met the requirements, but the yields were 58.9% and 56.9% respectively, which were lower than those in Experiment 2. Taking all factors into consideration, the elution gradient for a single purification step was determined to be 5% (0 min) - 12 ± 1% (5 min) - 32 ± 1% (95 min).

[0223] Example 8: Single Purification Sample Loading Test

[0224] In one purification process, 16g and 18g samples were loaded respectively to examine the purity and yield of the purified samples. The results are shown in Table 15 below:

[0225] Table 15

[0226] The results showed that when the sample loading amount was 16g, the sample purity was 99.7%, the maximum single impurity was 0.09% by weight, and the yield was 76.7%. When the sample loading amount was further increased to 18g, the sample purity was 99.7%, the maximum single impurity was 0.09% by weight, and the yield was 69.4%. Taking all factors into consideration, the optimal loading range for a single purification was determined to be 16–18g.

[0227] Example 9: Recovery and Purification Sample Loading Test

[0228] During the purification and recovery process, samples of 14.1 g, 11.6 g, and 16.8 g were loaded respectively to examine the purity and yield of the purified samples. The results are shown in Table 16 below:

[0229] Table 16

[0230] The results in the table above show that the loading in Experiment 2 was 11.6g, the purity of the sample was 99.8%, and the maximum single impurity was 0.08% by weight. When the loading was further increased to 14.1g and 16.8g, the maximum single impurity was greater than 0.1% by weight. Taking all factors into consideration, the recovery and purification loading was determined to be ≤11.6g.

[0231] Example 10: Salt Concentration Sample Loading Test

[0232] During the salt concentration process, samples of 47.4 g, 42.6 g, and 37.7 g were loaded respectively to examine the purity and yield of the purified samples. The results are shown in Table 17 below:

[0233] Table 17

[0234] The results show that when the sample loading amount is 37.7g, the sample purity is 99.9% and the yield is 95.7%. When the sample loading amount increases to 47.4g, the sample purity is 99.7% and the yield is 85.0%. Taking all factors into consideration, the salt concentration loading range is determined to be ≤42.6g.

[0235] Example 11 Study on freeze-drying parameters

[0236] 1. Freeze-dried thickness

[0237] The concentrated salt solution was collected and freeze-dried in a freeze-drying pan to a thickness of 8 mm, 10 mm, and 12 mm. The moisture and acetonitrile residues in the freeze-dried samples were examined. The experimental results are shown in Table 18 below:

[0238] Table 18

[0239] The experimental results above show that there are no significant differences in the moisture content and acetonitrile residue of the freeze-dried samples obtained from the three groups of experiments. Taking all factors into consideration, the freeze-drying thickness is determined to be 10±2mm.

[0240] 2. Freeze-drying concentration

[0241] Different concentrations of salt-concentrated collected solutions were loaded into lyophilization pans and lyophilized according to the same lyophilization curve. The moisture and acetonitrile residues in the lyophilized samples were examined. The experimental results are shown in Table 19 below:

[0242] Table 19

[0243] The results show that as the freeze-drying concentration increases, the moisture content and residual acetonitrile content of the freeze-dried sample gradually increase. When the freeze-drying concentration is 37.6 mg / mL, the moisture content of the freeze-dried sample is 5.9% and the residual acetonitrile content is 0.14%. If the freeze-drying concentration is further increased, there is a risk that the moisture content and residual acetonitrile content of the freeze-dried sample will continue to increase. Taking all factors into consideration, the freeze-drying concentration is determined to be ≤37.6 mg / mL.

[0244] 3. Freeze-drying curve

[0245] After the salt concentrate was collected and placed into a freeze-drying pan, it was freeze-dried according to different freeze-drying curves. The experimental procedures are shown in Table 20 below:

[0246] Table 20

[0247] The moisture and acetonitrile residues in the freeze-dried samples were tested, and the results are shown in Table 21 below:

[0248] Table 21

[0249] Experiment 2, based on Experiment 1, extended the first sublimation drying step and the holding time at -10℃ to 1600 min. The acetonitrile residue decreased from 0.14% to 0.04%, showing a significant removal effect. Experiment 3 lowered the first sublimation drying temperature to -15℃, and Experiment 4 lowered the pre-freezing temperature to -55℃. The resulting samples showed no significant decrease in moisture content or acetonitrile residue compared to Experiment 2. Considering all factors, the freeze-drying curve used in Experiment 2 was selected.

[0250] 4. Dispensing humidity

[0251] After the freeze-drying of the samples was completed, they were aliquoted under different humidity conditions, and the moisture content of the samples was measured. The results are shown in Table 22 below:

[0252] Table 22

[0253] The results show that the moisture content of the sample increases slowly with increasing ambient humidity, reaching 5.9% when the ambient humidity is 79.3%. To ensure that the sample moisture content is within acceptable limits, the repackaging humidity is set at ≤30%.

[0254] The above descriptions are merely several exemplary embodiments of this application and are not intended to limit this application in any way. Although this application discloses preferred embodiments as described above, they are not intended to limit this application. Any modifications or variations made by those skilled in the art using the disclosed technical content without departing from the scope of the technical solution of this application are equivalent to equivalent embodiments and fall within the scope of the technical solution of this application.

Claims

1. A lutetium [Lu] octreotide precursor compound of formula I ###0001### I 177 A method for purifying a lutetium [Lu] octreotide precursor compound of formula I, characterized in that, Includes the following steps: (1) The lutetium shown in Formula I [ 177 The crude product of the Lu]oxooctreotide precursor compound was dissolved in a mixed solvent of organic solvent and water. The pH of the resulting solution was adjusted to 2.00–4.00, and then adjusted to 7.08–7.

91. The solution was then filtered. (2) The filtrate obtained in step (1) is purified by high performance liquid chromatography (HPLC). The HPLC conditions include: using octadecylsilane-bonded silica gel as the column packing material; using an aqueous solution of KH2PO4 and K2HPO4 with a total concentration of 9-11 mM and a molar ratio of 1:9 as mobile phase A; using acetonitrile as mobile phase B; and eluting according to the following gradient program: From 0 to 5 minutes, the volume ratio of mobile phase A to mobile phase B is 95:5; For 5 to 95 minutes, the volume ratio of mobile phase A to mobile phase B is 87–89:13–11; 95 minutes after, the volume ratio of mobile phase A to mobile phase B is 67-69:33-31; 2. The purification method according to claim 1, characterized in that, The total concentration of KH2PO4 and K2HPO4 in the mobile phase A is 10 mM.

3. The purification method according to claim 1 or 2, characterized in that, The gradient procedure is as follows: From 0 to 5 minutes, the volume ratio of mobile phase A to mobile phase B is 95:5; From 5 to 95 minutes, the volume ratio of mobile phase A to mobile phase B was 88:12; After 95 minutes, the volume ratio of mobile phase A to mobile phase B was 68:

32.

4. The purification method of claim 1, wherein, In step (1), the organic solvent is acetonitrile and dimethyl sulfoxide.

5. The purification method of claim 4, wherein, In the mixed solvent of step (1), the volume ratio of acetonitrile, dimethyl sulfoxide and water is 1:2:

7.

6. The purification method according to claim 1 or 4, characterized by, In step (1), the obtained solution contains lutetium of formula I. 177 The concentration of the Lu]oxooctreotide precursor compound is 25–35 g / L.

7. The purification method of claim 6, wherein, In step (1), the obtained solution contains lutetium of formula I. 177 The concentration of the Lu]oxooctreotide precursor compound was 30 g / L.

8. The purification method of claim 1, wherein, In step (1), the pH of the obtained solution is adjusted to 2.00 to 4.00 at 20 to 30°C, stirred for 1.5 to 5 hours, then the pH is adjusted to 7.10 to 7.90, stirred for 2 to 18 hours, and an equal weight of 10% acetonitrile aqueous solution is added and filtered.

9. The purification method of claim 8, wherein, The pH value was adjusted using ammonia water with a volume ratio of 5%; filtration was performed using a 0.45μm nylon filter membrane.

10. The purification method of claim 1, wherein, In step (2), the chromatographic column packing is UniSil 10-100C18 packing.

11. The purification method of claim 1, wherein, In step (2), the sample loading amount of the chromatographic column is 16-18g.

12. The purification method of claim 1, wherein, In step (2), the pH value of the mobile phase A is 7.77 to 7.

97.

13. [Amended according to Rule 91 on 12.01.2026] The purification method according to claim 12, characterized in that, The pH value of the mobile phase A is 7.

87.

14. The purification method of claim 1, wherein, In step (2), the detection wavelength of the high performance liquid chromatography is 230 nm.

15. The purification method of claim 1, wherein, In step (2), the elution flow rate of mobile phase A and mobile phase B is 880 mL / min.

16. The purification method of claim 1, wherein, In step (2), a fraction with a purity ≥99.0% and a single impurity ≤0.10% by weight is collected. For a fraction with a purity ≥95% and ≤99.0% and a single impurity >0.10% by weight, a second purification is performed by the high performance liquid chromatography method to obtain a fraction with a purity ≥99.0% and a single impurity ≤0.10% by weight.

17. The purification method of claim 16, wherein, The fraction with a purity of ≥95% and ≤99.0% and a single impurity >0.10% by weight is diluted with an equal weight of water before secondary purification.

18. The purification method according to claim 16 or 17, characterized in that, The sample loading capacity of the chromatographic column used for secondary purification is ≤11.6g.

19. The purification method of claim 1, wherein, It also includes the following steps: (3) The fraction obtained in step (2) is concentrated by salt conversion by high performance liquid chromatography, wherein the conditions of the high performance liquid chromatography include: using octadecylsilane bonded silica gel as the chromatographic column packing. Using a 0.1% (v / v) aqueous solution of trifluoroacetic acid as mobile phase A and acetonitrile as mobile phase B, elution was carried out according to the following gradient: From 0 to 25 minutes, the volume ratio of mobile phase A to mobile phase B is 95:5; For 25 to 75 minutes, the volume ratio of mobile phase A to mobile phase B is 80:20; After 75 minutes, the volume ratio of mobile phase A to mobile phase B was 30:

70.

20. The purification method of claim 19, wherein, In step (3), the chromatographic column packing material is UniSil 10-100C18 packing material.

21. The purification method of claim 19, wherein, In step (3), the sample loading of the chromatographic column is ≤42.6g.

22. The purification method of claim 19, wherein, In step (3), the detection wavelength of the high performance liquid chromatography is 230 nm.

23. The purification method of claim 19, wherein, In step (3), the elution flow rate of mobile phase A and mobile phase B is 880 mL / min.

24. The purification method of claim 19, wherein, In step (3), the fraction obtained in step (2) is diluted with an equal weight of water before salt concentration.

25. The purification method of claim 19, wherein, In step (3), a fraction with a purity ≥ 99.0% and a single impurity ≤ 0.10% by weight is collected.

26. The purification method of claim 19 or 25, wherein, It also includes the following steps: (4) Freeze-dry the fraction collected in step (3).

27. The purification method of claim 26, wherein, In step (4), the freeze-drying includes the following steps: (4-1) Pre-freezing: Cool to -45 to -55°C within 30 minutes and hold for 210 minutes; (4-2) Sublimation: Under a vacuum of 0.2 to 0.25 mbar, the temperature is raised to -10 to -15 °C over 30 minutes and held for 510 to 1600 minutes; then the temperature is raised to 5 °C over 30 minutes and held for 1470 to 1500 minutes. (4-3) Desorption drying: Under a vacuum of 0.01 mbar, the temperature is raised to 25°C within 30 minutes and maintained for 790 to 2400 minutes.

28. The purification method of claim 27, wherein, In step (4-1), the temperature is lowered to -45°C within 30 minutes.

29. The purification method of claim 27, wherein, In step (4-3), the holding time is 790 to 800 minutes.

30. The purification method according to any one of claims 26 to 29, wherein, In step (4), the lutetium [represented by formula I] in the fraction before freeze-drying 177 The concentration of the Lu]oxooctreotide precursor compound is ≤37.6 mg / mL.

31. The purification method according to any one of claims 26 to 29, wherein, It also includes the following steps: (5) The freeze-dried fraction obtained in step (4) is packaged.

32. The purification method of claim 31, wherein, The dispensing is carried out at a humidity level of ≤30%.

33. A lutetium [Lu] octreotide preparation method characterized by, 177 Lu] octreotide preparation method characterized by, comprising a purified compound of Formula I: 177 Lu]octreotide precursor compound chelated with lutetium 177 Lu].

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