Purification method of lutetium [177Lu] oxygen octreotide precursor compound

Abstract

The invention provides a purification method of a lutetium [177Lu] oxyoctreotide precursor compound, which comprises the following steps: (1) dissolving a lutetium [177Lu] oxyoctreotide precursor compound crude product as shown in a formula I in a mixed solvent of an organic solvent and water, adjusting the pH value of the obtained solution to 2.00-4.00, then adjusting the pH value to 7.08-7.91, and filtering; (2) purifying the filtrate obtained in the step (1) through a high performance liquid chromatography, wherein the conditions of the high performance liquid chromatography are as follows: octadecylsilane chemically bonded silica is used as a chromatographic column filler; according to the method, an aqueous solution of KH2PO4 and K2HPO4 with the total concentration of 9-11mM, preferably 10mM, and the molar ratio of 1: 9 is taken as a mobile phase A, acetonitrile is taken as a mobile phase B, and gradient elution is carried out. According to the purification method, the purity of the lutetium [177Lu] oxyoctreotide precursor compound can be remarkably improved, the single impurity content of the product is reduced, and a foundation is laid for large-scale industrial production and clinical popularization and application of the lutetium [177Lu] oxyoctreotide.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology. Specifically, the present invention relates to a lutetium [ 177 A purification method and application of Lu]oxyoctreotide precursor compound. Background Art

[0002] In 2018, the U.S. FDA approved the lutetium [ 177 Lu]oxoctreotide (Lutathera) is now available for the treatment of SSTR-positive gastroenteropancreatic neuroendocrine tumors (GEP-NETs) that have progressed after standard therapy. 177 As the first radioactive drug approved for radioligand therapy (RLT), Lu]oxyoctreotide specifically binds to the somatostatin receptor on the surface of tumor cells, reaches the tumor cells and releases radionuclides to produce radiation to kill tumor cells. Recent clinical trial results also show that its clinical application range is expected to be further expanded.

[0003] Lutetium 177 Lu] oxyoctreotide is a radionuclide [ 177 Lu] is chelated with a precursor compound (MS011) shown in the following formula I as a chelating agent, wherein the precursor compound is composed of a polypeptide composed of 8 amino acids connected to a chelating agent DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), wherein the octadentate ligand formed by the four nitrogen atoms on the dodecane heterocycle plus the four acetic acid groups connected to the nitrogen atoms provides electrons to bind to the lutetium [ 177 Lu] atoms form a stable chelate.

[0004] The industrial production of radionuclide-conjugated drugs places high demands on the yield and product purity of the precursor compound preparation method, because impurities will seriously affect the subsequent radionuclide labeling process. 177 The preparation methods of lutetium [Lu] oxoctreotide precursor compounds generally have the problems of low yield and low product purity, which is difficult to achieve 177 The efficient and large-scale production of Lu]-oxoctreotide API to meet clinical drug demand poses a major challenge. 177 The purification process of Lu]oxyoctreotide precursor compounds has become one of the technical problems to be solved urgently in the pharmaceutical field. Summary of the invention

[0005] In view of this, the present invention aims to provide a lutetium [ 177 Lu] oxyoctreotide precursor compound purification method, the purification method can significantly improve the lutetium [ 177 Lu] oxyoctreotide precursor compound, reducing the impurity content, for Lutetium [ 177 This laid a foundation for the large-scale industrial production and clinical application of Lu]oxyoctreotide.

[0006] The above object of the present invention is achieved through the following technical solutions:

[0007] A lutetium represented by formula I 177 A method for purifying a precursor compound of Lu] oxyoctreotide, comprising the following steps:

[0008] (1) The lutetium [ 177 The crude product of Lu]oxoctreotide precursor compound is dissolved in a mixed solvent of an organic solvent and water, and the pH of the obtained solution is adjusted to 2.00-4.00, and then adjusted to 7.08-7.91, and filtered;

[0009] (2) Purifying the filtrate obtained in step (1) by high performance liquid chromatography, wherein the conditions of the high performance liquid chromatography include: using octadecylsilane bonded silica gel as the chromatographic column filler; using an aqueous solution of KH2PO4 and K2HPO4 with a molar ratio of 1:9 and a total concentration of 9 to 11 mM, preferably 10 mM as mobile phase A, and using 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 was 95:5;

[0011] 5 to 95 minutes, the volume ratio of mobile phase A to mobile phase B is 87-89:13-11, 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]

[0014] According to some embodiments of the present invention, in step (1), the mixed solvent of 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 [ 177 The concentration of the Lu]oxoctreotide precursor compound is 25 to 35 g / L, preferably 30 g / L.

[0015] According to some embodiments of the present invention, 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% by volume acetonitrile aqueous solution is added, and filtered; preferably, 5% by volume ammonia water is used to adjust the pH; preferably, 0.45 μm nylon filter membrane is used for filtration.

[0016] According to some embodiments of the present invention, in step (2), the chromatographic column filler is UniSil 10-100C18 filler.

[0017] According to some embodiments of the present invention, in step (2), the sample loading capacity of the chromatographic column is 16 to 18 g.

[0018] According to some embodiments of the present invention, in step (2), the pH of the mobile phase A is 7.77 to 7.97, preferably 7.87.

[0019] According to some embodiments of the present invention, in step (2), the detection wavelength of the high performance liquid chromatography is 230 nm.

[0020] According to some embodiments of the present invention, in step (2), the elution flow rate of the mobile phase is 880 mL / min.

[0021] According to some embodiments of the present invention, in step (2), fractions with a purity of ≥99.0% and a single impurity of ≤0.10% by weight are collected, and for fractions with a purity of ≥95% and ≤99.0% and a single impurity of >0.10% by weight, the high performance liquid chromatography secondary purification is repeated to obtain a fraction with a purity of ≥99.0% and a single impurity of ≤0.10% by weight; preferably, the fraction with a purity of ≥95% and ≤99.0% and a single impurity of >0.10% by weight is diluted with an equal weight of water before the secondary purification; preferably, the sample loading capacity of the chromatographic column for the secondary purification is ≤11.6 g.

[0022] According to some embodiments of the present invention, the purification method further comprises the following steps:

[0023] (3) The fraction obtained in step (2) is subjected to salt conversion and concentration by high performance liquid chromatography, wherein the conditions of the high performance liquid chromatography include: using octadecylsilane bonded silica gel as the chromatographic column filler; using a 0.1% by volume aqueous solution of trifluoroacetic acid as the mobile phase A, and using acetonitrile as the mobile phase B, and eluting according to the following gradient:

[0024] From 0 to 25 min, the volume ratio of mobile phase A to mobile phase B was 95:5;

[0025] 25 to 75 minutes, the volume ratio of mobile phase A to mobile phase B was 80:20;

[0026] After 75 minutes, the volume ratio of mobile phase A to mobile phase B was 30:70.

[0027] According to some embodiments of the present invention, in step (3), the chromatographic column filler is UniSil 10-100C18 filler.

[0028] According to some embodiments of the present invention, in step (3), the sample loading amount of the chromatographic column is ≤42.6 g.

[0029] According to some embodiments of the present invention, in step (3), the detection wavelength of the high performance liquid chromatography is 230 nm.

[0030] According to some embodiments of the present invention, in step (3), the elution flow rate of the mobile phase is 880 mL / min.

[0031] According to some embodiments of the present invention, in step (3), the fraction obtained in step (2) is diluted with an equal weight of water before salt conversion and concentration.

[0032] According to some embodiments of the present invention, in step (3), fractions with a purity of ≥99.0% and a single impurity of ≤0.10% by weight are collected.

[0033] According to some embodiments of the present invention, the purification method further comprises the following steps:

[0034] (4) Lyophilizing the fraction obtained in step (3).

[0035] According to some embodiments of the present invention, in step (4), the freeze-drying comprises the following steps:

[0036] (4-1) Prefreezing: cooling to -45 to -55°C, preferably -45°C, within 30 minutes and maintaining for 210 minutes;

[0037] (4-2) primary sublimation: under a vacuum degree of 0.2 to 0.25 mbar, preferably 0.2 mbar, heating to -10 to -15°C, preferably -10°C within 30 minutes, and maintaining for 510 to 1600 minutes, preferably 1600 minutes; then heating to 5°C within 30 minutes, and maintaining for 1470-1500 minutes, preferably 1500 minutes;

[0038] (4-3) Desorption drying: under a vacuum degree 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.

[0039] According to some embodiments of the present invention, in step (4), the thickness of the freeze-dried fraction is 8 to 12 mm.

[0040] According to some embodiments of the present invention, in step (4), the lutetium shown in formula I in the pre-lyophilized fraction is 177 The concentration of Lu]oxyoctreotide precursor compound is ≤37.6 mg / mL.

[0041] According to some embodiments of the present invention, the purification method further comprises the following steps:

[0042] (5) Packaging the freeze-dried fraction obtained in step (4); preferably, the packaging is carried out at a humidity of ≤30%.

[0043] In addition, the present invention also provides a lutetium [ 177 Lu] oxyoctreotide preparation method, comprising the steps of: 177 The lutetium [Lu] obtained by the purification method of oxyoctreotide precursor compound 177 Lu] oxyoctreotide precursor compound and lutetium [ 177 Lu] chelation.

[0044] The present invention has at least the following beneficial effects:

[0045] The present invention provides an optimized lutetium [ 177 The invention discloses a method for purifying a precursor compound of lutetium [Lu], wherein the single impurity content in the crude precursor compound is further reduced by HPLC purification and the purity of the precursor compound product is improved, so that the single impurity content is ≤0.10% by weight and the product purity is ≥99.0%. 177 This laid the foundation for the large-scale industrial production and clinical application of Lu]oxyoctreotide. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0047] Figure 1 The lutetium prepared in Example 1 [ 177 NMR spectrum of Lu]-oxyoctreotide precursor compound MS011.

[0048] Figure 2 The lutetium prepared in Example 1 [ 177 LC-MS spectrum of Lu]-oxyoctreotide precursor compound MS011.

[0049] Figure 3 The lutetium prepared in Example 1 [ 177 Infrared spectrum of Lu]oxyoctreotide precursor compound MS011.

[0050] Figure 4-6 is the lutetium in Example 2 [ 177 HPLC spectrum of crude solution of Lu]-oxyoctreotide precursor compound MS011.

[0051] Figure 7-9 is the lutetium in Example 2 [ 177 HPLC spectrum of the first purification fraction collection solution (MP) of Lu]-oxoctreotide precursor compound MS011.

[0052] Figure 10-12 is the lutetium in Example 2 [ 177 HPLC spectrum of the first purification recovery solution (RP) of Lu]-oxoctreotide precursor compound MS011.

[0053] Figure 13-15 is the lutetium in Example 2 [ 177 HPLC spectrum of the collected fractions of the first purification recovery of Lu]-oxoctreotide precursor compound MS011.

[0054] Figure 16-18 is the lutetium in Example 2 [ 177 HPLC profile of the main fractions (combined MP) from the first purification of the [Lu]-oxoctreotide precursor compound MS011.

[0055] Figure 19-21 is the lutetium in Example 2 [ 177 HPLC spectrum of the concentrated collection solution of the [Lu]-oxyoctreotide precursor compound MS011. DETAILED DESCRIPTION

[0056] The present invention is further described in detail below in conjunction with specific embodiments. The given examples are only for illustrating the present invention, but not for limiting the scope of the present invention.

[0057] 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 unless otherwise specified.

[0058] Abbreviations

[0059]

[0060]

[0061] Example 1 Lutetium 177 Preparation of Lu]-oxyoctreotide precursor compound

[0062] 1. Lutetium 177 Synthesis route of Lu]oxyoctreotide precursor compound (MS011)

[0063] Key materials: Wang Resin, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Lys(Boc)-OH,

[0064] Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-D-Phe-OH, DOTA-(tBu)3-CO2H

[0065] Reagents: PyBOP, HOBt, DIEA, Ac2O, DIC, TFA, DODT, Tis, water, piperidine, iodine, vitamin C

[0066] Solvent:DMF, MeOH, DCM, MTBE, ACN

[0067] 2. Synthesis process of MS011

[0068] 2.1 Coupling of the first amino acid Fmoc-Thr(tBu)-OH

[0069] Using blank Wang resin (Wang Resin, Xi'an Lanxiao Technology Co., Ltd., cross-linking degree of 1%, mesh size of 100-200) with a substitution degree of 1.0-1.2 mmol / g, 300±1 g of Wang resin (1 eq) and 3.0±0.1 L of DMF (10 vol, v / w) were added to the synthesis tank R-2105 in sequence under nitrogen protection, and nitrogen bubbling was stirred for 30-35 minutes. The stirring was stopped and the solvent in the kettle was emptied.

[0070] Add 2.1±0.1 L DMF (7 vol, v / w) into synthesis tank R-2105, stir with nitrogen bubbling for 3-5 minutes, drain the solvent; repeat this step once more.

[0071] 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 to the activation tank R-0304 in sequence, stirred to dissolve, and cooled to 5±5°C.

[0072] The temperature inside the activation tank R-0304 was controlled at 0-10°C, and 366.0±1 g DIEA (8.0 eq) was added dropwise to the activation tank R-0304. After the addition was completed, the mixture was stirred for 3-5 minutes.

[0073] The solution in the activation tank R-0304 is added dropwise to the synthesis tank R-2105, and the activation tank R-0304 is eluted with 0.20±0.01Kg DMF, and the elution liquid is transferred to the synthesis tank R-2105.

[0074] The temperature inside the synthesis tank R-2105 was controlled at 25-30°C and the reaction was kept warm for 2 hours. After the reaction was completed, the solvent in the synthesis tank R-2105 was emptied.

[0075] Add 6.0±0.1L DMF to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; add 2.1±0.1L DMF (7vol, v / w) to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step once; add 2.1±0.1L DMF (7vol, v / w) to the synthesis tank R-2105 for spraying, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step twice.

[0076] Under nitrogen protection, 0.85±0.1 Kg DMF, 722.8±1 g Ac2O (20 eq), and 91.5±1 g DIEA (2.0 eq) were added to the activation tank R-0304 in sequence and stirred for 5 to 10 minutes.

[0077] Under nitrogen protection, transfer the solution in activation tank R-0304 to synthesis tank R-2105, control the temperature in synthesis tank R-2105 at 20-30°C, and keep the reaction for 15-20 hours; after the insulation is completed, drain the solvent in synthesis tank R-2105.

[0078] Add 6.0±0.1L DMF to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; add 2.1±0.1L DMF (7vol, v / w) to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step once; add 2.1±0.1L DMF (7vol, v / w) to the synthesis tank R-2105 for spraying, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step twice.

[0079] Add 2.78±0.1Kg DCM (7vol, v / w) into the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; add 1.55±0.1Kg MTBE (7vol, v / w), stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; add 2.78±0.1Kg DCM (7vol, v / w), stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; add 1.55±0.1Kg MTBE (7vol, v / w), stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent, and repeat this step 3 times.

[0080] Take out the Fmoc-Thr(tBu)-Wang Resin from the synthesis tank R-2105, transfer it to the vacuum drying oven D-2101, control the jacket temperature at 30-35°C, vacuum degree ≤-0.08MPa, dry for 10 hours, turn the material over every 3-4 hours, weigh it from the 10th hour, weigh it every 2 hours, until the first process control test (In-Process Control 01, IPC01) loss on drying (LOD) ≤10%, unload the material, put it in a double-layer LDPE bag, weigh it, and label it. Take samples for central control and test the degree of substitution (degree of substitution Sub = 0.4-0.7mmol / g). During the test, the material is placed in a refrigerator at 2-8°C.

[0081] In the above operation steps of 2.1, the material feeding amount is calculated based on Wang resin; the stirring speed under nitrogen bubbling is set to 100 rpm.

[0082] In the following operation steps 2.2 to 2.10, the material feeding amount is calculated based on Fmoc-Thr(tBu)-Wang Resin; the stirring speed under nitrogen bubbling is set to 100 rpm.

[0083] 2.2 Fmoc-Cys(Trt)-OH coupling

[0084] Under nitrogen protection, 320±10 g Fmoc-Thr(tBu)-Wang Resin (1 eq) and 3.2±0.1 L DMF (10 vol, v / w) were added to the synthesis tank R-2105 in sequence, and the mixture was stirred with nitrogen bubbling for 30 to 35 minutes. The stirring was stopped and the solvent in the kettle was emptied.

[0085] Add 2.24±0.1L DMF (7vol, v / w) into synthesis tank R-2105, stir and wash with nitrogen bubbling for 3-5 minutes, drain the solvent; repeat this step once more.

[0086] Under nitrogen protection, add 6.98±0.1Kg PIP and 30.56±0.1Kg DMF to the buffer tank R-2107 in sequence, stir evenly, and set aside (a total of 8 times of use).

[0087] Release about 2.08±0.1Kg (6.5vol, w / w) from buffer tank R-2107 into PP barrel, control the temperature inside synthesis tank R-2105 at 20-30℃, add the solution in PP barrel into synthesis tank R-2105, stir with nitrogen bubbling for 20 minutes, drain the solvent in synthesis tank R-2105; repeat this step once more.

[0088] Add 2.24±0.1L DMF (7vol, v / w) into synthesis tank R-2105, stir and wash with nitrogen bubbling for 3-5 minutes, and drain the solvent; repeat this step 5 times (take samples for Kaiser test, the resin solution should be blue).

[0089] Under nitrogen protection, 307.5±1 g Fmoc-Cys(Trt)-OH (3.0 eq), 74.5±1 g HOBT (3.15 eq), and 0.76±0.1 Kg DMF were added to the activation tank R-0304 in sequence, stirred to dissolve, and cooled to 5±5°C.

[0090] The temperature inside the activation tank R-0304 was controlled at 0-10°C, and 69.6±1g DIC (3.15eq) was added dropwise to the activation tank R-0304. After the addition was completed, the mixture was stirred for 5-10 minutes.

[0091] The solution in the activation tank R-0304 was added dropwise to the synthesis tank R-2105, and the activation tank R-0304 was eluted with 0.20±0.1Kg DMF, and the elution liquid was transferred to the synthesis tank R-2105; after the addition was completed, the temperature in the synthesis tank R-2105 was controlled at 20-30°C, nitrogen was bubbled, and the mixture was kept warm and stirred for 2-3 hours (samples were taken for Kaiser test, and the resin and solution were colorless and transparent; ninhydrin was used to test every 1 hour until negative); after the reaction was completed, the solvent in the synthesis tank R-2105 was emptied.

[0092] Add 6.0±0.1L DMF to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; add 2.24±0.1L DMF (7vol, v / w) to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step once; add 2.24±0.1L DMF (7vol, v / w) to the synthesis tank R-2105 for spraying, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step twice.

[0093] 2.3 Fmoc-Thr(tBu)-OH coupling

[0094] Release about 2.08±0.1Kg (6.5vol, w / w) from buffer tank R-2107 into PP barrel, control the temperature inside synthesis tank R-2105 at 20-30°C, add the solution in PP barrel into synthesis tank R-2105, stir with nitrogen bubbling for 20 minutes, drain the solvent in synthesis tank R-2105; repeat this step once more.

[0095] Add 2.24±0.1L DMF (7vol, v / w) into synthesis tank R-2105, stir and wash with nitrogen bubbling for 3-5 minutes, and drain the solvent; repeat this step 5 times (take samples for Kaiser test, the resin solution should be blue).

[0096] Under nitrogen protection, 208.7±1 g Fmoc-Thr(tBu)-OH (3.0 eq), 74.5±1 g HOBT (3.15 eq), and 0.56±0.1 Kg DMF were added to the activation tank R-0304 in sequence, stirred to dissolve, and cooled to 5±5°C.

[0097] The temperature inside the activation tank R-0304 was controlled at 0-10°C, and 69.6±1g DIC (3.15eq) was added dropwise to the activation tank R-0304. After the addition was completed, the mixture was stirred for 5-10 minutes.

[0098] The solution in the activation tank R-0304 was added dropwise to the synthesis tank R-2105, and the activation tank R-0304 was eluted with 0.20±0.1Kg DMF, and the elution liquid was transferred to the synthesis tank R-2105; after the addition was completed, the temperature in the synthesis tank R-2105 was controlled at 20-30°C, nitrogen was bubbled, and the mixture was kept warm and stirred for 2-3 hours (samples were taken for Kaiser test, and the resin and solution were colorless and transparent; ninhydrin was used to test every 1 hour until negative); after the reaction was completed, the solvent in the synthesis tank R-2105 was emptied.

[0099] Add 6.0±0.1L DMF to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; add 2.24±0.1L DMF (7vol, v / w) to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step once; add 2.24±0.1L DMF (7vol, v / w) to the synthesis tank R-2105 for spraying, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step twice.

[0100] 2.4 Fmoc-Lys(Boc)-OH coupling

[0101] Release about 2.08±0.1Kg (6.5vol, w / w) from buffer tank R-2107 into PP barrel, control the temperature inside synthesis tank R-2105 at 20-30°C, add the solution in PP barrel into synthesis tank R-2105, stir with nitrogen bubbling for 20 minutes, drain the solvent in synthesis tank R-2105; repeat this step once more.

[0102] Add 2.24±0.1L DMF (7vol, v / w) into synthesis tank R-2105, stir and wash with nitrogen bubbling for 3-5 minutes, and drain the solvent; repeat this step 5 times (take samples for Kaiser test, the resin solution should be blue).

[0103] Under nitrogen protection, add 246±1g Fmoc-Lys(Boc)-OH (3.0eq), 74.5±1g HOBT (3.15eq) and 0.56±0.1Kg DMF into activation tank R-0304 in sequence, stir to dissolve, and cool to 5±5°C.

[0104] The temperature inside the activation tank R-0304 was controlled at 0-10°C, and 69.6±1g DIC (3.15eq) was added dropwise to the activation tank R-0304. After the addition was completed, the mixture was stirred for 5-10 minutes.

[0105] The solution in the activation tank R-0304 was added dropwise to the synthesis tank R-2105, and the activation tank R-0304 was eluted with 0.20±0.1Kg DMF, and the elution liquid was transferred to the synthesis tank R-2105; after the addition was completed, the temperature in the synthesis tank R-2105 was controlled at 20-30°C, nitrogen was bubbled, and the mixture was kept warm and stirred for 2-3 hours (samples were taken for Kaiser test, and the resin and solution were colorless and transparent; ninhydrin was used to test every 1 hour until negative); after the reaction was completed, the solvent in the synthesis tank R-2105 was emptied.

[0106] Add 6.0±0.1L DMF to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; add 2.24±0.1L DMF (7vol, v / w) to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step once. Add 2.24±0.1L DMF (7vol, v / w) to the synthesis tank R-2105 for spraying, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step twice.

[0107] 2.5 Fmoc-D-Trp(Boc)-OH coupling

[0108] Release about 2.08±0.1Kg (6.5vol, w / w) from buffer tank R-2107 into PP barrel, control the temperature inside synthesis tank R-2105 at 20-30℃, add the solution in PP barrel into synthesis tank R-2105, stir with nitrogen bubbling for 20 minutes, drain the solvent in synthesis tank R-2105; repeat this step once more.

[0109] Add 2.24±0.1L DMF (7vol, v / w) into synthesis tank R-2105, stir and wash with nitrogen bubbling for 3-5 minutes, and drain the solvent; repeat this step 5 times (take samples for Kaiser test, the resin solution should be blue).

[0110] Under nitrogen protection, 276.5±1 g Fmoc-D-Trp(Boc)-OH (3.0 eq), 74.5±1 g HOBT (3.15 eq), and 0.62±0.1 Kg DMF were added to the activation tank R-0304 in sequence, stirred to dissolve, and cooled to 5±5°C.

[0111] The temperature inside the activation tank R-0304 was controlled at 0-10°C, and 69.6±1g DIC (3.15eq) was added dropwise to the activation tank R-0304. After the addition was completed, the mixture was stirred for 5-10 minutes.

[0112] The solution in the activation tank R-0304 was added dropwise to the synthesis tank R-2105, and the activation tank R-0304 was eluted with 0.20±0.1Kg DMF, and the elution liquid was transferred to the synthesis tank R-2105; after the addition was completed, the temperature in the synthesis tank R-2105 was controlled at 20-30°C, nitrogen was bubbled, and the mixture was kept warm and stirred for 2-3 hours (samples were taken for Kaiser test, and the resin and solution were colorless and transparent; ninhydrin was used to test every 1 hour until negative); after the reaction was completed, the solvent in the synthesis tank R-2105 was emptied.

[0113] Add 6.0±0.1L DMF to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; add 2.24±0.1L DMF (7vol, v / w) to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step once; add 2.24±0.1L DMF (7vol, v / w) to the synthesis tank R-2105 for spraying, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step twice.

[0114] 2.6Fmoc-Tyr(tBu)-OH coupling

[0115] Release about 2.08±0.1Kg (6.5vol, w / w) from buffer tank R-2107 into PP barrel, control the temperature inside synthesis tank R-2105 at 20-30℃, add the solution in PP barrel into synthesis tank R-2105, stir with nitrogen bubbling for 20 minutes, drain the solvent in synthesis tank R-2105; repeat this step once more.

[0116] Add 2.24±0.1L DMF (7vol, v / w) into synthesis tank R-2105, stir and wash with nitrogen bubbling for 3-5 minutes, and drain the solvent; repeat this step 5 times (take samples for Kaiser test, the resin solution should be blue).

[0117] Under nitrogen protection, 241.3±1 g Fmoc-Tyr(tBu)-OH (3.0 eq), 74.5±1 g HOBT (3.15 eq), and 0.62±0.1 Kg DMF were added to the activation tank R-0304 in sequence, stirred to dissolve, and cooled to 5±5°C.

[0118] The temperature inside the activation tank R-0304 was controlled at 0-10°C, and 69.6±1g DIC (3.15eq) was added dropwise to the activation tank R-0304. After the addition was completed, the mixture was stirred for 5-10 minutes.

[0119] The solution in the activation tank R-0304 was added dropwise to the synthesis tank R-2105, and the activation tank R-0304 was eluted with 0.20±0.1Kg DMF, and the elution liquid was transferred to the synthesis tank R-2105; after the addition was completed, the temperature in the synthesis tank R-2105 was controlled at 20-30°C, nitrogen was bubbled, and the mixture was kept warm and stirred for 2-3 hours (samples were taken for Kaiser test, and the resin and solution were colorless and transparent; ninhydrin was used to test every 1 hour until negative); after the reaction was completed, the solvent in the synthesis tank R-2105 was emptied.

[0120] Add 6.0±0.1L DMF to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; add 2.24±0.1L DMF (7vol, v / w) to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step once; add 2.24±0.1L DMF (7vol, v / w) to the synthesis tank R-2105 for spraying, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step twice.

[0121] 2.7 Fmoc-Cys(Trt)-OH coupling

[0122] Release about 2.08±0.1Kg (6.5vol, w / w) from buffer tank R-2107 into PP barrel, control the temperature inside synthesis tank R-2105 at 20-30℃, add the solution in PP barrel into synthesis tank R-2105, stir with nitrogen bubbling for 20 minutes, drain the solvent in synthesis tank R-2105; repeat this step once more.

[0123] Add 2.24±0.1L DMF (7vol, v / w) into synthesis tank R-2105, stir and wash with nitrogen bubbling for 3-5 minutes, and drain the solvent; repeat this step 5 times (take samples for Kaiser test, the resin solution should be blue).

[0124] Under nitrogen protection, 307.5±1 g Fmoc-Cys(Trt)-OH (3.0 eq), 74.5±1 g HOBT (3.15 eq), and 0.62±0.1 L DMF were added to the activation tank R-0304 in sequence, stirred to dissolve, and cooled to 5±5°C.

[0125] The temperature inside the activation tank R-0304 was controlled at 0-10°C, and 69.6±1g DIC (3.15eq) was added dropwise to the activation tank R-0304. After the addition was completed, the mixture was stirred for 5-10 minutes.

[0126] The solution in the activation tank R-0304 was added dropwise to the synthesis tank R-2105, and the activation tank R-0304 was eluted with 0.20±0.1Kg DMF, and the elution liquid was transferred to the synthesis tank R-2105; after the addition was completed, the temperature in the synthesis tank R-2105 was controlled at 20-30°C, nitrogen was bubbled, and the mixture was kept warm and stirred for 2-3 hours (samples were taken for Kaiser test, and the resin and solution were colorless and transparent; ninhydrin was used to test every 1 hour until negative); after the reaction was completed, the solvent in the synthesis tank R-2105 was emptied.

[0127] Add 6.0±0.1L DMF to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; add 2.24±0.1L DMF (7vol, v / w) to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step once; add 2.24±0.1L DMF (7vol, v / w) to the synthesis tank R-2105 for spraying, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step twice.

[0128] 2.8 Fmoc-D-Phe-OH coupling

[0129] Release about 2.08±0.1Kg (6.5vol, w / w) from buffer tank R-2107 into PP barrel, control the temperature inside synthesis tank R-2105 at 20-30℃, add the solution in PP barrel into synthesis tank R-2105, stir with nitrogen bubbling for 20 minutes, drain the solvent in synthesis tank R-2105; repeat this step once more.

[0130] Add 2.24±0.1L DMF (7vol, v / w) into synthesis tank R-2105, stir and wash with nitrogen bubbling for 3-5 minutes, and drain the solvent; repeat this step 5 times (take samples for Kaiser test, the resin solution should be blue).

[0131] Under nitrogen protection, 203.4±1 g Fmoc-D-Phe-OH (3.0 eq), 74.5±1 g HOBT (3.15 eq), and 0.62±0.1 L DMF were added to the activation tank R-0304 in sequence, stirred to dissolve, and cooled to 5±5°C.

[0132] The temperature inside the activation tank R-0304 was controlled at 0-10°C, and 69.6±1g DIC (3.15eq) was added dropwise to the activation tank R-0304. After the addition was completed, the mixture was stirred for 5-10 minutes.

[0133] The solution in the activation tank R-0304 was added dropwise to the synthesis tank R-2105, and the activation tank R-0304 was eluted with 0.20±0.1Kg DMF, and the elution liquid was transferred to the synthesis tank R-2105; after the addition was completed, the temperature in the synthesis tank R-2105 was controlled at 20-30°C, nitrogen was bubbled, and the mixture was kept warm and stirred for 2-3 hours (samples were taken for Kaiser test, and the resin and solution were colorless and transparent; ninhydrin was used to test every 1 hour until negative); after the reaction was completed, the solvent in the synthesis tank R-2105 was emptied.

[0134] Add 6.0±0.1L DMF to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; add 2.24±0.1L DMF (7vol, v / w) to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step once; add 2.24±0.1L DMF (7vol, v / w) to the synthesis tank R-2105 for spraying, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step twice.

[0135] 2.9 DOTA-(tBu)3-CO2H coupling

[0136] Release about 2.08±0.1Kg (6.5vol, w / w) from buffer tank R-2107 into PP barrel, control the temperature inside synthesis tank R-2105 at 20-30℃, add the solution in PP barrel into synthesis tank R-2105, stir with nitrogen bubbling for 20 minutes, drain the solvent in synthesis tank R-2105; repeat this step once more.

[0137] Add 2.24±0.1L DMF (7vol, v / w) into synthesis tank R-2105, stir and wash with nitrogen bubbling for 3-5 minutes, and drain the solvent; repeat this step 5 times (take samples for Kaiser test, the resin solution should be blue).

[0138] 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 to the activation tank R-0304 in sequence, stirred to dissolve, and cooled to 5±5°C.

[0139] The temperature inside the activation tank R-0304 was controlled at 0-10°C, and 90.5±1g DIEA (4.0eq) was added dropwise to the activation tank R-0304. After the addition was completed, the mixture was stirred for 5-10 minutes.

[0140] The solution in the activation tank R-0304 was added dropwise to the synthesis tank R-2105, and the activation tank R-0304 was eluted with 0.20±0.1Kg DMF, and the elution liquid was transferred to the synthesis tank R-2105; after the addition was completed, the temperature in the synthesis tank R-2105 was controlled at 20-30°C, nitrogen was bubbled, and the mixture was kept warm and stirred for 2-3 hours (samples were taken for Kaiser test, and the resin and solution were colorless and transparent; ninhydrin was used to test every 1 hour until negative); after the reaction was completed, the solvent in the synthesis tank R-2105 was emptied.

[0141] Add 6.0±0.1L DMF to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; add 2.24±0.1L DMF (7vol, v / w) to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step once; add 2.24±0.1L DMF (7vol, v / w) to the synthesis tank R-2105 for spraying, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step twice.

[0142] 2.10 Solid phase cyclization

[0143] Under nitrogen protection, add 3.3±0.1 Kg DMF and 177.7±1 g iodine (4.0 eq) into the activation tank R-0304 in sequence and stir evenly.

[0144] Add the solution in the activation tank R-0304 dropwise into the synthesis tank R-2105, control the temperature in the synthesis tank R-2105 at 27-32°C, keep the reaction warm for 40±5 minutes, and drain the solvent in the synthesis tank R-2105.

[0145] Add 6.0±0.1L DMF to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; add 2.24±0.1L DMF (7vol, v / w) to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step once; add 2.24±0.1L DMF (7vol, v / w) to the synthesis tank R-2105 for spraying, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step twice.

[0146] Under nitrogen protection, 184.9±1 g vitamin C (3.0 eq), 0.99±0.05 kg methanol and 5.9±0.1 kg DMF were added to the activation tank R-0304 in sequence and stirred to dissolve.

[0147] Control the temperature inside the synthesis tank R-2105 at 20-30°C, add half of the solution (about 2.24±0.05L) in the activation tank R-0304 into the synthesis tank R-2105, keep warm and stir for 10 minutes, and drain the solvent in the synthesis tank R-2105.

[0148] Control the temperature inside the synthesis tank R-2105 at 20-30°C, add the remaining solution (about 2.24±0.05L) in the activation tank R-0304 into the synthesis tank R-2105, keep warm and stir for 10 minutes, and drain the solvent in the synthesis tank R-2105.

[0149] Add 6.0±0.1L DMF to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; add 2.24±0.1LDMF (7vol, v / w) to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step once; add 2.24±0.1LDMF (7vol, v / w) to the synthesis tank R-2105 for spraying, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step twice.

[0150] Add 2.98±0.2Kg DCM (7vol, v / w) into the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; add 1.66±0.1Kg MTBE (7vol, v / w) into the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; add 2.98±0.2Kg DCM (7vol, v / w) into the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step once; add 1.66±0.1Kg MTBE (7vol, v / w) into the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step once.

[0151] Take out the peptide resin in the synthesis tank R-2105, transfer it to the vacuum drying oven D-2101, control the jacket temperature at 30-35°C, vacuum degree ≤-0.08MPa, dry for 10 hours, turn the material every 3-4 hours, weigh it from the 10th hour, weigh it every 2 hours until LOD ≤ 10%, unload it, put it into a double-layer LDPE bag, weigh it, and label it. The material is placed in a refrigerator at 2-8°C.

[0152] 2.11 Peptide resin cleavage

[0153] Under nitrogen protection, add 153±1 kg MTBE (300 vol, v / w) into the settling tank, cool to -20 to -10°C, start stirring at a speed of 100 rpm.

[0154] 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 to the cracking kettle in sequence, the internal temperature was reduced to -10~-5℃, and the rotation speed was 100 rpm.

[0155] Under nitrogen protection, add 690±1g of peptide resin into the cleavage kettle, control the internal temperature at -10~0℃, and rinse the feed port with a small amount of cleavage solution.

[0156] Within 20 minutes, the temperature in the cracking reactor was raised to 5-10°C and maintained at 5-10°C for 1 hour with stirring.

[0157] Within 30 minutes, the temperature in the cracking reactor was raised to 26-30°C and maintained at 26-30°C for 3 hours with stirring.

[0158] Filter, transfer the filtrate into the settling tank R-2101 (control the temperature of the settling tank not exceeding 20°C), wash the resin in the cracking kettle with TFA 3 times, stir for 3 to 5 minutes each time, use 0.22±0.02Kg TFA for each washing, and transfer the washing liquid into the settling tank together.

[0159] The sedimentation tank is stirred for 2 to 4 hours and allowed to settle for 15 to 20 hours.

[0160] The material in the settling tank was transferred to a filter with a screen (three-in-one M-2101) for filtration and washed with MTBE, using 1.5±0.1 Kg MTBE (2.94 vol, v / w) for each washing; each washing lasted 10 to 30 minutes; and the washing operation was repeated 3 times.

[0161] The filter cake in the three-in-one was dried under reduced pressure for 12 hours, stirred every 1 to 2 hours, the jacket temperature was 25 to 35°C, and the vacuum degree was ≤-0.085MPa. After drying, the material in the three-in-one was transferred to the tray drying oven D-2101, and continued to be dried under reduced pressure, weighed every 2 hours, until LOD ≤ 10%.

[0162] After drying, the material is transferred into a double-layer LDPE bag and thermoformed; then put into an aluminum foil bag and thermoformed; a desiccant is added between the LDPE bag and the aluminum foil bag, and the bag is weighed, labeled, and stored at -20±5°C to obtain the crude MS011 (MS011-A).

[0163] In the operation step 2.11, the material feeding amount is calculated based on the peptide resin; the stirring speed under nitrogen bubbling is set to 100 rpm.

[0164] The NMR spectrum, LC-MS spectrum and IR spectrum of the synthesized product are shown in Figure 1-3 .

[0165] Example 2 Lutetium 177 Purification of Lu]-oxyoctreotide precursor compound

[0166] 1. Primary purification

[0167] A certain amount of crude MS011-A (prepared in Example 1) was added to the reactor, and 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 solution was about 30 g / L, and the mixture was stirred until no solid particles were visible.

[0168] Control the temperature in the autoclave at 20-30°C, adjust the pH to 2.00-4.00 with 5% ammonia water (pre-prepared) or 5% trifluoroacetic acid aqueous solution (pre-prepared), stir for 1.5-5 hours, continue to drop 5% ammonia water, adjust the pH to 7.10-7.90, stir for 2-18 hours, and add approximately equal weight of 10% acetonitrile aqueous solution (acetonitrile: pure water = 0.79:9 (w / w)) to the reactor at 20-30°C.

[0169] Filter the material in the kettle with a 0.45μm nylon filter membrane, put the filtrate into a barrel for later use, and detect the relevant substances and their content.

[0170] The prepared sample solution was purified according to the primary purification method. The information of the primary purification method is shown in Table 1:

[0171] Table 1

[0172]

[0173]

[0174] During purification, each fraction is sampled and tested to test the purity of the purified fraction.

[0175] All fractions meeting the MP standard (purity ≥ 99.0% and single impurity ≤ 0.10% by weight) were combined and temporarily stored at 2-8°C for salt conversion and concentration.

[0176] All fractions meeting the RP criteria (purity > 95.0% but not meeting the MP requirement) were combined.

[0177] 2. Recovery and purification

[0178] Add an equal weight of purified water to the fraction barrel that meets the RP standard for distillation and then perform recovery and purification according to the recovery and purification method. The recovery and purification method information is shown in Table 2:

[0179] Table 2

[0180]

[0181]

[0182] During the recovery and purification, each fraction was sampled and Kaiser was used to test the resin color.

[0183] All fractions that meet the MP standard (purity ≥ 99.0% and single impurity ≤ 0.10% by weight) are combined and temporarily stored in a 2-8°C warehouse for salt conversion and concentration; the fractions that do not meet the MP standard are discarded.

[0184] 3. Salt Concentration

[0185] Add an equal amount of purified water to the MP standard distillate barrel to dilute it and then perform salt transfer and concentration according to the salt transfer and concentration method. The salt transfer and concentration method information is shown in Table 3:

[0186]

[0187]

[0188] During the salt conversion and concentration period, each fraction was sampled and tested.

[0189] All fractions that meet the FP standard (purity ≥ 99.0% and single impurity ≤ 0.10% by weight) are combined and filtered through a 0.22μm hydrophobic polytetrafluoroethylene filter element, and the filtrate is sampled to test the resin color. The test result concentration must not exceed 37.6mg / ml. If the test result exceeds 37.6mg / ml, 0.1% trifluoroacetic acid aqueous solution must be added to dilute it to within 37.6mg / ml.

[0190] 4. Freeze-drying

[0191] All FP solutions were freeze-dried according to the freeze-drying procedure, and the height of each tray containing the sample solution was 10±2 mm. The freeze-drying procedure is shown in Table 4:

[0192] Table 4

[0193]

[0194] After freeze-drying, re-press, unpack and package (packaging must be done at a humidity of ≤30%), and notify QC to take samples.

[0195] After sampling, pack according to the packing instructions and put into storage.

[0196] 5. Purification results

[0197] Table 5

[0198]

[0199]

[0200] IPC sample storage conditions: room temperature. The purity test method of MS011 crude solution is as follows:

[0201]

[0202]

[0203] Example 3 Screening of crude product dissolution conditions

[0204] 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, the concentration is about 30 mg / mL, and the stability is examined according to the conditions in Table 6 below:

[0205] Table 6

[0206]

[0207] 2. Take 2 g of the crude product (batch number: MS011-20240126-E0057-02, prepared according to the method of Example 1), add 66 mL of mixed solvent (DMSO / H2O / ACN=2 / 1 / 7, volume ratio) to dissolve, the concentration is about 30 mg / ml, and the stability is examined according to the conditions in Table 7 below:

[0208] Table 7

[0209]

[0210] From experiments 1 and 2, it can be seen that the crude peptide solution was stirred at 30±1°C for 1.5h to 5h at pH 2.28 and 3.98, and the purity of the crude product did not change significantly, and it was basically stable; from experiments 3 and 4, it can be seen that the crude peptide solution was stirred at 20±1°C for 1.5h to 5h at pH 1.98 and 4.00, and the purity of the crude product did not change significantly, and it was basically stable. Therefore, the pH of the crude peptide solution in the first stage of primary purification was set at 2.00-4.00, and the stirring temperature was 20-30°C.

[0211] 3. The solutions of Experiment 3 and Experiment 4 were equally divided into two parts, and the stability was investigated according to the conditions in Table 8 below:

[0212] Table 8

[0213]

[0214] From experiments 5, 6, 7, and 8, it can be seen that after the crude peptide solution of pH 2.00-4.00 is stirred at 20-30°C for 5h, the pH of the crude peptide solution is adjusted to 7.08-7.91, and then stirred at 20-30°C for 2h to 4h, the purity of the crude product has no obvious change and is basically stable. Continue stirring at 20-30°C for 18h, the purity of the crude peptide is slightly reduced, and the impurity (RRT0.18) increases by about 1%, but due to its large difference in properties with the product, it does not affect subsequent purification. Therefore, after the crude peptide solution of pH 2.00-4.00 is stirred at 20-30°C for 1.5h-5h, the pH is adjusted to 7.08-7.91 (for example, 7.10-7.90) using 5% ammonia water, and continued stirring at 20-30°C for 2h-18h, the crude product is basically stable.

[0215] Example 4 Screening of primary purification buffer (Phase A)

[0216] 1. Type of primary purification buffer (phase A)

[0217] The crude solution was purified by using 0.1% TFA-H2O, 0.1% H3PO4-H2O, 0.1% H3PO4+triethylamine, 10mM ammonium bicarbonate and 10% KH2PO4+90% K2HPO4 (10mM, 10mM KH2PO4 and 10mM K2HPO4 solutions were uniformly mixed in a volume ratio of 1:9) aqueous solution 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:

[0218] Table 9

[0219]

[0220]

[0221] According to the results in the above table, it can be seen that the samples obtained in Experiments 4 and 5 have the highest purity, both reaching more than 99%. The yield of Experiment 5 is 87.7%, which is higher than the yield of Experiment 4 (78.5%). Taking comprehensive considerations, the primary purification buffer (phase A) is determined to be 10mM10%KH2PO4+90%K2HPO4 (pH7.87).

[0222] 2. Primary purification buffer (phase A) concentration

[0223] 10% KH2PO4 + 90% K2HPO4 (8mM), 10% KH2PO4 + 90% K2HPO4 (10mM) and 10% KH2PO4 + 90% K2HPO4 (12mM) solutions were used as phase A and acetonitrile was used as phase B to purify the crude solution. The purity and yield of the purified samples were compared. The experimental results are shown in Table 10 below:

[0224] Table 10

[0225]

[0226] According to the above table, the purity of the three groups of experiments was above 99%, the maximum single impurity was ≤0.1% by weight, and the yield was highest when the buffer concentration was 10mM. After comprehensive consideration, the buffer concentration was determined to be 10±1mM.

[0227] 3. pH of primary purification buffer (phase A)

[0228] 10mM 10% KH2PO4+90% K2HPO4 (pH7.60), 10mM 10% KH2PO4+90% K2HPO4 (pH7.87) and 10mM 10% KH2PO4+90% K2HPO4 (pH8.00) buffers were used as phase A and acetonitrile was used as phase B to purify the crude solution. The purity and yield of the purified samples were compared. The experimental results are shown in Table 11 below:

[0229] Table 11

[0230]

[0231] According to the results in the table above, the purity of the samples obtained from the three groups of experiments was all above 99%, the maximum single impurity was ≤0.1% by weight, the yield was all above 80% with no significant difference, and the pH of the buffer solution was determined to be 7.87±0.1 based on comprehensive considerations.

[0232] Example 5 Chromatographic Filler Screening

[0233] Using 10% KH2PO4 + 90% K2HPO4 (10mM) as buffer, the samples were analyzed using UniSil10-100C18, UniHybrid 10-120C18 and UniSil 10-120C4 fillers, and the main peak purity and separation of the samples under different fillers were compared. The results are shown in Table 12 below:

[0234] Table 12

[0235]

[0236] The front impurities next to the main peak of the crude peptide are large and difficult to remove. According to the results in the above table, the separation degree of UniSil 10-100 C18 filler for the front impurities is 1.3, which is the largest among the three groups of experiments, and the purity of the main peak is 74.8%, which is the smallest among the three groups of experiments. Based on the above considerations, UniSil 10-100 C18 filler was determined to be the chromatographic filler.

[0237] Example 6 Screening of primary purification buffer (Phase B)

[0238] Using 10% 10mM KH2PO4 + 90% 10mM K2HPO4 as a buffer, methanol and acetonitrile were added to purify the sample, and the purity and yield of the purified sample were investigated. The experimental results are shown in Table 13 below:

[0239] Table 13

[0240]

[0241]

[0242] According to the results in the above table, it can be seen that the purity of the sample obtained in Experiment 2 is 99.4%, and the yield is 87.7%, both of which are higher than the purity and yield of the sample obtained in Experiment 1. Taking comprehensive consideration, it is determined that the primary purified organic phase is acetonitrile.

[0243] Example 7 Primary purification elution gradient optimization

[0244] The crude solution was purified using different elution gradients: 5% (0 min) -10% (5 min) -30% (95 min), 5% (0 min) -12% (5 min) -32% (95 min), 5% (0 min) -14% (5 min) -34% (95 min) based on the volume percentage of phase B. The purity and yield of the purified samples were compared. The experimental results are shown in Table 14 below:

[0245] Table 14

[0246]

[0247] According to the results in the table above, the sample purity obtained in Experiment 2 is 99.4% and the yield is 87.2%. The sample purity and maximum single impurity obtained in Experiments 1 and 3 also meet the requirements, but the yields are 58.9% and 56.9%, respectively, which are lower than those in Experiment 2. After comprehensive consideration, the primary purification elution gradient is determined to be 5% (0 min) -12±1% (5 min) -32±1% (95 min).

[0248] Example 8: Primary purification loading test

[0249] During the first purification process, 16 g and 18 g of the sample were loaded respectively to investigate the purity and yield of the purified samples. The results are shown in Table 15 below:

[0250] Table 15

[0251]

[0252]

[0253] From the results, we know that when the sample amount is 16g, the sample purity is 99.7%, the maximum single impurity is 0.09% by weight, and the yield is 76.7%. When the sample amount is further increased to 18g, the sample purity is 99.7%, the maximum single impurity is 0.09% by weight, and the yield is 69.4%. Taking all factors into consideration, it is determined that the purification load range is 16 to 18g.

[0254] Example 9 Recovery and purification sample loading test

[0255] During the first purification and recovery process, 14.1 g, 11.6 g and 16.8 g were loaded respectively to investigate the purity and yield of the purified samples. The results are shown in Table 16 below:

[0256] Table 16

[0257]

[0258] From the results in the above table, we know that the loading capacity of Experiment 2 is 11.6g, the purity of the obtained sample is 99.8%, and the maximum single impurity is 0.08% by weight. When the loading capacity is further increased to 14.1g and 16.8g, the maximum single impurity is greater than 0.1% by weight. Taking comprehensive considerations, it is determined that the recovery and purification loading capacity is ≤11.6g.

[0259] Example 10 Salt Concentration Sample Loading Test

[0260] During the salt conversion and concentration process, 47.4 g, 42.6 g and 37.7 g of samples were loaded respectively to investigate the purity and yield of the purified samples. The results are shown in Table 17 below:

[0261] Table 17

[0262]

[0263] From the results, we can see that when the sample amount is 37.7g, the sample purity is 99.9% and the yield is 95.7%. When the sample amount increases to 47.4g, the sample purity is 99.7% and the yield is 85.0%. Taking all factors into consideration, the salt conversion and concentration loading range is determined to be ≤42.6g.

[0264] Example 11 Study of freeze-drying parameters

[0265] 1. Freeze-dried thickness

[0266] The salt conversion concentrated collection solution was placed in a freeze drying tray for freeze drying. The freeze drying thickness was 8 mm, 10 mm and 12 mm respectively. The moisture and acetonitrile residues of the samples obtained after freeze drying were investigated. The experimental results are shown in Table 18 below:

[0267] Table 18

[0268]

[0269] From the above experimental results, it can be seen that there is no significant difference in the moisture content and acetonitrile residue of the freeze-dried samples obtained in the three groups of experiments. After comprehensive consideration, the freeze-dried thickness was determined to be 10±2mm.

[0270] 2. Freeze-dried concentration

[0271] The concentrated collection solutions of different concentrations of salt conversion were placed in freeze-drying trays and freeze-dried according to the same freeze-drying curve. The water content and acetonitrile residue of the samples obtained after freeze-drying were investigated. The experimental results are shown in Table 19 below:

[0272] Table 19

[0273]

[0274] From the results, it can be seen that with the increase of freeze-drying concentration, the moisture content and residual acetonitrile content of the freeze-dried sample showed a gradual increasing trend. When the freeze-drying concentration was 37.6 mg / mL, the moisture content of the freeze-dried sample was 5.9%, and the residual acetonitrile content was 0.14%. If the freeze-drying concentration continues to increase, the moisture content and residual acetonitrile content of the freeze-dried sample will have the risk of continuing to increase. After comprehensive consideration, the freeze-drying concentration was determined to be ≤37.6 mg / mL.

[0275] 3. Freeze-drying curve

[0276] The salt-converted concentrated collection solution was placed in a freeze-drying tray and freeze-dried according to different freeze-drying curves. The experimental process is shown in Table 20 below:

[0277] Table 20

[0278]

[0279] The moisture and acetonitrile residues of the freeze-dried samples were tested, and the results are shown in Table 21 below:

[0280] Table 21

[0281]

[0282] Experiment 2 extended the sublimation drying on the basis of Experiment 1, and the -10℃ insulation time was extended to 1600min. The acetonitrile residue decreased from 0.14% to 0.04%, and the acetonitrile residue removal effect was obvious; Experiment 3 reduced the sublimation drying temperature to -15℃, and Experiment 4 reduced the pre-freezing temperature to -55℃. The moisture content and acetonitrile residue of the obtained samples did not decrease significantly compared with Experiment 2. Comprehensive consideration determined that the freeze-drying curve was the freeze-drying curve used in Experiment 2.

[0283] 4. Packaging humidity

[0284] After the samples were freeze-dried, they were packaged under different humidity conditions and the moisture content of the samples was tested. The results are shown in Table 22 below:

[0285] Table 22

[0286]

[0287] From the above results, it can be seen that with the increase of environmental humidity, the moisture content of the sample shows a trend of slowly increasing. When the environmental humidity is 79.3%, the moisture content of the sample is 5.9%. In order to ensure that the moisture content of the sample is qualified, the packaging humidity is determined to be ≤30%.

[0288] The above are only several exemplary embodiments of the present invention, and are not intended to limit the present invention in any form. Although the present invention is disclosed as above with preferred embodiments, it is not intended to limit the present invention. Any technician familiar with the profession, without departing from the scope of the technical solution of the present invention, using the technical content disclosed above to make some changes or modifications are equivalent to equivalent embodiments and fall within the scope of the technical solution of the present invention.

Claims

1. A lutetium represented by formula I [ 177 A method for purifying a precursor compound of Lu] oxyoctreotide, comprising the following steps: (1) Lutetium [ 177 The crude product of Lu]oxoctreotide precursor compound is dissolved in a mixed solvent of an organic solvent and water, and the pH of the obtained solution is adjusted to 2.00-4.00, and then adjusted to 7.08-7.91, and filtered; (2) Purifying the filtrate obtained in step (1) by high performance liquid chromatography, wherein the conditions of the high performance liquid chromatography include: using octadecylsilane bonded silica gel as the chromatographic column filler; using an aqueous solution of KH2PO4 and K2HPO4 with a molar ratio of 1:9 and a total concentration of 9 to 11 mM, preferably 10 mM as mobile phase A, and using acetonitrile as mobile phase B, and eluting according to the following gradient: From 0 to 5 minutes, the volume ratio of mobile phase A to mobile phase B was 95:5; 5 to 95 minutes, the volume ratio of mobile phase A to mobile phase B is 87-89:13-11, preferably 88:12; After 95 minutes, the volume ratio of mobile phase A to mobile phase B is 67-69:33-31, preferably 68:32; 2. The purification method according to claim 1, wherein In step (1), the mixed solvent of 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 [ 177 The concentration of Lu] oxoctreotide precursor compound is 25 to 35 g / L, preferably 30 g / L; Preferably, 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% by volume acetonitrile aqueous solution is added, and filtered; preferably, 5% by volume ammonia water is used to adjust the pH; preferably, 0.45 μm nylon filter membrane is used for filtration.

3. The purification method according to claim 1, wherein In step (2), the chromatographic column filler is UniSil 10-100C18 filler; Preferably, in step (2), the sample loading amount of the chromatographic column is 16 to 18 g; Preferably, in step (2), the pH of the mobile phase A is 7.77 to 7.97, preferably 7.87; Preferably, in step (2), the detection wavelength of the high performance liquid chromatography is 230 nm; Preferably, in step (2), the elution flow rate of the mobile phase is 880 mL / min; Preferably, in step (2), fractions with a purity of ≥99.0% and a single impurity of ≤0.10% by weight are collected, and for fractions with a purity of ≥95% and ≤99.0% and a single impurity of >0.10% by weight, the high performance liquid chromatography secondary purification is repeated to obtain a fraction with a purity of ≥99.0% and a single impurity of ≤0.10% by weight; preferably, the fraction with a purity of ≥95% and ≤99.0% and a single impurity of >0.10% by weight is diluted with an equal weight of water before the secondary purification; preferably, the sample loading capacity of the chromatographic column for the secondary purification is ≤11.6 g.

4. The purification method according to claim 1, further comprising the following steps: (3) The fraction obtained in step (2) is subjected to salt conversion and concentration by high performance liquid chromatography, wherein the conditions of the high performance liquid chromatography include: using octadecylsilane bonded silica gel as the chromatographic column filler; using a 0.1% by volume aqueous solution of trifluoroacetic acid as the mobile phase A, and using acetonitrile as the mobile phase B, and eluting according to the following gradient: From 0 to 25 min, the volume ratio of mobile phase A to mobile phase B was 95:5; 25 to 75 minutes, the volume ratio of mobile phase A to mobile phase B was 80:20; After 75 minutes, the volume ratio of mobile phase A to mobile phase B was 30:

70.

5. The purification method according to claim 4, wherein In step (3), the chromatographic column filler is UniSil 10-100C18 filler; Preferably, in step (3), the sample loading amount of the chromatographic column is ≤42.6 g; Preferably, in step (3), the detection wavelength of the high performance liquid chromatography is 230 nm; Preferably, in step (3), the elution flow rate of the mobile phase is 880 mL / min; Preferably, in step (3), the fraction obtained in step (2) is diluted by adding an equal weight of water before salt conversion and concentration; Preferably, in step (3), a fraction having a purity of ≥99.0% and a single impurity of ≤0.10% by weight is collected.

6. The purification method according to claim 4, further comprising the following steps: (4) Lyophilizing the fraction obtained in step (3).

7. The purification method according to claim 6, wherein In step (4), the freeze-drying comprises the following steps: (4-1) Prefreezing: cooling to -45 to -55°C, preferably -45°C, within 30 minutes and maintaining for 210 minutes; (4-2) primary sublimation: under a vacuum degree of 0.2 to 0.25 mbar, preferably 0.2 mbar, heating to -10 to -15°C, preferably -10°C, within 30 minutes, and maintaining for 510 to 1600 minutes, preferably 1600 minutes; then heating to 5°C within 30 minutes, and maintaining for 1470 to 1500 minutes, preferably 1500 minutes; (4-3) Desorption drying: under a vacuum degree 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.

8. The purification method according to claim 6 or 7, wherein: In step (4), the thickness of the freeze-dried fraction is 8 to 12 mm; Preferably, in step (4), the lutetium represented by formula I in the fraction before freeze-drying is 177 The concentration of Lu]oxyoctreotide precursor compound is ≤37.6 mg / mL.

9. The purification method according to any one of claims 6 to 8, further comprising the following steps: (5) packaging the freeze-dried fraction obtained in step (4); Preferably, the packaging is carried out at a humidity of ≤30%.

10. A lutetium 177 A method for preparing Lu] oxyoctreotide, comprising treating the lutetium [ 177 Lu] oxyoctreotide precursor compound and lutetium [ 177 Lu] chelation.

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