Polypeptide drug VAP-DOX salt form and preparation method thereof

Abstract

The invention discloses a polypeptide drug VAP-DOX salt form and a preparation method thereof. The preparation method comprises the following steps: carrying out salt conversion on a solution containing the polypeptide drug VAP-DOX to obtain the polypeptide drug VAP-DOX, wherein the salt conversion adopts a Pre-HPLC (High Performance Liquid Chromatography) method, the mobile phase A comprises acetonitrile or methanol, and the mobile phase B comprises an aqueous solution containing ammonium salt or alkali metal salt; and the pH value of the mobile phase B is 6-8. The polypeptide drug VAP-DOX salt form is hydrochloride, sulfate or acetate. According to the preparation method, the specific VAP-DOX salt form with good stability can be effectively obtained, the high yield is achieved, large-scale production of VAP-DOX can be achieved, and a foundation is laid for product quality and industrialization of VAP-DOX raw material medicine.

Description

Technical Field

[0001] The present invention relates to the field of drug synthesis, and particularly to a salt form of polypeptide drug VAP-DOX and a preparation method thereof. Background Art

[0002] CN108164584A has clarified the targeted diagnosis and treatment effect of VAP-DOX (structural formula is shown below) on tumors; among them, this molecule has a typical PDC drug structure, the linker is a maleimide structure, the cytotoxic molecule is doxorubicin, the polypeptide segment is coupled through the sulfur atom of the cysteine side chain, and the cytotoxic molecule is coupled to the linker through an imine structure; due to the presence of a hydrazone structure in this molecular structure, there are often problems of extremely poor stability.

[0003] Based on the above characteristics, it is necessary to further study the preparation process of this molecule to realize its industrialization.

[0004] Summary of the Invention

[0005] To solve the problem of poor stability of the polypeptide drug VAP-DOX, the present invention provides a salt form of the polypeptide drug VAP-DOX and a preparation method thereof. This preparation method can effectively obtain a specific salt form of VAP-DOX, and the salt form of VAP-DOX has good stability; in addition, this preparation method has a high yield, which helps to realize the scale-up production of VAP-DOX and lays a foundation for the product quality and industrialization of VAP-DOX raw materials.

[0006] To achieve the above invention purpose, the present invention provides the following technical solutions:

[0007] The first aspect of the present invention provides a preparation method of a salt form of a polypeptide drug VAP-DOX, which comprises the following steps:

[0008] Carry out salt conversion on the solution containing the polypeptide drug VAP-DOX to obtain it;

[0009] Wherein, the salt conversion adopts the Pre-HPLC method, mobile phase A includes acetonitrile or methanol, and mobile phase B includes an aqueous solution containing an ammonium salt or an alkali metal salt; the pH of mobile phase B is 6-8.

[0010] In the present invention, the ammonium salt in mobile phase B can be selected from one or more of ammonium acetate, ammonium chloride and ammonium bicarbonate.

[0011] In the present invention, the alkali metal salt in mobile phase B can be selected from sodium salt and / or potassium salt; the sodium salt is preferably selected from one or more of sodium acetate, sodium chloride and sodium bicarbonate; the potassium salt is preferably selected from one or more of potassium acetate, potassium chloride and potassium bicarbonate.

[0012] In the present invention, the concentration of the ammonium salt or alkali metal salt in the mobile phase B can be 5 - 50 mM, preferably 30 mM.

[0013] In the present invention, the pH of the mobile phase B can be 6.5 - 7.5, preferably 7.1.

[0014] In the present invention, the pH of the mobile phase B can be adjusted by a pH regulator; the pH regulator is preferably selected from one or more of sulfuric acid, hydrochloric acid, acetic acid, phosphoric acid, and formic acid, more preferably one or more of sulfuric acid, hydrochloric acid, and acetic acid.

[0015] In some embodiments, the mobile phase B is an aqueous solution containing an ammonium salt, the ammonium salt is ammonium acetate, and the pH regulator is acetic acid; preferably, the concentration of ammonium acetate is 5 - 50 mM, and the pH of the mobile phase B is 6.5 - 7.5.

[0016] In some embodiments, the mobile phase B is an aqueous solution containing an ammonium salt, the ammonium salt is ammonium chloride, and the pH regulator is hydrochloric acid; preferably, the concentration of ammonium chloride is 5 - 50 mM, and the pH of the mobile phase B is 6.5 - 7.5.

[0017] In some embodiments, the mobile phase B is an aqueous solution containing an ammonium salt, the ammonium salt is ammonium bicarbonate, and the pH regulator is sulfuric acid; preferably, the concentration of ammonium bicarbonate is 5 - 50 mM, and the pH of the mobile phase B is 6.5 - 7.5.

[0018] In the present invention, in the step of salt conversion, the Pre - HPLC method can be carried out in a gradient elution manner, and the gradient elution is preferably a two - stage gradient elution.

[0019] Preferably, in the Pre - HPLC method, the elution mode of the two - stage gradient elution is as follows:

[0020] Time (min) Mobile phase A (%) Mobile phase B (%) 0 5～10 95～80 5～10 15～20 85～80 30～35 45～50 55～50

[0021] Wherein, the meaning of % is the percentage of the mobile phase A or mobile phase B in the sum of the volumes of the mobile phase A and mobile phase B.

[0022] In the present invention, the solution containing the polypeptide drug VAP - DOX may further include a purification step before the step of salt conversion.

[0023] Preferably, the purification is carried out by the Pre - HPLC method, the mobile phase A includes acetonitrile or methanol, and the mobile phase B includes an aqueous solution containing an ammonium salt or an alkali metal salt.

[0024] More preferably, the types of ammonium salts and alkali metal salts in mobile phase B in the purification step are the same as those in mobile phase B in the salt conversion step.

[0025] Even more preferably, the ammonium salt in mobile phase B in the purification step further includes ammonium sulfate.

[0026] In some embodiments, in the purification step, mobile phase B is an aqueous solution containing ammonium salts, and the ammonium salts are ammonium acetate and ammonium sulfate; preferably, the concentrations of ammonium acetate and ammonium sulfate are each independently 5 - 100 mM, more preferably 30 - 100 mM, such as 60 mM; more preferably, the concentrations of ammonium acetate and ammonium sulfate are the same.

[0027] In some embodiments, in the purification step, mobile phase B is an aqueous solution containing ammonium salts, and the ammonium salts are ammonium chloride and ammonium sulfate; preferably, the concentrations of ammonium chloride and ammonium sulfate are each independently 5 - 100 mM, more preferably 30 - 100 mM, such as 60 mM; more preferably, the concentrations of ammonium chloride and ammonium sulfate are the same.

[0028] In some embodiments, in the purification step, mobile phase B is an aqueous solution containing ammonium salts, and the ammonium salts are ammonium bicarbonate and ammonium sulfate; preferably, the concentrations of ammonium bicarbonate and ammonium sulfate are each independently 5 - 100 mM, more preferably 30 - 100 mM, such as 60 mM; more preferably, the concentrations of ammonium bicarbonate and ammonium sulfate are the same.

[0029] In the present invention, in the purification step, the Pre - HPLC method can be carried out in a gradient elution manner, and the gradient elution is preferably a two - stage gradient elution.

[0030] Preferably, in the Pre - HPLC method, the elution mode of the two - stage gradient elution is as follows:

[0031] Time (min) Mobile phase A (%) Mobile phase B (%) 0 5～10 95～90 5～15 13～18 87～82 65～80 43～48 57～52

[0032] Wherein, the meaning of % is the percentage of mobile phase A or mobile phase B in the sum of the volumes of mobile phase A and mobile phase B.

[0033] In the present invention, in the salt conversion step, the chromatographic column can be a reversed - phase column; preferably, the packing of the reversed - phase column is C4, C8, or C18; more preferably, the parameter of the packing is 10 μm

[0034] In the present invention, in the salt conversion step, the diameter of the chromatographic column can be 20 mm.

[0035] In the present invention, in the step of salt conversion, the flow rate can be 5 - 15 mL / min, preferably 8 - 12 mL / min.

[0036] In the present invention, in the step of purification, the chromatographic column can be a reversed-phase column; preferably, the packing material of the reversed-phase column is C4, C8, or C18; more preferably, the parameter of the packing material is 10 μm

[0037] In the present invention, in the step of purification, the diameter of the chromatographic column can be 20 mm.

[0038] In the present invention, in the step of purification, the flow rate can be 5 - 15 mL / min, preferably 10 mL / min.

[0039] In the present invention, in the step of purification, the sample loading amount can be 0.5% - 1%, where the meaning of % is the mass ratio of the sample to the packing material.

[0040] In the present invention, the "solution containing polypeptide drug VAP-DOX" can be an aqueous solution containing polypeptide drug VAP-DOX.

[0041] In the present invention, the preparation of the "solution containing polypeptide drug VAP-DOX" can include the following steps:

[0042] Dissolve and filter the polypeptide drug VAP-DOX to obtain it.

[0043] Among them, the filtration can be conventional in the art, preferably filtered using a mixed cellulose membrane; the pore size of the mixed cellulose membrane is preferably 0.45 μm.

[0044] In the present invention, the freeze-drying can be conventional in the art, preferably including the steps of pre-freezing, primary drying, and secondary drying.

[0045] Among them, the conditions for pre-freezing are, for example, pre-freezing at -60 °C for 3 h.

[0046] Among them, the conditions for primary drying are, for example, drying at -10 °C for 16 h, and then drying at -5 °C for 16 h.

[0047] Among them, the conditions for secondary drying are, for example, drying at 5 °C for 53 h, and then drying at 15 °C for 6 h.

[0048] In the present invention, the polypeptide drug VAP-DOX can be prepared by a conventional method in the art.

[0049] In some preferred embodiments, the preparation method of the polypeptide drug VAP-DOX includes the following steps:

[0050] S1. In tetrahydrofuran, in the presence of EDCI·HCl, maleimidocaproic acid and tert-butyl hydrazinecarboxylate are reacted, and after purification, N-tert-butoxycarbonyl-6-maleimidocaproyl hydrazide is obtained;

[0051] S2. In dichloromethane, N-tert-butoxycarbonyl-6-maleimidocaproyl hydrazide and trifluoroacetic acid are reacted, and after purification, 6-maleimidocaproyl hydrazide is obtained;

[0052] S3. 6-Maleimidocaproyl hydrazide is coupled with doxorubicin hydrochloride, and after purification, maleimidocaproyl hydrazide doxorubicin is obtained;

[0053] S4. Maleimidocaproyl hydrazide doxorubicin is coupled with an octapeptide ( D VAP-Cys), centrifuged, washed, and dried to obtain a crude product of the polypeptide drug VAP-DOX; wherein, the D chemical structure of VAP-Cys is NH2-D-Pro-D-Ala-D-Val-D-Arg-D-Thr-D-Asn-D-Ser-D-Cys-OH.

[0054] The present invention also provides a salt form of the polypeptide drug VAP-DOX, which is prepared according to the preparation method of the salt form of the polypeptide drug VAP-DOX as described above.

[0055] The present invention also provides a salt form of the polypeptide drug VAP-DOX, and its structure is shown in the following formula:

[0056]

[0057] Wherein, the X is selected from Cl - , SO4 2- or CH3COO - .

[0058] On the basis of conforming to the common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

[0059] The reagents and raw materials used in the present invention are all commercially available.

[0060] The positive and progressive effects of the present invention are as follows:

[0061] The present invention prepares a stable VAP-DOX salt form through a specific Pre-HPLC salt conversion method, obtaining various stable forms of VAP-DOX, providing a greater selection space for the requirements of VAP-DOX as raw materials for different pharmaceutical preparations; this preparation method has a high yield, helps to realize the large-scale production of VAP-DOX, lays a foundation for the product quality and industrialization of VAP-DOX raw materials, and makes the large-scale production of the targeted therapeutic polypeptide drug VAP-DOX a reality. Description of the Drawings

[0062] Figure 1 It is the chemical structural formula of VAP-DOX.

[0063] Figure 2 It is the process route diagram for the preparation of the VAP-DOX salt form of the present invention.

[0064] Figure 3 It is the chromatogram of the VAP-DOX acetate prepared in Example 1 of the present invention.

[0065] Figure 4 It is the chromatogram of the VAP-DOX hydrochloride prepared in Example 2 of the present invention.

[0066] Figure 5 It is the chromatogram of the VAP-DOX sulfate prepared in Example 3 of the present invention. Detailed Description of the Invention

[0067] The present invention discloses a method for preparing a salt form of a polypeptide drug VAP-DOX and its preparation method. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make changes or appropriate modifications and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0068] The meanings of some abbreviations used in the present invention are shown in Table 1 as follows:

[0069] Table 1. Related descriptions of the abbreviations involved in the present invention

[0070]

[0071]

[0072] The present invention provides a method for preparing a stable salt form of a polypeptide drug VAP-DOX (the structure is as Figure 1 shown), including the following steps:

[0073] 1. Preparation of DOX-3:

[0074] Add tetrahydrofuran to a reactor, add raw material DOX-2 and stir to dissolve it; cool it to 0 - 10°C in an ice bath, add tert-butyl carbazate and purified water and stir until clear; control the reaction system at 0 - 10°C, and slowly add EDCI·HCl in batches. After the addition of EDCI·HCl is completed, react at 0 - 10°C for 1 - 2 hours.

[0075] After the reaction is completed, add ethyl acetate to the reaction solution, stir for 10 minutes and let it stand for 5 - 10 minutes, and separate and drain the lower aqueous phase. Add purified water to the organic phase again, stir for 10 minutes, and separate and drain the lower aqueous phase. Add saturated sodium chloride aqueous solution to the organic phase again, stir for 10 minutes, and separate and drain the lower aqueous phase. Dry the organic phase with anhydrous sodium sulfate. Filter, wash the filter cake with ethyl acetate and combine the filtrates. Concentrate the filtrates under reduced pressure until no obvious solvent drips out, and stop to obtain the target compound DOX-3.

[0076] 2. Preparation of DOX-4:

[0077] Add dichloromethane and DOX-3 to a reaction flask, control the temperature at 20 - 30°C and dropwise add trifluoroacetic acid. After the addition is completed, maintain the reaction at 20 - 30°C for 2 - 3 hours. After the reaction is completed, concentrate the reaction solution under reduced pressure (stop when no obvious solvent drips out). Add ethyl acetate to the residue and stir to disperse it, then cool it to 0 - 10°C, add n-heptane, and a large amount of solid precipitates; raise the temperature to 20 - 30°C and stir for 1 hour. Filter, wash the filter cake with a mixed solvent of ethyl acetate and n-heptane, filter to obtain the wet product, and dry the wet product under reduced pressure at 30 - 40°C to constant weight to obtain DOX-4.

[0078] 3. Preparation of DOX-5:

[0079] Add methanol and DOX-4 to a reaction flask, stir to dissolve, then add DOX, and react at 20 - 30°C for 2 - 3 hours. After the reaction is completed, add the reaction solution to methyl tert-butyl ether and control the internal temperature at 20 - 30°C. A large amount of solid precipitates, stir for 30 minutes, and filter. Add the filter cake to acetonitrile, slurry at 20 - 30°C for 1 hour, centrifuge to obtain the wet product, and dry the wet product under reduced pressure at 30 - 40°C to constant weight to obtain DOX-5.

[0080] 4. Preparation of crude VAP-DOX:

[0081] Add purified water to a reaction flask, DVAP-Cys and ammonium salt were stirred and dissolved, and the temperature was lowered to 0 - 10 °C. Methanol was added to the reaction flask, and the temperature was lowered to 0 - 10 °C, then the raw material DOX-5 was added. The temperature inside the reaction flask was controlled at 0 - 10 °C, and the above aqueous solution was added dropwise to the reaction flask. After the addition was completed, the reaction was carried out at 0 - 10 °C for 1 hour. The reaction solution was centrifuged, and the filter cake was washed with ethanol. The filter cake was dried under vacuum at 30 - 40 °C until constant weight to obtain the crude product of LN005.

[0082] 5. Preparation of VAP-DOX salt form:

[0083] (1) Dissolution and filtration

[0084] The crude product was dissolved in purified water to prepare a solution with a concentration of about 10 mg / mL, and then filtered through a 0.45 μm mixed cellulose membrane CA-CN to remove insoluble substances. The filtrate was directly used for the subsequent process steps.

[0085] (2) Prep-HPLC purification

[0086] The sample injection amount for a single purification was about sample / filler ≤ 1.1%.

[0087] Mobile phase: Mobile phase A (acetonitrile), Mobile phase B (aqueous solution containing 60 mmol / L specific ammonium salt and 60 mmol / L ammonium sulfate).

[0088] Gradient (as shown in Table 2):

[0089] Table 2. Changes in the concentration of the mobile phase with time during the Prep-HPLC purification step

[0090]

[0091] The meaning of % in the table is the percentage of Mobile phase A or Mobile phase B in the sum of the volumes of Mobile phase A and Mobile phase B.

[0092] Flow rate: 10 mL / min;

[0093] Chromatographic column: 20 columns (250 mm * 20 mm);

[0094] Filler: Conventional C18 10 μm Reverse-phase high-performance liquid chromatography filler;

[0095] Sample collection: According to the main peak of the Prep-HPLC detection chart, 2 - 3 sections of components were collected in segments, and samples were taken to detect the HPLC purity. Components with an HPLC purity ≥ 98% were qualified components, and the collected sample solutions were stored frozen at -20 °C.

[0096] (3) Salt conversion by Prep-HPLC

[0097] Combine all qualified components and dilute with water to twice the original volume, and load all qualified components for salt conversion.

[0098] Mobile phase: Mobile phase A (acetonitrile), Mobile phase B (aqueous solution containing 30 mmol / L specific ammonium salt, adjusted to pH 7.1);

[0099] Gradient (as shown in Table 3):

[0100] Table 3. Changes in mobile phase concentration over time during the salt conversion step of Prep-HPLC

[0101]

[0102] In the table, the meaning of % is the percentage of Mobile phase A or Mobile phase B in the sum of the volumes of Mobile phase A and Mobile phase B.

[0103] Flow rate: 8 mL / min;

[0104] Chromatographic column: 20 columns (250 mm * 20 mm);

[0105] Packing: Conventional C18 10 μm Reverse-phase high-performance liquid chromatography packing;

[0106] Collect the main peak solution, sample and detect the HPLC purity, and the qualified samples enter the subsequent processes.

[0107] (4) Freeze-drying

[0108] Immediately subject the collected salt-converted solution to freeze-drying. The detailed freeze-drying procedure is as follows:

[0109] Pre-freezing: Lower the shelf temperature to -60 °C within 1 h after starting the freeze-dryer and maintain for 3 h;

[0110] First-stage drying: Raise the shelf temperature from -60 °C to -10 °C within 1 h and maintain for 16 h, then raise the shelf temperature from -10 °C to -5 °C within 1 h and maintain for 16 h (pre-vacuum: 0.2 mbar, vacuum alarm: 0.4 mbar, cold trap temperature: -70 °C);

[0111] Second-stage drying: Raise the shelf temperature from -5 °C to 5 °C within 1 h and maintain for 53 h, then raise the shelf temperature from -5 °C to 15 °C within 1 h and maintain for 6 h (pre-vacuum: 0.2 mbar, vacuum alarm: 0.4 mbar, cold trap temperature: -70 °C).

[0112] After drying, the VAP-DOX salt form is obtained.

[0113] The reagents or instruments used in the present invention can all be purchased from the market.

[0114] The present invention will be further described below in conjunction with embodiments:

[0115] Example 1 Preparation of VAP-DOX acetate

[0116] As Figure 2 shown, the preparation method of this example specifically includes the following steps:

[0117] 1. Preparation of DOX-3:

[0118] Add tetrahydrofuran to the reactor, add the raw material DOX-2 (17.03 g) and stir to dissolve; cool to 0 - 10 °C in an ice bath, add tert-butyl carbazate (10.14 g) and purified water and stir until clear; control the reaction system at 0 - 10 °C, and slowly add EDCI.HCl (23.19 g) in batches. After the addition of EDCI.HCl is completed, react at 0 - 10 °C for 1 - 2 hours. After the reaction is completed, add ethyl acetate to the reaction solution, stir for 10 minutes and let stand for 5 - 10 minutes, and separate and drain the lower aqueous phase. Add purified water to the organic phase again, stir for 10 minutes, and separate and drain the lower aqueous phase. Add saturated sodium chloride aqueous solution to the organic phase again, stir for 10 minutes, and separate and drain the lower aqueous phase. The organic phase is dried with anhydrous sodium sulfate. Filter, wash the filter cake with ethyl acetate and combine the filtrate. The filtrate is concentrated under vacuum until no obvious solvent drips out, and the target compound DOX-3 is obtained.

[0119] 2. Preparation of DOX-4:

[0120] Add dichloromethane and DOX-3 (25.70 g) to the reaction flask, control the temperature at 20 - 30 °C and dropwise add trifluoroacetic acid (59.30 g). After the addition is completed, maintain the reaction at 20 - 30 °C for 2 - 3 hours. After the reaction is completed, the reaction solution is concentrated under reduced pressure (stop when no obvious solvent drips out). Add ethyl acetate to the residue and stir to disperse, then cool to 0 - 10 °C, add n-heptane, and a large amount of solid precipitates; raise the temperature to 20 - 30 °C and stir for 1 hour. Filter, wash the filter cake with a mixed solvent of ethyl acetate and n-heptane, filter to obtain the wet product, and dry the wet product under vacuum at 30 - 40 °C to constant weight to obtain DOX-4.

[0121] 3. Preparation of DOX-5:

[0122] Add methanol and DOX-4 (17.20 g) to the reaction flask, stir to dissolve, then add DOX (10.88 g), and react at 20 - 30 °C for 2 - 3 hours. After the reaction is completed, add the reaction solution to methyl tert-butyl ether and control the internal temperature at 20 - 30 °C. A large amount of solid precipitates, stir for 30 minutes, and filter. Add the filter cake to acetonitrile, slurry at 20 - 30 °C for 1 hour, centrifuge to obtain the wet product, and dry the wet product under vacuum at 30 - 40 °C to constant weight to obtain DOX-5.

[0123] 4. Preparation of crude VAP-DOX:

[0124] Add purified water, DVAP-Cys (9.97 g), and ammonium acetate to a reaction flask and stir to dissolve. Cool the temperature to 0 - 10°C. Add methanol to the reaction flask, cool the temperature to 0 - 10°C, and add the raw material DOX-5 (7.44 g). Control the temperature inside the reaction flask at 0 - 10°C, and add the above aqueous solution dropwise to the reaction flask. After the dropwise addition, react at 0 - 10°C for 1 hour. Centrifuge the reaction solution, and wash the filter cake with ethanol. Dry the filter cake under vacuum at 30 - 40°C until it reaches a constant weight to obtain the crude LN005.

[0125] 5. Preparation of VAP-DOX acetate:

[0126] (1) Dissolution and filtration

[0127] Take the crude VAP-DOX (0.535 g) and prepare a crude product solution with purified water as the solvent at about 10 mg / mL. Then filter to remove insoluble substances using a 0.45 μm mixed cellulose membrane CA-CN. The filtrate is directly used for the subsequent process steps.

[0128] (2) Prep-HPLC purification

[0129] The single injection for purification is about sample / filler ≤ 1.1%.

[0130] Mobile phase: Mobile phase A (acetonitrile), Mobile phase B (aqueous solution containing 60 mmol / L ammonium acetate and 60 mmol / L ammonium sulfate).

[0131] Gradient (as shown in Table 4):

[0132] Table 4. Variation of mobile phase concentration with time in the Prep-HPLC purification step

[0133]

[0134] The meaning of % in the table is the percentage of Mobile phase A or Mobile phase B in the sum of the volumes of Mobile phase A and Mobile phase B.

[0135] Flow rate: 10 mL / min;

[0136] Chromatographic column: 20 columns (250 mm * 20 mm);

[0137] Filler: Conventional C18 10 μm Reverse-phase high-performance liquid chromatography filler;

[0138] Sample collection: Collect 2 - 3 fractions by segmenting the main peak in the Prep-HPLC detection chart, and take samples to detect the HPLC purity. Control the HPLC purity of the fractions ≥ 98% as qualified fractions, and store the collected sample solution at -20°C for freezing preservation.

[0139] (3) Salt conversion by Prep-HPLC

[0140] Combine all qualified fractions and dilute them with water to twice the original volume, and then load all qualified fractions for salt conversion.

[0141] Mobile phase: Mobile phase A (acetonitrile), Mobile phase B (aqueous solution containing 30 mmol / L ammonium acetate, adjusted to pH 7.1 with acetic acid);

[0142] Gradient (as shown in Table 5):

[0143] Table 5. Variation of mobile phase concentration with time in the Prep-HPLC salt conversion step

[0144]

[0145] The meaning of % in the table is the percentage of Mobile phase A or Mobile phase B in the sum of the volumes of Mobile phase A and Mobile phase B.

[0146] Chromatographic column: 20 columns (250 mm * 20 mm);

[0147] Packing: Conventional C18 10μm Reverse-phase high-performance liquid chromatography packing;

[0148] Collect the main peak solution, take samples to detect the HPLC purity, and the qualified samples enter the subsequent processes.

[0149] (4) Freeze-drying

[0150] Immediately perform freeze-drying on the collected salt conversion solution, and the detailed freeze-drying procedure is as follows:

[0151] Pre-freezing: Lower the shelf temperature to -60°C within 1 h after starting the freeze-dryer and maintain it for 3 h;

[0152] First-stage drying: Raise the shelf temperature from -60°C to -10°C within 1 h and maintain it for 16 h, and then raise the shelf temperature from -10°C to -5°C within 1 h and maintain it for 16 h (Pre-vacuum: 0.2 mbar, Vacuum alarm: 0.4 mbar, Trapping temperature: -70°C);

[0153] Second-stage drying: Raise the shelf temperature from -5°C to 5°C within 1 h and maintain it for 53 h, and then raise the shelf temperature from -5°C to 15°C within 1 h and maintain it for 6 h (Pre-vacuum: 0.2 mbar, Vacuum alarm: 0.4 mbar, Trapping temperature: -70°C).

[0154] After drying, 0.245 g of VAP-DOX acetate with a purity of 98.92% (merged peak, corresponding peak numbers 7 and 8) was obtained, as Figure 3 shown in Table 6; the purification yield was 75.0%; the total yield was 45.8%.

[0155] Table 6. Chromatographic data of VAP-DOX acetate obtained in Example 1

[0156]

[0157]

[0158] Preparation of VAP-DOX hydrochloride in Example 2

[0159] As Figure 2 shown, the preparation method of this example specifically includes the following steps:

[0160] 1. Preparation of DOX-3:

[0161] Add tetrahydrofuran to the reactor, add the raw material DOX-2 (17.13 g) and stir to dissolve; cool to 0-10 °C in an ice bath, add tert-butyl carbazate (10.24 g) and purified water and stir until clear; control the reaction system at 0-10 °C, and slowly add EDCI.HCl (23.39 g) in batches. After the addition of EDCI.HCl is complete, react at 0-10 °C for 1-2 hours. After the reaction is complete, add ethyl acetate to the reaction solution, stir for 10 minutes and let stand for 5-10 minutes, and separate and drain the lower aqueous phase. Add purified water to the organic phase again, stir for 10 minutes, and separate and drain the lower aqueous phase. Add saturated sodium chloride aqueous solution to the organic phase again, stir for 10 minutes, and separate and drain the lower aqueous phase. Dry the organic phase with anhydrous sodium sulfate. Filter, wash the filter cake with ethyl acetate and combine the filtrate. The filtrate is concentrated under vacuum until no obvious solvent drips out, and the target compound DOX-3 is obtained.

[0162] 2. Preparation of DOX-4:

[0163] Add dichloromethane and DOX-3 (25.83 g) to the reaction flask, control the temperature at 20-30 °C and dropwise add trifluoroacetic acid (59.38 g). After the addition is complete, maintain the reaction at 20-30 °C for 2-3 hours. After the reaction is complete, concentrate the reaction solution under reduced pressure (stop when no obvious solvent drips out). Add ethyl acetate to the residue and stir to disperse, then cool to 0-10 °C, add n-heptane, and a large amount of solid precipitates; heat to 20-30 °C and stir for 1 hour. Filter, wash the filter cake with a mixed solvent of ethyl acetate and n-heptane, filter to obtain the wet product, and dry the wet product under vacuum at 30-40 °C to constant weight to obtain DOX-4.

[0164] 3. Preparation of DOX-5:

[0165] Add methanol and DOX-4 (17.33 g) to a reaction flask, stir to dissolve, then add DOX (10.97 g). React at 20 - 30 °C for 2 - 3 hours. After the reaction is completed, add the reaction solution to methyl tert-butyl ether and control the internal temperature at 20 - 30 °C. A large amount of solid precipitates. Stir for 30 minutes and filter. Add the filter cake to acetonitrile, slurry at 20 °C - 30 °C for 1 hour, centrifuge to obtain the wet product, and place the wet product in a vacuum drying oven at 30 - 40 °C to dry to constant weight to obtain DOX-5.

[0166] 4. Preparation of crude VAP-DOX:

[0167] Add purified water, DVAP-Cys (9.99 g), and ammonium chloride to a reaction flask, stir to dissolve, and cool to 0 - 10 °C. Add methanol to the reaction flask, cool to 0 - 10 °C, and add the raw material DOX-5 (7.48 g). Control the temperature in the reaction flask at 0 - 10 °C, and drop the above aqueous solution into the reaction flask. After dropping, react at 0 - 10 °C for 1 hour. Centrifuge the reaction solution, and wash the filter cake with ethanol. Dry the filter cake in a vacuum drying oven at 30 °C - 40 °C to constant weight to obtain the crude LN005.

[0168] 5. Preparation of VAP-DOX hydrochloride:

[0169] (1) Dissolution and filtration

[0170] Take the crude VAP-DOX (0.561 g), prepare a crude product solution with purified water as the solvent at about 10 mg / mL, and then filter to remove insoluble substances using a 0.45 μm mixed cellulose membrane CA-CN. The filtrate is directly used for the subsequent process steps.

[0171] (2) Prep-HPLC purification

[0172] The single injection amount for purification is about sample / filler ≤ 1.1%.

[0173] Mobile phase: Mobile phase A (acetonitrile), Mobile phase B (aqueous solution containing 60 mmol / L ammonium chloride and 60 mmol / L ammonium sulfate).

[0174] Gradient (as shown in Table 7):

[0175] Table 7. Changes in the concentration of the mobile phase with time during the Prep-HPLC purification step

[0176]

[0177] The meaning of % in the table is the percentage of Mobile phase A or Mobile phase B in the sum of the volumes of Mobile phase A and Mobile phase B.

[0178] Flow rate: 10 mL / min;

[0179] Chromatographic column: 20 columns (250 mm * 20 mm);

[0180] Packing: Conventional C18 10 μm Reverse-phase high-performance liquid chromatography packing;

[0181] Sample collection: Collect 2 - 3 segments of components by peak segmentation according to the Prep-HPLC detection chart, and take samples to detect the HPLC purity. Control the HPLC purity of the components ≥ 98% as qualified components, and store the collected sample solution at -20 °C for freezing preservation.

[0182] (3) Prep-HPLC salt conversion

[0183] Combine all qualified components and dilute them with water to 2 times the original volume, and load all qualified components for salt conversion.

[0184] Mobile phase: Mobile phase A (acetonitrile), Mobile phase B (aqueous solution containing 30 mmol / L ammonium chloride, adjusted to pH 7.1 with hydrochloric acid);

[0185] Gradient (as shown in Table 8):

[0186] Table 8. Changes in mobile phase concentration over time during the Prep-HPLC salt conversion step

[0187]

[0188]

[0189] The meaning of % in the table is the percentage of mobile phase A or mobile phase B in the sum of the volumes of mobile phase A and mobile phase B.

[0190] Chromatographic column: 20 columns (250 mm * 20 mm);

[0191] Packing: Conventional C18 10 μm Reverse-phase high-performance liquid chromatography packing;

[0192] Collect the main peak solution, take samples to detect the HPLC purity, and qualified samples enter the subsequent process.

[0193] (4) Freeze-drying

[0194] Immediately perform freeze-drying on the collected salt conversion solution. The detailed freeze-drying procedure is as follows:

[0195] Preliminary freezing: Lower the shelf temperature to -60 °C within 1 h after starting the freeze-dryer and maintain it for 3 h;

[0196] First-stage drying: Raise the shelf temperature from -60°C to -10°C within 1 h and maintain for 16 h, then raise the shelf temperature from -10°C to -5°C within 1 h and maintain for 16 h (pre-vacuum pumping: 0.2 mbar, vacuum alarm: 0.4 mbar, condenser temperature: -70°C);

[0197] Second-stage drying: Raise the shelf temperature from -5°C to 5°C within 1 h and maintain for 53 h, then raise the shelf temperature from -5°C to 15°C within 1 h and maintain for 6 h (pre-vacuum pumping: 0.2 mbar, vacuum alarm: 0.4 mbar, condenser temperature: -70°C).

[0198] After drying, 0.267 g of VAP-DOX hydrochloride with a purity of 98.91% (merged peak, corresponding peak numbers 9 and 10) was obtained, as Figure 4 shown in Table 9; the purification yield was 75.7%; the total yield was 45.8%.

[0199] Table 9. Chromatographic data of VAP-DOX hydrochloride obtained in Example 2

[0200]

[0201] Preparation of VAP-DOX sulfate in Example 3

[0202] As Figure 2 shown, the preparation method of this example specifically includes the following steps:

[0203] 1. Preparation of DOX-3:

[0204] Add tetrahydrofuran to the reactor, add the raw material DOX-2 (17.15 g) and stir to dissolve; cool in an ice bath to 0 - 10°C, add tert-butyl carbazate (10.55 g) and purified water and stir until clear; control the reaction system at 0 - 10°C, and slowly add EDCI·HCl (23.48 g) in batches. After the addition of EDCI·HCl is complete, react at 0 - 10°C for 1 - 2 hours. After the reaction is complete, add ethyl acetate to the reaction solution, stir for 10 minutes and let stand for 5 - 10 minutes, and separate and drain the lower aqueous phase. Add purified water to the organic phase again, stir for 10 minutes, and separate and drain the lower aqueous phase. Add saturated sodium chloride aqueous solution to the organic phase again, stir for 10 minutes, and separate and drain the lower aqueous phase. Dry the organic phase with anhydrous sodium sulfate. Filter, wash the filter cake with ethyl acetate and combine the filtrates. Stop the vacuum distillation of the filtrates when no obvious solvent drips out, and obtain the target compound DOX-3.

[0205] 2. Preparation of DOX-4:

[0206] Add dichloromethane and DOX-3 (25.99 g) into the reaction flask. While controlling the temperature at 20 - 30°C, add trifluoroacetic acid (59.58 g) dropwise. After addition, maintain the temperature at 20 - 30°C and react for 2 - 3 hours. After the reaction is completed, concentrate the reaction solution under reduced pressure (stop when there is no obvious solvent dripping out). Add ethyl acetate to the residue and stir to disperse, then cool down to 0 - 10°C. Add n-heptane, and a large amount of solid will precipitate. Raise the temperature to 20 - 30°C and stir for 1 hour. Filter, wash the filter cake with a mixed solvent of ethyl acetate and n-heptane, filter to obtain the wet product, and place the wet product in a vacuum drying oven at 30 - 40°C to dry to constant weight to obtain DOX-4.

[0207] 3. Preparation of DOX-5:

[0208] Add methanol and DOX-4 (17.45 g) into the reaction flask, stir to dissolve, then add DOX (10.90 g), and react at 20 - 30°C for 2 - 3 hours. After the reaction is completed, add the reaction solution to methyl tert-butyl ether and control the internal temperature at 20 - 30°C. A large amount of solid will precipitate. Stir for 30 minutes and filter. Add the filter cake to acetonitrile, slurry at 20 - 30°C for 1 hour, centrifuge to obtain the wet product, and place the wet product in a vacuum drying oven at 30 - 40°C to dry to constant weight to obtain DOX-5.

[0209] 4. Preparation of VAP-DOX crude product:

[0210] Add purified water, DVAP-Cys (10.09 g) and ammonium bicarbonate into the reaction flask, stir to dissolve, and cool down to 0 - 10°C. Add methanol to the reaction flask, cool down to 0 - 10°C, and add the raw material DOX-5 (7.67 g). Control the temperature inside the reaction flask at 0 - 10°C, and drip the above aqueous solution into the reaction flask. After dripping, react at 0 - 10°C for 1 hour. Centrifuge the reaction solution, wash the filter cake with ethanol; dry the filter cake in a vacuum drying oven at 30 - 40°C to constant weight to obtain the LN005 crude product.

[0211] 5. Preparation of VAP-DOX sulfate:

[0212] (1) Dissolution and filtration

[0213] Take the VAP-DOX crude product (0.598 g) and prepare a crude product solution of about 10 mg / mL using purified water as the solvent, then filter to remove insoluble substances using a 0.45 μm mixed cellulose membrane CA-CN. The filtrate is directly used for the subsequent process steps.

[0214] (2) Prep-HPLC purification

[0215] The single injection for purification is about sample / filler ≤ 1.1%.

[0216] Mobile phase: Mobile phase A (acetonitrile), Mobile phase B (aqueous solution containing 60 mmol / L ammonium bicarbonate and 60 mmol / L ammonium sulfate).

[0217] Gradient (as shown in Table 10):

[0218] Table 10. Variation of mobile phase concentration with time in the Prep-HPLC purification step

[0219]

[0220] The meaning of % in the table is the percentage of Mobile phase A or Mobile phase B in the sum of the volumes of Mobile phase A and Mobile phase B.

[0221] Flow rate: 10 mL / min;

[0222] Chromatographic column: 20 columns (250 mm * 20 mm);

[0223] Packing: Conventional C18 10 μm Reverse-phase high-performance liquid chromatography packing;

[0224] Sample collection: Collect 2 - 3 segments of components by peak segmentation according to the Prep-HPLC detection chart, and take samples to detect the HPLC purity. Control the HPLC purity of the components ≥ 98% as qualified components, and store the collected sample solution at -20 °C for freezing preservation.

[0225] (3) Salt conversion in Prep-HPLC

[0226] Combine all qualified components and dilute them with water to 2 times the original volume, and load all qualified components for salt conversion.

[0227] Mobile phase: Mobile phase A (acetonitrile), Mobile phase B (aqueous solution containing 30 mmol / L ammonium bicarbonate, adjusted to pH 7.1 with sulfuric acid);

[0228] Gradient (as shown in Table 11):

[0229] Table 11. Variation of mobile phase concentration with time in the Prep-HPLC salt conversion step

[0230]

[0231] The meaning of % in the table is the percentage of Mobile phase A or Mobile phase B in the sum of the volumes of Mobile phase A and Mobile phase B.

[0232] Chromatographic column: 20 columns (250 mm * 20 mm);

[0233] Packing: Conventional C18 10 μm Reverse-phase high-performance liquid chromatography packing;

[0234] Collect the main peak solution, take samples for HPLC purity detection, and qualified samples enter the subsequent processes.

[0235] (4) Freeze-drying

[0236] Immediately subject the collected salt-converted solution to freeze-drying. The detailed freeze-drying procedure is as follows:

[0237] Pre-freezing: Lower the shelf temperature to -60°C within 1 h after starting the freeze-dryer and maintain it for 3 h;

[0238] First-stage drying: Raise the shelf temperature from -60°C to -10°C within 1 h and maintain it for 16 h, and then raise the shelf temperature from -10°C to -5°C within 1 h and maintain it for 16 h (pre-vacuum pumping: 0.2 mbar, vacuum alarm: 0.4 mbar, condenser temperature: -70°C);

[0239] Second-stage drying: Raise the shelf temperature from -5°C to 5°C within 1 h and maintain it for 53 h, and then raise the shelf temperature from -5°C to 15°C within 1 h and maintain it for 6 h (pre-vacuum pumping: 0.2 mbar, vacuum alarm: 0.4 mbar, condenser temperature: -70°C).

[0240] After drying, 0.277 g of VAP-DOX sulfate is obtained, with a purity of 99.11% (merged peak, corresponding peak numbers 9 and 10), as Figure 5 shown in Table 12; the purification yield is 75.3%; the total yield is 46.7%.

[0241] Table 12. Chromatographic data of VAP-DOX sulfate obtained in Example 3

[0242]

[0243] Effect examples

[0244] Examine the relevant properties of the different salt forms of VAP-DOX obtained in Examples 1-3 respectively, as follows:

[0245] 1. Moisture determination

[0246] Respectively take the different salt forms of VAP-DOX in Examples 1-3 and determine them according to the moisture determination method (General Chapter 0832, Method 1, Part IV of the Chinese Pharmacopoeia 2020 Edition).

[0247] 2. Impurity determination

[0248] Respectively take the different salt forms of VAP-DOX in Examples 1-3 and determine them according to the high performance liquid chromatography method (General Chapter 0512, Part IV of the Chinese Pharmacopoeia 2020 Edition). Prepare freshly before use.

[0249] (1) Test solution

[0250] Take different salt forms of VAP-DOX in Examples 1-3 respectively, weigh accurately, dissolve with diluent (methanol: water = 20:80 (volume ratio)), and quantitatively dilute to make a solution containing about 1 mg per 1 mL. Prepare freshly before use.

[0251] (2) Chromatographic conditions

[0252] Use octadecylsilane chemically bonded silica gel as the filler (Chromcore 120 C18-T, 4.6×250 mm, 5 μm; or a chromatographic column with equivalent efficiency); use a 0.02 mol / L dipotassium hydrogen phosphate solution containing 0.1% triethylamine (adjust the pH value to 7.0 with phosphoric acid)-acetonitrile (volume ratio 92:8) as mobile phase A; use a 0.02 mol / L dipotassium hydrogen phosphate solution containing 0.1% triethylamine (adjust the pH value to 7.0 with phosphoric acid)-acetonitrile (volume ratio 40:60) as mobile phase B, and perform gradient elution according to the conditions shown in Table 13; the detection wavelength is 220 nm; the injection volume is 20 μL.

[0253] Table 13. Chromatographic gradient elution conditions for impurity detection in Example 1 of the effect

[0254]

[0255] The meaning of % in the table is the percentage of mobile phase A or mobile phase B in the sum of the volumes of mobile phase A and mobile phase B.

[0256] (3) System suitability requirements

[0257] The resolution between the LN005 peak and the adjacent peak should be not less than 1.0; the number of theoretical plates calculated based on the LN005 peak should be not less than 5000.

[0258] (4) Assay method

[0259] Accurately measure the test solution, inject it into the liquid chromatograph, and record the chromatogram.

[0260] 3. Proportion of free salt

[0261] Take 0.5 g of different salt forms of VAP-DOX in Examples 1-3 respectively, and calculate the proportion of free salt by the residue determination method in the inspection (General Rules 0841, Volume IV, Chinese Pharmacopoeia 2020 Edition).

[0262] In the present invention, the meaning of free salt is the inorganic matter remaining after the salt conversion step when VAP-DOX is completely converted into the corresponding salt form; the lower the proportion of free salt, the more beneficial.

[0263] The relevant properties were investigated on the 0th day, 5th day, and 10th day respectively, and the obtained results are shown in Table 14, where % are all mass percentages.

[0264] Table 14. Related performance parameters of different VAP-DOX salt forms obtained in Examples 1-3

[0265]

[0266]

[0267] As can be seen from the data in Table 14, for the VAP-DOX acetate, VAP-DOX hydrochloride, and VAP-DOX sulfate prepared in Examples 1-3 of the present invention, their properties did not change during different storage times, and the contents of substances such as moisture and impurities did not increase significantly, indicating that all three salt forms have excellent stability.

[0268] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A preparation method of the salt form of polypeptide drug VAP-DOX, characterized in that, It includes the following steps: It is obtained by salting out the solution containing the polypeptide drug VAP-DOX; Among them, the salting out is carried out by the Pre-HPLC method. Mobile phase A includes acetonitrile or methanol, and mobile phase B includes an aqueous solution containing an ammonium salt or an alkali metal salt; the pH of mobile phase B is 6-8.

2. The preparation method of the salt form of polypeptide drug VAP-DOX according to claim 1, characterized in that, The step of salting out satisfies one or more of the following conditions (1)-(5): (1) The ammonium salt in mobile phase B is selected from one or more of ammonium acetate, ammonium chloride, and ammonium bicarbonate; (2) The alkali metal salt in mobile phase B is selected from sodium salt and / or potassium salt; the sodium salt is selected from one or more of sodium acetate, sodium chloride, and sodium bicarbonate; the potassium salt is selected from one or more of potassium acetate, potassium chloride, and potassium bicarbonate; (3) The concentration of the ammonium salt or alkali metal salt in mobile phase B is 5-50 mM, preferably 30 mM; (4) The pH of mobile phase B is 6.5-7.5, preferably 7.1; (5) The pH of mobile phase B is adjusted by a pH regulator; the pH regulator is preferably selected from one or more of sulfuric acid, hydrochloric acid, acetic acid, phosphoric acid, and formic acid, more preferably one or more of sulfuric acid, hydrochloric acid, and acetic acid.

3. The preparation method of the salt form of polypeptide drug VAP-DOX according to claim 2, characterized in that, The step of salting out satisfies one of the following conditions (1)-(3): (1) Mobile phase B is an aqueous solution containing an ammonium salt, the ammonium salt is ammonium acetate, and the pH regulator is acetic acid; preferably, the concentration of ammonium acetate is 5-50 mM, and the pH of mobile phase B is 6.5-7.5; (2) Mobile phase B is an aqueous solution containing an ammonium salt, the ammonium salt is ammonium chloride, and the pH regulator is hydrochloric acid; preferably, the concentration of ammonium chloride is 5-50 mM, and the pH of mobile phase B is 6.5-7.5; (3) Mobile phase B is an aqueous solution containing an ammonium salt, the ammonium salt is ammonium bicarbonate, and the pH regulator is sulfuric acid; preferably, the concentration of ammonium bicarbonate is 5-50 mM, and the pH of mobile phase B is 6.5-7.

5.

4. The preparation method of the salt form of polypeptide drug VAP-DOX according to claim 1, characterized in that, In the step of salting out, the Pre-HPLC method is carried out in a gradient elution manner, and the gradient elution is preferably a two-stage gradient elution; More preferably, in the Pre-HPLC method, the elution mode of the two-stage gradient elution is as follows: Among them, the meaning of % is the percentage of mobile phase A or mobile phase B in the sum of the volumes of mobile phase A and mobile phase B.

5. The preparation method of the salt form of polypeptide drug VAP-DOX according to claim 1, characterized in that, Before the step of salting out, the solution containing the polypeptide drug VAP-DOX also includes a purification step; Preferably, the purification is carried out by the Pre-HPLC method. Mobile phase A includes acetonitrile or methanol, and mobile phase B includes an aqueous solution containing an ammonium salt or an alkali metal salt; More preferably, the types of ammonium salts and alkali metal salts in mobile phase B in the purification step are the same as the types of ammonium salts and alkali metal salts in mobile phase B in the salting out step; Even more preferably, ammonium sulfate is also included in the ammonium salts in mobile phase B in the purification step.

6. The preparation method of the salt form of polypeptide drug VAP-DOX according to claim 5, characterized in that, The purification step satisfies one of the following conditions (1)-(3): (1) The mobile phase B is an aqueous solution containing an ammonium salt, and the ammonium salt is ammonium acetate and ammonium sulfate; preferably, the concentration of ammonium acetate and the concentration of ammonium sulfate are each independently 5 - 100 mM, more preferably 30 - 100 mM, such as 60 mM; more preferably, the concentration of ammonium acetate is the same as the concentration of ammonium sulfate; (2) The mobile phase B is an aqueous solution containing an ammonium salt, and the ammonium salt is ammonium chloride and ammonium sulfate; preferably, the concentration of ammonium chloride and the concentration of ammonium sulfate are each independently 5 - 100 mM, more preferably 30 - 100 mM, such as 60 mM; more preferably, the concentration of ammonium chloride is the same as the concentration of ammonium sulfate; (3) The mobile phase B is an aqueous solution containing an ammonium salt, and the ammonium salt is ammonium bicarbonate and ammonium sulfate; preferably, the concentration of ammonium bicarbonate and the concentration of ammonium sulfate are each independently 5 - 100 mM, more preferably 30 - 100 mM, such as 60 mM; more preferably, the concentration of ammonium bicarbonate is the same as the concentration of ammonium sulfate.

7. The preparation method of the salt form of polypeptide drug VAP-DOX according to claim 5, characterized in that, In the purification step, the Pre - HPLC method is carried out by gradient elution, and the gradient elution is preferably a two - stage gradient elution; Preferably, in the Pre - HPLC method, the elution mode of the two - stage gradient elution is as follows: Wherein, the meaning of % is the percentage of mobile phase A or mobile phase B in the sum of the volumes of mobile phase A and mobile phase B.

8. The preparation method of the polypeptide drug VAP-DOX salt form according to any one of claims 1-7, characterized in that, The preparation method satisfies one or more of the following conditions (1) - (10): (1) In the step of salt transfer, the chromatographic column is a reverse-phase column. Preferably, the packing material of the reverse-phase column is C4, C8, or C18; more preferably, the parameters of the packing material are (2) In the salt conversion step, the diameter of the chromatographic column is 20 mm; (3) In the salt conversion step, the flow rate is 5 - 15 mL / min, preferably 8 - 12 mL / min; (4) When a purification step is further included, in the purification step, the chromatographic column is a reverse-phase column. Preferably, the packing material of the reverse-phase column is C4, C8, or C18; more preferably, the parameters of the packing material are (5) When a purification step is further included, in the purification step, the diameter of the chromatographic column is 20 mm; (6) When a purification step is further included, in the purification step, the flow rate is 5 - 15 mL / min, preferably 10 mL / min; (7) When a purification step is further included, in the purification step, the sample loading amount is 0.5% - 1%, where % means the mass ratio of the sample to the packing material; (8) The "solution containing polypeptide drug VAP - DOX" is an aqueous solution containing polypeptide drug VAP - DOX; (9) The preparation of the "solution containing polypeptide drug VAP - DOX" includes the following steps: dissolving and filtering the polypeptide drug VAP - DOX to obtain it; wherein, the filtration is preferably carried out using a mixed cellulose membrane, and the pore size of the mixed cellulose membrane is preferably 0.45 μm; (10) After the salt conversion step, a freeze - drying step is further included.

9. A polypeptide drug VAP-DOX salt form, characterized in that, It is prepared by the preparation method of the polypeptide drug VAP - DOX salt form according to any one of claims 1 - 8.

10. A polypeptide drug VAP-DOX salt form, characterized in that, Its structure is shown in the following formula: Among them, the X is selected from Cl - , SO4 2- or CH3COO - .

Images