Antibody-cytotoxic small molecule drug conjugates and their preparation and application

Abstract

The present invention provides an antibody-cytotoxic small molecule drug conjugate and its preparation and application. The method comprises preparing the antibody-cytotoxic small molecule drug conjugate by chemically modifying a hydrophilic heterobifunctional non-cleavable linker to connect an antibody and an active cytotoxic small molecule chemical drug; wherein the hydrophilic heterobifunctional non-cleavable linker has a structure shown in the following formula (I) or formula (II): #imgabs0# wherein L1 is selected from phenylene, #imgabs1# or -(CH2) m ‑C(=O)‑NHC2H4‑(OC2H4) n ‑; R1 is selected from H or ‑SO3Na; m is 0, 1, 2 or 3; n is 1 to 30.

Description

Technical Field

[0001] The present invention relates to the field of medicine, in particular to cancer treatment, and specifically to an antibody-cytotoxic small molecule drug conjugate drug and its preparation and application. Background Art

[0002] For the currently disclosed antibody-drug chemical coupling technology, the linkers mainly used are all non-aqueous organic compounds. In the production and preparation process of antibody-drug conjugates (ADCs), a certain proportion of organic solvents (such as DMAC, DMF, DMSO, etc.) are required to dissolve the linkers for antibody chemical coupling reactions. The volume ratio of organic solvents even reaches 30% (V:V). The use of a large amount of organic solvents not only has the danger of using organic solvents in the production process (the workshop may need explosion-proof design), but also the production waste liquid will cause a certain degree of environmental pollution or make it difficult to recycle a large amount of waste mixed with organic solvents. In addition, the use of a certain proportion of organic solvents (such as DMAC, DMF, DMSO, etc.) in the antibody chemical coupling reaction process has a great influence on the physical and chemical properties of antibody protein drugs (it may cause antibody protein denaturation and precipitation), and may even cause antibody protein precipitation or denaturation, directly leading to the failure of antibody drug production. At the same time, in the drug production process and the finished product release test, it is necessary to control the residual organic solvent in the test drug. All these have caused antibody drug conjugates (antibody-drug) to be produced. The production and preparation process of ADCs is difficult to scale up for commercialization and industrialization, does not conform to the concept of environmentally friendly production, and has high processing costs.

[0003] For example, US5208020 and US5416064 reported various methods for preparing antibody-maytansine alkaloid conjugates. First, a monoclonal antibody was chemically modified with a heterobifunctional linker, and then a Sephadex TM The monoclonal antibody connected to the linker was purified by G25 column, and then the monoclonal antibody connected to the linker was coupled with a certain amount of chemical toxin containing thiol maytansine derivatives, and then Sephadex was used again. TM G25 column or ultrafiltration purification.

[0004] CN101087611A also reports a method for preparing an antibody-maytansine alkaloid conjugate by the same method as US5208020 and US5416064, that is, first chemically modifying a monoclonal antibody with a heterobifunctional linker, purifying to obtain a first step chemical modification reaction solution mixture of the antibody, and then coupling the antibody modification product with a maytansine derivative chemical toxin, and then purifying to obtain a conjugate. The antibody-drug conjugate obtained in this way has a low purity and uses a large amount of organic solvent. For example, the monomer purity of the trastuzumab-SMCC-DM1 conjugate disclosed in the embodiment of CN101087611A is only about 95%, and the polymer content of the antibody drug can reach more than 4.2%. A certain proportion of organic solvents are used in the production process. The presence of such a high polymer antibody drug may cause a high risk of immunogenicity during medication treatment, and the residual organic solvent of the drug will also affect drug safety.

[0005] CN103254311A discloses that a certain proportion of surfactant is added during the antibody chemical coupling process to improve the chemical coupling rate, and an anion chromatography purification step is added during the purification step, thereby improving the purity of the product and reducing the endotoxin content of the drug. Organic solvents such as dimethylacetamide (DMAC), dimethylformamide (DMF), and dimethyl sulfoxide (DMSO) are required in the production process.

[0006] In view of the above reasons, the present invention is proposed. Summary of the invention

[0007] One object of the present invention is to provide an antibody-cytotoxic small molecule drug conjugate;

[0008] Another object of the present invention is to provide a method for preparing an antibody-cytotoxic small molecule drug conjugate drug using a novel hydrophilic heterobifunctional linker;

[0009] Another object of the present invention is to provide the use of the antibody-cytotoxic small molecule drug conjugate drug.

[0010] To achieve the above-mentioned object, on the one hand, the present invention provides a method for preparing an antibody-cytotoxic small molecule drug conjugate, wherein the method comprises preparing the antibody-cytotoxic small molecule drug conjugate by chemically modifying an antibody and an active cytotoxic small molecule chemical drug with a hydrophilic heterobifunctional non-cleavable linker;

[0011] Wherein, the hydrophilic heterobifunctional non-cleavable linker has a structure as shown in the following formula (I) or formula (II):

[0012]

[0013] Wherein, L1 is selected from phenylene, or -(CH2) m -C(=O)-NHC2H4-(OC2H4) n -(Among them, -(CH2) m -C(=O)-NHC2H4-(OC2H4) n - May also be in linear or branched form, including PEG n branched and straight chain forms);

[0014] R1 is selected from H or -SO3Na;

[0015] m is 0, 1, 2 or 3; n is 1-30.

[0016] According to some specific embodiments of the present invention, the hydrophilic heterobifunctional non-cleavable linker has a structure as shown in the following formula (1) to formula (4):

[0017]

[0018] wherein formula (1) is 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester sodium salt;

[0019] Formula (2) is SM(PEG)n (PEGylated, long-chain SMCC crosslinker);

[0020] Formula (3) is SM(PEG)n-SO3Na(PEGylated, long-chain SMCC crosslinker);

[0021] Formula (4) is Sulfo-MBS;

[0022] Formula (II) is Sulfo-SIAB.

[0023] According to some specific embodiments of the present invention, the method comprises:

[0024] (A) first chemically modifying an antibody using a hydrophilic heterobifunctional non-cleavable linker to obtain a modified antibody, and then chemically coupling the modified antibody with an active cytotoxic small molecule chemical drug to obtain the antibody-cytotoxic small molecule drug conjugate drug; or,

[0025] (B) firstly chemically modifying an active cytotoxic small molecule chemical drug with a hydrophilic heterobifunctional non-cleavable linker to obtain a modified small molecule chemical drug, and then chemically coupling the modified small molecule chemical drug with an antibody to obtain the antibody-cytotoxic small molecule drug conjugate drug.

[0026] According to some specific embodiments of the present invention, the method comprises replacing the antibody in a coupling reaction buffer system by ultrafiltration concentration or desalting chromatography, and then chemically modifying the antibody with a hydrophilic heterobifunctional non-cleavable linker to obtain a modified antibody, or chemically coupling a modified small molecule chemical drug and an antibody to obtain the antibody-cytotoxic small molecule drug conjugate drug.

[0027] According to some specific embodiments of the present invention, the coupling reaction buffer system is 20-100 mM sodium hydrogen phosphate buffer or 10-30 mM sodium acetate buffer.

[0028] According to some specific embodiments of the present invention, the coupling reaction buffer system is 50 mM sodium hydrogen phosphate buffer or 20 mM sodium acetate buffer.

[0029] According to some specific embodiments of the present invention, in method (A), the method comprises obtaining the modified antibody, purifying the modified antibody by chromatography column or ultrafiltration, and then chemically coupling it with an active cytotoxic small molecule chemical drug.

[0030] According to some specific embodiments of the present invention, in method (A), the method comprises obtaining the modified antibody, purifying the modified antibody by G25 chromatography column or ultrafiltration, and then chemically coupling it with an active cytotoxic small molecule chemical drug.

[0031] According to some specific embodiments of the present invention, the method comprises subjecting the obtained antibody-cytotoxic small molecule drug conjugate to cation / anion chromatography purification and / or ultrafiltration displacement purification.

[0032] According to some specific embodiments of the present invention, in method (A), the modified antibody and the active cytotoxic small molecule chemical drug are chemically coupled in the presence of an emulsifier to obtain the antibody-cytotoxic small molecule drug conjugate drug; based on the total volume of the reaction solution of the chemical coupling reaction as 100%, the amount of the emulsifier used is 0.1-1 v / v%.

[0033] The present invention reduces the volume content of the organic solvent in the chemical coupling reaction system by utilizing an emulsifier, thereby improving the chemical coupling efficiency.

[0034] According to some specific embodiments of the present invention, the amount of the emulsifier used is 0.2-0.6 v / v%, based on the total volume of the reaction solution of the chemical coupling reaction being 100%.

[0035] According to some specific embodiments of the present invention, the method comprises the following steps:

[0036] (1) Preparation of antibody reaction buffer:

[0037] The antibody is replaced by ultrafiltration concentration or desalting chromatography to a coupling reaction buffer system, and then the coupling reaction buffer system is concentrated to an antibody concentration of 10 to 40 mg / ml to obtain the replaced antibody;

[0038] (2) Chemical modification of antibody reaction with linker:

[0039] adding a hydrophilic heterobifunctional non-cleavable linker to the replaced antibody solution to perform a chemical modification reaction to obtain a reaction solution containing a modified antibody;

[0040] (3) Purification of antibody-linker modified products:

[0041] Purifying the reaction solution containing the modified antibody obtained in step (2) by column chromatography or ultrafiltration;

[0042] (4) Chemical coupling reaction:

[0043] The purified modified antibody obtained in step (3), the active cytotoxic small molecule chemical drug and the emulsifier are mixed and then subjected to a chemical coupling reaction;

[0044] (5) Cationic / anionic column chromatography purification:

[0045] Purify the chemical coupling reaction solution obtained in step (4) by cation / anion column chromatography and collect the eluate;

[0046] (6) Ultrafiltration replacement and concentration:

[0047] The eluate obtained in step (5) is concentrated by ultrafiltration using a preparation buffer, and the concentrate is collected to obtain the antibody-cytotoxic small molecule drug conjugate.

[0048] According to some specific embodiments of the present invention, step (1) is to concentrate the coupling reaction buffer system to an antibody concentration of 20-25 mg / ml.

[0049] According to some specific embodiments of the present invention, in step (3), the reaction solution containing the modified antibody obtained in step (2) is loaded onto a G25 column for column chromatography or ultrafiltration purification.

[0050] According to some specific embodiments of the present invention, step (3) further comprises flushing the chromatography column or ultrafiltration system with an equilibrium buffer.

[0051] According to some specific embodiments of the present invention, the column chromatography in step (5) is G25-COO column chromatography.

[0052] According to some specific embodiments of the present invention, the eluent for the column chromatography in step (5) is 5-30 mM sodium succinate buffer or 5-70 mM sodium phosphate buffer.

[0053] According to some specific embodiments of the present invention, the preparation buffer in step (6) is sodium succinate buffer or sodium phosphate buffer.

[0054] According to some specific embodiments of the present invention, the preparation buffer in step (6) is 5-30 mM sodium succinate buffer or 5-70 mM sodium phosphate buffer.

[0055] According to some specific embodiments of the present invention, the method further comprises adding other preparation components to the eluate obtained in step (6).

[0056] According to some specific embodiments of the present invention, the other formulation components are a mixed solution of 5-30 mM succinate sodium salt buffer (or hydrochloric acid-histidine buffer) or 5-70 mM sodium phosphate buffer-sucrose or trehalose-0.02% Tween 20 or 80.

[0057] It can be understood that the "mixed solution of 5-30mM sodium succinate buffer (or hydrochloric acid-histidine buffer) or 5-70mM sodium phosphate buffer-sucrose or trehalose-0.02% Tween 20 or 80" mentioned here refers to a mixed solution of sodium succinate buffer (or sodium phosphate buffer)-sucrose (or trehalose)-0.02% Tween 20 (or Tween 80).

[0058] According to some specific embodiments of the present invention, the other formulation ingredients are a mixed solution of 5-10 mM sodium succinate or hydrochloric acid-histidine buffer)-2-10% sucrose-0.01-0.1% Tween 20 or Tween 80, and the pH value of the mixed solution is 4.0-7.0.

[0059] It can be understood that the "mixed solution of 5-10mM sodium succinate or hydrochloric acid-histidine buffer)-2-10% sucrose-0.01-0.1% Tween 20 or Tween 80" mentioned here refers to a mixed solution of 5-10mM sodium succinate or hydrochloric acid-histidine buffer), 2-10% sucrose, and 0.01-0.1% Tween 20 (or Tween 80).

[0060] According to some specific embodiments of the present invention, the molar ratio of the antibody and the hydrophilic heterobifunctional non-cleavable linker is (2-10):1; the molar ratio of the active cytotoxic small molecule chemical drug to the modified antibody is (4-7):1.

[0061] According to some specific embodiments of the present invention, the reaction conditions for chemically modifying antibodies with hydrophilic heterobifunctional non-cleavable linkers include: reaction temperature of 10°C-40°C, reaction time of 0.5 to 5 hours (preferably 60 min to 150 min), and pH value of the reaction system solution of 4-9; the reaction conditions for chemical coupling of modified antibodies and active cytotoxic small molecule chemical drugs include: reaction temperature of 15°C-35°C, reaction time of 0.5 to 6 hours, and pH value of the reaction system solution of 3-10.

[0062] According to some specific embodiments of the present invention, the antibody is selected from a combination of one or more of any immunoglobulin, any immunoglobulin fragment, antibody, single-chain antibody, monoclonal antibody fragment, polyclonal antibody fragment, and chimeric antibody that specifically and targets the surface of cancer cells; and the active cytotoxic small molecule chemical drug is maytansine alkaloid and its derivatives.

[0063] According to some specific embodiments of the present invention, the antibody is a humanized monoclonal antibody.

[0064] According to some specific embodiments of the present invention, the antibody is an anti-her2 antibody, an anti-CD79b antibody, an anti-CD22 antibody, an anti-CD30 antibody, an anti-CD33 antibody and an anti-EGFR antibody.

[0065] According to some specific embodiments of the present invention, the active cytotoxic small molecule chemical drug is selected from N 2 '-Deacetyl-N 2 '-(3-mercapto-1-oxopropyl) maytansine (DM1), N (2 ' ) -Deacetyl-N 2 -(4-mercapto-4-methyl-1-oxopentyl)-maytansine (DM4), and N 2’ -Deacetyl-N 2 '-(4-mercapto-1-oxopentyl)-maytansine (DM3) or a combination of one or more thereof or the MMAE / F series.

[0066] According to some specific embodiments of the present invention, the emulsifier is selected from a combination of one or more of polysorbate 20, polysorbate 40, polysorbate 60 and polysorbate 80.

[0067] According to some specific embodiments of the present invention, the coupling reaction buffer system is a phosphate buffer with a pH value of 4.0-9.0.

[0068] According to some specific embodiments of the present invention, the coupling reaction buffer system is sodium phosphate buffer, potassium phosphate buffer, Tris-HCl buffer or disodium hydrogen phosphate-citrate buffer.

[0069] According to some specific embodiments of the present invention, the ultrafiltration concentration or desalting chromatography method can adopt existing technology, for example, ultrafiltration concentration can be performed by a Pellicon system, or desalting chromatography can be performed by using a SephadexTMG25 gel chromatography column.

[0070] According to some specific embodiments of the present invention, the chemical coupling reaction in step (4) is carried out in an organic solvent selected from dimethylacetamide (DMAC), dimethylformamide (DMF), and dimethyl sulfoxide (DMSO).

[0071] According to some specific embodiments of the present invention, the method of the present invention specifically comprises the following steps:

[0072] (1) Preparation of antibody reaction buffer for preparing antibody-maytansine conjugates

[0073] The antibody stock solution is replaced by ultrafiltration concentration or desalting chromatography into the first step chemical modification reaction buffer, the reaction buffer is phosphate buffer, and concentrated to an antibody concentration of 20 to 30 mg / ml to obtain the replaced antibody;

[0074] (2) Chemical modification of antibodies using water-soluble (hydrophilic) non-cleavable heterobifunctional linkers (the first step of chemical modification of antibodies)

[0075] Accurately weigh a water-soluble (hydrophilic) non-cleavable heterobifunctional linker, add it to the above-mentioned replaced antibody solution for chemical modification reaction for 60min to 150min; the reaction temperature is 15 to 30°C. Preferably, the water-soluble (hydrophilic) non-cleavable heterobifunctional linker is 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester sodium salt.

[0076] (3) Ultrafiltration or G25 purification to prepare antibody-linker modified products

[0077] The antibody chemical modification reaction solution is loaded onto a G25 column or ultrafiltration for purification, and the flow-through is collected. After loading, the G25 column or ultrafiltration system is flushed with an equilibrium buffer until the collection is completed;

[0078] (4) Chemical coupling reaction of antibody-linker modified product with maytansine derivative (DM1) (second step chemical coupling reaction of antibody)

[0079] The replaced antibody obtained in step (3) is mixed with the mother solution of maytansine alkaloid in a ratio of 4 to 7:1 of the molar ratio of the maytansine derivative (DM1) to the antibody-linker modified product, and 0.1% to 1% V / V of an emulsifier is added to the chemical coupling reaction solution to carry out an antibody chemical coupling reaction at a reaction temperature of 15 to 30° C. for a reaction time of 3 to 6 hours;

[0080] (5) Cationic chromatography purification

[0081] The antibody chemical coupling reaction solution is purified by cationic chromatography, and the eluate is collected.

[0082] (6) Ultrafiltration, replacement and concentration

[0083] The cationic chromatography purified product of the antibody chemical coupling reaction solution was ultrafiltrated and concentrated using the component of the preparation buffer, and the eluate was collected.

[0084] (7) Preparation of Antibody-Maytansine Derivative Conjugate Drug Solution

[0085] The above antibody chemical coupling reaction solution is ultrafiltered and concentrated into a product solution, and other preparation components are added in a certain proportion to adjust to a certain concentration to prepare an antibody-maytansine derivative coupling drug stock solution.

[0086] On the other hand, the present invention also provides an antibody-cytotoxic small molecule drug conjugate, wherein the antibody-cytotoxic small molecule drug conjugate is obtained by connecting an antibody and an active cytotoxic small molecule chemical drug by a hydrophilic heterobifunctional non-cleavable linker; the hydrophilic heterobifunctional non-cleavable linker has a structure shown in the following formula (I) or formula (II):

[0087]

[0088] Wherein, L is selected from phenylene, or -(CH2) m -C(=O)-NHC2H4-(OC2H4) n -;

[0089] R1 is selected from H or -SO3Na;

[0090] m is 0, 1, 2 or 3; n is 1 to 30;

[0091] Preferably, the hydrophilic heterobifunctional non-cleavable linker has a structure as shown in the following formula (1) to formula (4):

[0092]

[0093] According to some specific embodiments of the present invention, the antibody is selected from a combination of one or more of any immunoglobulin, any immunoglobulin fragment, antibody, single-chain antibody, monoclonal antibody fragment, polyclonal antibody fragment, and chimeric antibody that specifically and targets the surface of cancer cells; and the active cytotoxic small molecule chemical drug is maytansine alkaloid and its derivatives.

[0094] According to some specific embodiments of the present invention, the antibody is a humanized monoclonal antibody.

[0095] According to some specific embodiments of the present invention, the antibodies are anti-her2 antibodies, anti-CD79b antibodies, anti-CD30 antibodies, anti-CD33 antibodies and anti-CD22 antibodies.

[0096] According to some specific embodiments of the present invention, the active cytotoxic small molecule chemical drug is selected from N 2 '-Deacetyl-N 2 '-(3-mercapto-1-oxopropyl) maytansine, N (2 ' ) -Deacetyl-N 2 -(4-mercapto-4-methyl-1-oxopentyl)-maytansine, and N 2’ -Deacetyl-N 2 '-(4-mercapto-1-oxopentyl)-maytansine, a combination of one or more thereof and a MMAE / F composition series.

[0097] According to some specific embodiments of the present invention, the antibody-cytotoxic small molecule drug conjugate is prepared by any one of the preparation methods described above in the present invention.

[0098] In another aspect, the present invention also provides the use of the antibody-cytotoxic small molecule drug conjugate in the preparation of anti-tumor drugs.

[0099] According to some specific embodiments of the present invention, the tumor is selected from HER2-positive overexpressing cancer or bladder cancer.

[0100] According to some specific embodiments of the present invention, the tumor is selected from HER2-positive overexpressing cancers, namely breast cancer, colorectal cancer or gastric cancer.

[0101] In summary, the present invention provides an antibody-cytotoxic small molecule drug conjugate and a preparation method. The technical solution of the present invention has the following advantages:

[0102] 1. In the process of producing antibody-cytotoxic small molecule drug (maytansine) conjugate drugs, a hydrophilic linker (such as 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfonyl succinimidyl ester sodium salt) is selected. No organic solvent is required in the first step of the antibody chemical modification process, making the pilot process more suitable for industrial and commercial production. At the same time, the change of the physical and chemical properties of the antibody protein by the presence of organic solvents in the production process is avoided. In the drug production process, there is no need to detect the residual organic solvent in the intermediate and the finished product release test. For example, taking the anti-HER2 antibody-MCC-DM1 production process as an example, under the same conditions, the first step of the antibody chemical modification reaction does not require an organic solvent. The hydrophilic heterobifunctional non-cleavable linker 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfonyl succinimidyl ester sodium salt is used to improve the efficiency of the antibody chemical modification reaction, reduce the production cost and waste treatment cost, and there is no organic solvent or a lower content of organic solvent in the production process, which is safe, reduces costs and conforms to the concept of environmental friendliness.

[0103] 2. The second step of chemical coupling reaction liquid purification of antibodies adds a cationic chromatography purification process, and the antibody-cytotoxic small molecule drug (maytansine) conjugate drug produced has higher purity, lower endotoxin content and lower free small molecule drug content (FREE DRUG). Taking the anti-HER2 antibody-MCC-DM1 production process as an example, the addition of a cationic chromatography purification process to the second step of chemical coupling reaction liquid purification of antibodies can reduce the antibody drug aggregate from about 4% to about 1.5% or lower, and the SEC-HPLC purity can be increased to about 99% or higher. At the same time, it can reduce the drug endotoxin content and reduce the residue of free small molecule drugs in the drug.

[0104] 3. Add 0.1% to 1% V / V emulsifier (such as Tween 20 or Tween 80) to the second step chemical coupling reaction solution of the antibody. The emulsifier is also the main component of the drug preparation. There is no need to detect the residue in the finished product. It improves the efficiency of the antibody chemical coupling reaction and the drug monomer content, reduces the relative usage of cytotoxic small molecule drugs and the drug aggregate content, and the reaction conditions are milder, which is more suitable for production process expansion and commercialization.

[0105] 4. A series of new water-soluble heterobifunctional linkers are used to enhance the stability of antibody-drug conjugates, further improve drug purity, safety and efficacy, and broaden the drug treatment window.

[0106] 5. This preparation method is suitable for the production of antibody-drug conjugates targeting different targets, especially the production of antibody-drug conjugates wherein the antibodies are anti-her2 antibodies, anti-CD79b antibodies, anti-CD30 antibodies, anti-CD33 antibodies and anti-CD22 antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0107] Figure 1 The mass spectrum (DAR) of the anti-HER2 antibody-MCC-DM1 conjugate drug of Example 1;

[0108] Figure 2 The antibody-antigen binding activity of the anti-HER2 antibody-MCC-DM1 drug conjugate of Example 1;

[0109] Brief description of the experimental process and conclusion:

[0110] Coat the ELISA plate with antigen, add the gradient diluted samples to the coated ELISA plate, and incubate for 0.5-2h; then add the enzyme-labeled secondary antibody (Anti-human IgG (γchain specific)-peroxidase conjugate, from Sigma) dilution solution and incubate for 0.5-3h; after washing the plate, incubate at room temperature for 10-100min; after terminating the reaction with the stop solution, measure the absorbance at a wavelength of 450nm, and fit the four-parameter curve.

[0111] Conclusion: The anti-Her2 antibody-MCC-DM1 conjugate drug has high activity and specific binding activity with the antigen target.

[0112] Figure 3 The anti-HER2 antibody-MCC-DM1 drug conjugate of Example 1 inhibits the biological activity of cancer cells in vitro;

[0113] Brief description of the experimental process and conclusion:

[0114] According to the anti-cancer mechanism of the drug, cells with high expression of Her2 were selected as target cells. Through method optimization, experimental conditions such as cell amount, incubation time, and sample concentration were determined.

[0115] Detection method:

[0116] Preparation of cell suspension: adjust the cell concentration to 0.4-1*105 cells / mL, plate and incubate for 1-5 hours.

[0117] Sample processing: dilute the reference and the sample to be tested with culture medium in a gradient manner and add them to the cell plate. Set up a blank control well and incubate at 37 degrees for 60-120 hours. After staining for 5-9 hours, read the fluorescence readings with an ELISA reader at 530nm excitation light and 590nm emission light, and record the results. Use the parallel analysis method to process the results.

[0118] Results: The anti-Her2 antibody-MCC-DM1 conjugate drug had obvious biological activity in inhibiting cancer cells.

[0119] Figure 4This is the purity (monomer content) detection spectrum (SEC-HPLC) of the anti-HER2 antibody-MCC-DM1 drug conjugate of Example 1;

[0120] Brief description of the experimental process and conclusion:

[0121] Detection was performed according to the molecular exclusion chromatography method in the general rule 0514 of the third part of the 2015 edition of the Chinese Pharmacopoeia. The chromatographic gel used in the chromatographic column for separating proteins is a filler (such as TSK gel G3000 SWXL 7.8mm from TOSOH); the mobile phase is phosphate buffer (potassium dihydrogen phosphate-potassium chloride solution, pH 6.0±1)-isopropanol; the detection wavelength is 280nm. The test sample was diluted with the mobile phase to contain about 10mg per 1mL, as the test sample solution, and 20μL of the test sample solution was injected into the liquid chromatograph. Calculated by the area normalization method, the monomer content should be ≥93.0%, and the polymer content should be ≤7.0%.

[0122] Figure 5 The efficacy of the anti-Her2 antibody-MCC-DM1 conjugate drug of Example 1 on human breast cancer BT-474 nude mouse transplanted tumors (n=10);

[0123] Figure 6 This is the mass spectrum (DAR) of the anti-her2 antibody - MCC-DM1 conjugated drug synthesized using a hydrophilic linker in Example 2. DETAILED DESCRIPTION

[0124] The following describes in detail the implementation process of the present invention and the beneficial effects produced by the specific embodiments, which is intended to help readers better understand the essence and characteristics of the present invention, and is not intended to limit the scope of implementation of the present invention.

[0125] Method for determining coupling rate: Determining coupling rate refers to determining the number of maytansine derivative alkaloid drug molecules or other toxin molecules connected to each monoclonal antibody molecule, that is, the ratio of the molar number of maytansine alkaloid drugs or other cytotoxic small molecules to the molar number of monoclonal antibodies. It can be determined by dual-wavelength ultraviolet spectrophotometry and liquid phase-mass spectrometry. Dual-wavelength ultraviolet spectrophotometry: The coupling rate (DAR) is calculated by measuring the ultraviolet absorption value of the antibody-maytansine alkaloid conjugate at 252nM and 280nM, and the Lambert ratio law is used to calculate; mass spectrometry: ESI-MS analysis of antibody-maytansine derivative alkaloid conjugate drugs can obtain peak diagrams and distribution diagrams of different numbers of drugs connected, and the coupling rate (DAR) is calculated based on the area of ​​each peak. The coupling rates obtained by the two methods have good consistency and mutual correction.

[0126] Determination of antibody-drug conjugate concentration: Dual-wavelength ultraviolet spectrophotometry is used to determine the ultraviolet absorption values ​​at 252nM and 280nM, and the concentrations of the antibody and the drug can be calculated by the Lambert-Beer law; the antibody-drug conjugate concentration described in the present invention refers to the sum of the monoclonal antibody concentration and the maytansine derivative alkaloid drug conjugated thereto.

[0127] SEC-HPLC analysis: TSKgelG3000 high-efficiency molecular sieve was used to analyze the antibody-maytansine alkaloid conjugate drug, and the mobile phase was KH2PO4+KCl, 20% (V:V) acetonitrile or isopropanol, pH 6.0. The proportion of conjugate aggregates and conjugate monomers was calculated by the area normalization method.

[0128] Endotoxin content detection: refer to the bacterial endotoxin detection method of the "Chinese Pharmacopoeia 2015 Edition".

[0129] The linker in the embodiment is a heterobifunctional non-cleavable linker 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfonic acid succinimide ester sodium salt. Among them, 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfonic acid succinimide ester sodium salt was purchased from Sigma or Suzhou Haofan Biotechnology Co., Ltd., SMCC was purchased from Suzhou Haofan Biotechnology Co., Ltd., DM1 was purchased from Lianning Biopharmaceutical Co., Ltd., and antibodies (anti-HER2) and water for injection were produced and provided by Dongyao Pharmaceutical Co., Ltd.

[0130] The "a", "b", "c", "d", "e", "f", "g" and "h" that appear in front of each step in the embodiments indicate that different parallel technical solutions are adopted respectively for parallel comparison.

[0131] Example 1

[0132] The specific steps for preparing the anti-HER2 antibody-MCC-DM1 conjugate drug are as follows:

[0133] (1) Preparation of antibody replacement buffer system for coupling reaction

[0134] The anti-HER2 antibody stock solution (antibody A, i.e., anti-her2 antibody) was replaced into 50 mM sodium phosphate buffer (pH 4.0-8.0) by ultrafiltration replacement with 50 mM sodium phosphate buffer, and concentrated to an antibody concentration of 20-40 mg / ml to obtain an antibody solution after replacement, with a pH of 4-8.

[0135] (2) Preparation of 10 mg / ml sulfo-SMCC reaction solution

[0136] According to the molar ratio of sulfo-SMCC:antibody substance of 8:1, accurately weigh the required water-soluble (hydrophilic) heterobifunctional non-cleavable linker 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimidyl ester sodium salt solid powder, dissolve it in water for injection to prepare a 10 mg / ml sulfo-SMCC solution for use;

[0137] (3) First step of antibody chemical modification reaction

[0138] The water-soluble (hydrophilic) heterobifunctional non-cleavable linker 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester sodium salt solution prepared in step (2) is added to the antibody (antibody A) solution prepared in step (1), and the mixture is stirred until fully mixed. The reaction temperature is controlled at 10 to 30° C. and the reaction time is 1 to 3 hours.

[0139] (4) Purification of the first step of antibody chemical modification reaction solution

[0140] The reaction solution in step (3) was purified by ultrafiltration with 8-15 times the volume of the reaction buffer in step (5) and 20 mM sodium acetate (pH 4.0-8.0), and the purified solutions of the antibody (antibody A)-MCC modified products were collected respectively.

[0141] (5) Antibody second step chemical coupling reaction

[0142] According to the ratio of DM1: (antibody A)-MCC = 1-5:1, the DM1 product is accurately weighed and dissolved in DMAC (dimethylacetamide) to prepare a 20 mg / ml DM1 / DMAC (dimethylacetamide) organic solution; 0.1% to 1% V / V emulsifier (Tween 20 or Tween 80) is added to the antibody (antibody A)-MCC modified product solution prepared in step (4), and 1-5% (volume ratio) of 20 mg / ml DM1 / DMAC (dimethylacetamide) solution reaction liquid is added, the reaction temperature is controlled at 15 to 30° C., and the reaction time is 1 h to 6 h or overnight.

[0143] (6) Antibody second step chemical coupling reaction solution purification

[0144] The reaction solution in step (5) is first purified by 20Mm sodium acetate buffer or sodium phosphate buffer cation (or anion) chromatographic exchange (Fractogel EMD COO-, eluent 20Mm sodium acetate buffer) at a linear flow rate of ≤300cm / h to remove small molecule drugs and drug aggregates, and then ultrafiltration and displacement purification is performed using 5-10mM sodium succinate buffer or phosphate buffer preparation buffer 8-15 times the volume to collect the purified solution of the anti-HER2 antibody-MCC-DM1 conjugate drug.

[0145] (7) Preparation of anti-HER2 antibody-MCC-DM1 conjugate drug stock solution

[0146] The purified solution of the anti-HER2 antibody-MCC-DM1 drug conjugate collected in step (6) is respectively added with a certain proportion of other formulation buffer components (preferably the formulation components and content are 5-10mM sodium succinate-2-10% sucrose-0.01-0.1% Tween 20 or Tween 80, pH 4.0-7.0) to prepare the anti-HER2 antibody-MCC-DM1 drug conjugate stock solution. Pharmaceutical detection spectrum is as follows Figure 1 The purity of the drug is shown in Figure 4 The quality of the prepared drug is shown in Table 1. The activity of the prepared drug is shown in Figure 2 , Figure 3 and Figure 5 shown.

[0147] Table 1 Quality analysis of anti-HER2 antibody-MCC-DM1 conjugate drug

[0148]

[0149]

[0150] Example 2

[0151] To prepare anti-HER2 antibody-MCC-DM1 drug conjugates, and to verify and evaluate the effect of using a water-soluble (hydrophilic) heterobifunctional non-cleavable linker, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimidyl ester sodium salt, on the quality of the antibody-drug conjugates.

[0152] The preparation method of the anti-HER2 antibody-MCC-DM1 conjugate drug comprises the following specific steps:

[0153] (1) Preparation of antibody replacement buffer system for coupling reaction

[0154] The anti-HER2 antibody stock solution (antibody A) was replaced with 50Mm sodium phosphate buffer (pH 4.0-8.0) by ultrafiltration replacement with 50Mm sodium phosphate buffer or gel G25 column (FF) exchange chromatography (flow rate not higher than 300cm / h), and concentrated to an antibody concentration of 20-40mg / ml to obtain a replaced antibody solution with a pH of 4-8.

[0155] (2) a: Prepare 10 mg / ml SMCC / DMSO (dimethyl sulfoxide) organic solution reaction solution

[0156] According to the molar ratio of SMCC:antibody of 8:1, the required SMCC solid powder was accurately weighed and dissolved in the organic solvent DMSO (dimethyl sulfoxide) to prepare a 10 mg / ml SMCC / DMSO (dimethyl sulfoxide) solution.

[0157] b: According to the molar ratio of sulfo-SMCC:antibody of 8:1, accurately weigh the required water-soluble (hydrophilic) heterobifunctional non-cleavable linker 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimidyl ester sodium salt solid powder, and dissolve it in water for injection to prepare a 10 mg / ml sulfo-SMCC solution;

[0158] (3) First step of antibody chemical modification reaction

[0159] a: Add the SMCC / DMSO (dimethyl sulfoxide) organic solution reaction liquid of step (2a) to the antibody (antibody A) solution prepared in step (1), stir and mix thoroughly, and control the chemical modification reaction temperature at 10-30° C. for a reaction time of 1 h to 3 h.

[0160] b: Add the same amount of water-soluble (hydrophilic) heterobifunctional non-cleavable linker 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester sodium salt solution as in part b of step (2) to the antibody (antibody A) solution prepared in step (1), stir until fully mixed, control the reaction temperature at 10-30°C, and the reaction time is 1h-3h.

[0161] (4) Purification of the first step of antibody chemical modification reaction solution

[0162] The reaction solutions of groups a and b in step (3) were respectively purified by ultrafiltration with 10 times the volume of the reaction buffer in step (5) and 20 mM sodium acetate (pH 4.0-8.0), and the purified solutions of the antibody (antibody A)-MCC modified products were collected respectively.

[0163] (5) Antibody second step chemical coupling reaction

[0164] a: Add 5-10% (volume ratio) of 10 mg / ml DM1 / DMAC (dimethylacetamide) organic solution reaction liquid to the antibody (antibody A)-MCC modified product solution prepared in step (4), stir and mix thoroughly, control the chemical modification reaction temperature at 10-30° C., and the reaction time is 1 h to 5 h or overnight.

[0165] b: Add 5-10% (volume ratio) of DM1 / DMAC (dimethylacetamide) reaction solution to the antibody (antibody A)-MCC modified product solution prepared in step (4), the reaction temperature is 15-30° C., and the reaction time is 1 h to 5 h or overnight.

[0166] (6) Antibody second step chemical coupling reaction solution purification

[0167] The reaction solutions of groups a and b in step (5) were purified by ultrafiltration with 10 times the volume of formulation buffer (i.e., 5-10 mM sodium succinate buffer or phosphate buffer), and the purified solutions of anti-HER2 antibody-MCC-DM1 drug conjugate were collected respectively.

[0168] (7) Preparation of anti-HER2 antibody-MCC-DM1 conjugate drug stock solution

[0169] The purified solutions of the anti-HER2 antibody-MCC-DM1 drug conjugate collected in groups a and b in step (6) are respectively added with a certain proportion of other formulation buffer components (preferably the formulation components and content are 5-10mM sodium succinate-2-10% sucrose-0.01-0.1% Tween 20 or Tween 80, pH 4.0-7.0) to prepare the anti-HER2 antibody-MCC-DM1 drug conjugate stock solution. Pharmaceutical detection spectrum is as follows Figure 6 The quality of the prepared medicine is shown in Table 2 below.

[0170] Table 2 Comparison of quality analysis of anti-HER2 antibody-MCC-DM1 conjugate drugs using water-soluble (hydrophilic) heterobifunctional non-cleavable linker 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfonic acid succinimidyl ester sodium:

[0171]

[0172]

[0173] Example 3

[0174] To prepare anti-HER2 antibody-MCC-DM1 conjugate drugs, and to study and evaluate the effect of adding a cationic chromatography purification process to the second step of antibody chemical conjugation reaction purification on the quality of antibody conjugate drugs.

[0175] The preparation method of the anti-HER2 antibody-MCC-DM1 conjugate drug comprises the following specific steps:

[0176] (1) Preparation of antibody replacement buffer system for coupling reaction

[0177] The anti-HER2 antibody stock solution (antibody A) is replaced with 50 mM sodium phosphate reaction buffer by ultrafiltration replacement or G25 column exchange chromatography, and concentrated to an antibody concentration of 20 to 30 mg / ml to obtain a replaced antibody solution with a pH value of 4 to 8.

[0178] (2) According to the theoretical calculated value of the molar ratio of sulfo-SMCC:antibody of 8:1, accurately weigh the solid powder of water-soluble (hydrophilic) heterobifunctional non-cleavable linker 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimidyl ester sodium salt, and dissolve it in water for injection to prepare a 10 mg / ml sulfo-SMCC solution.

[0179] (3) First step of antibody chemical modification reaction

[0180] Add the 10 mg / ml (hydrophilic) heterobifunctional non-cleavable linker 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester sodium salt solution prepared in step (2) to the antibody (antibody A) solution prepared in step (1), stir well, control the reaction temperature to 10-30° C., and the reaction time to 1 h-3 h.

[0181] (4) Purification of the first step of antibody chemical modification reaction solution

[0182] The first step chemical modification reaction solution of the antibody in step (3) was purified by ultrafiltration with 10 times the volume of the reaction buffer (20Mm sodium acetate buffer or sodium phosphate buffer) in step (5) to collect antibody (antibody A)-MCC

[0183] Purified solution of modified product.

[0184] (5) Antibody second step chemical coupling reaction

[0185] Add 5-10% (volume ratio) of 10 mg / ml DM1 / DMAC (dimethylacetamide) reaction solution to the antibody (antibody A)-MCC modified product solution prepared in step (4), control the reaction temperature to 15-30° C., and the reaction time to 1 h-6 h or overnight.

[0186] (6) Antibody second step chemical coupling reaction solution purification

[0187] c: The reaction solution in step (5) is directly purified by ultrafiltration with 10 times the volume of 20 mM sodium succinate preparation buffer to collect the purified solution of the anti-HER2 antibody-MCC-DM1 drug conjugate;

[0188] d: The reaction solution in step (5) is first purified by 20Mm sodium acetate buffer or sodium phosphate buffer cation (or anion) chromatographic exchange (Fractogel EMD COO-, eluent 20Mm sodium acetate buffer) at a linear flow rate of ≤300cm / h to remove small molecule drugs and drug aggregates, and then ultrafiltration and displacement purification is performed using 5-10mM sodium succinate buffer or phosphate buffer preparation buffer with 10 times the volume to collect the purified solution of the anti-HER2 antibody-MCC-DM1 conjugate drug.

[0189] (7) Preparation of anti-HER2 antibody-MCC-DM1 conjugate drug stock solution

[0190] The purified solutions of the anti-HER2 antibody-MCC-DM1 drug conjugate collected in groups c and d in step (6) were respectively added with a certain proportion of other formulation buffer components (preferably the formulation components and contents are 5-10 mM sodium succinate-2-10% sucrose-0.01-0.1% Tween 20 or Tween 80, pH 4.0-7.0) to prepare the anti-HER2 antibody-MCC-DM1 drug conjugate stock solution. The quality of the prepared drug is shown in Table 3:

[0191] Table 3 Effect of adding cationic chromatography purification process to the second step of antibody chemical coupling reaction purification on the quality of antibody-drug conjugates: Comparison of quality analysis of anti-HER2 antibody-MCC-DM1 conjugate drugs:

[0192]

[0193]

[0194] Example 4

[0195] Preparation of anti-HER2 antibody-MCC-DM1 conjugates to verify the effect of adding 0.1% to 1% V / V emulsifier (Tween 20) in the second step of antibody chemical conjugation reaction on the quality of antibody conjugates

[0196] The preparation method of the anti-HER2 antibody-MCC-DM1 conjugate drug comprises the following specific steps:

[0197] (1) Preparation of antibody replacement buffer system for coupling reaction

[0198] The anti-HER2 antibody stock solution (antibody A) is replaced with 50 mM sodium phosphate reaction buffer by ultrafiltration replacement or G25 column exchange chromatography, and concentrated to an antibody concentration of 20 to 30 mg / ml to obtain a replaced antibody solution with a pH value of 4 to 8.

[0199] (2) According to the theoretical calculated value of the molar ratio of sulfo-SMCC:antibody of 8:1, accurately weigh the solid powder of water-soluble (hydrophilic) heterobifunctional non-cleavable linker 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimidyl ester sodium salt, and dissolve it in water for injection to prepare a 10 mg / ml sulfo-SMCC solution.

[0200] (3) First step of antibody chemical modification reaction

[0201] Add the 10 mg / ml (hydrophilic) heterobifunctional non-cleavable linker 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester sodium salt solution prepared in step (2) to the antibody (antibody A) solution prepared in step (1), stir well, control the reaction temperature to 10-30° C., and the reaction time to 1 h-3 h.

[0202] (4) Purification of the first step of antibody chemical modification reaction solution

[0203] The first step chemical modification reaction solution of the antibody in step (3) was purified by ultrafiltration with 10 times the volume of the reaction buffer (20Mm sodium acetate buffer or sodium phosphate buffer) in step (5) to collect antibody (antibody A)-MCC

[0204] Purified solution of modified product.

[0205] (5) Antibody second step chemical coupling reaction

[0206] e: Accurately weigh the DM1 product according to the ratio of DM1: (antibody A)-MCC = 5-10:1, and dissolve it in DMAC (dimethylacetamide) to prepare a 10 mg / ml DM1 / DMAC (dimethylacetamide) organic solution; add 5-10% (volume ratio) of 10 mg / ml DM1 / DMAC (dimethylacetamide) organic solution reaction liquid to the antibody (antibody A)-MCC modified product solution prepared in step (4), control the reaction temperature to 15-30° C., and the reaction time to 1 h-6 h or overnight.

[0207] f: accurately weigh the DM1 product according to the ratio of DM1: (antibody A)-MCC = 1-5, and dissolve it in DMAC (dimethylacetamide) to prepare a 20 mg / ml DM1 / DMAC (dimethylacetamide) organic solution; add 0.1% to 1% V / V emulsifier (Tween 20 or Tween 80) to the antibody (antibody A)-MCC modified product solution prepared in step (4), add 1-5% (volume ratio) of 20 mg / ml DM1 / DMAC (dimethylacetamide) solution reaction liquid, control the reaction temperature to 15-30° C., and the reaction time to 1 h to 6 h or overnight.

[0208] (6) Antibody second step chemical coupling reaction solution purification

[0209] The reaction solution in step (5) is first purified by exchange with 20Mm sodium acetate buffer or sodium phosphate buffer cation (or anion) chromatography (Fractogel EMD COO-) at a linear flow rate of ≤300cm / h to remove small molecule drugs and drug aggregates, and then purified by ultrafiltration with 10 times the volume of 5-10mM sodium succinate buffer or phosphate buffer preparation buffer to collect the purified solution of anti-HER2 antibody-MCC-DM1 conjugate drug.

[0210] (7) Preparation of anti-HER2 antibody-MCC-DM1 conjugate drug stock solution

[0211] The purified solutions of the anti-HER2 antibody-MCC-DM1 drug conjugate collected in groups e and f in step (6) were respectively added with a certain proportion of other formulation buffer components (preferably the formulation components and contents are 5-10 mM sodium succinate-2-10% sucrose-0.01-0.1% Tween 20 or Tween 80, pH 4.0-7.0) to prepare the anti-HER2 antibody-MCC-DM1 drug conjugate stock solution. The obtained drug mass is shown in Table 4:

[0212] Table 4 Comparison of the efficiency and quality of the anti-HER2 antibody-MCC-DM1 conjugate drug conjugates with the addition of 0.1% to 1% V / V of emulsifier (Tween 20) in the second step of the antibody chemical conjugation reaction:

[0213]

[0214]

[0215] Example 5

[0216] Preparation of anti-CD79b antibody-SM(PEG) 24 -DM1 drug conjugates, verifying the effects of novel antibodies and novel PEGylated water-soluble linkers on the quality of antibody-drug conjugates

[0217]

[0218] Anti-CD79b Antibody-SM(PEG) 24 -DM1 conjugated drug preparation method, the specific steps are as follows:

[0219] (1) Preparation of antibody replacement buffer system for coupling reaction

[0220] The anti-CD79b antibody stock solution is replaced with 50mM sodium phosphate reaction buffer by ultrafiltration replacement or G25 column exchange chromatography, and concentrated to an antibody concentration of 20-30mg / ml to obtain a replaced antibody solution, the pH value of which is 4-8.

[0221] (2) Press -SM(PEG) 24 : Antibody molar ratio 8:1 theoretical calculated value, accurately weigh water-soluble (hydrophilic) heterobifunctional non-cleavable linker SM (PEG) 24 Solid powder, dissolved in water for injection to prepare 10mg / ml SM (PEG) 24 Solution.

[0222] (3) First step of antibody chemical modification reaction

[0223] Add the 10 mg / ml (hydrophilic) heterobifunctional non-cleavable linker SM (PEG) 24 solution prepared in step (2) to the anti-CD79b antibody solution prepared in step (1), stir thoroughly, control the reaction temperature to 10-30° C., and the reaction time to 1 h to 5 h.

[0224] (4) Purification of the first step of antibody chemical modification reaction solution

[0225] The first step chemical modification reaction solution of the antibody in step (3) is purified by ultrafiltration replacement with 10 times the volume of the reaction buffer (20Mm sodium acetate buffer or sodium phosphate buffer) in step (5), and the purified solution of the anti-CD79b antibody-SM (PEG) 24 modified product is collected.

[0226] (5) Antibody second step chemical coupling reaction

[0227] g: Accurately weigh the DM1 product according to the ratio of DM1: anti-CD79b antibody-SM (PEG) 24 = 5-10, and dissolve it in DMAC (dimethylacetamide) to prepare a 10 mg / ml DM1 / DMAC (dimethylacetamide) organic solution; add 5-10% (volume ratio) of 10 mg / ml DM1 / DMAC (dimethylacetamide) organic solution reaction liquid to the anti-CD79b antibody-SM (PEG) 24 modified product solution prepared in step (4), control the reaction temperature to 15-30° C., and the reaction time to 1 h to 6 h or overnight.

[0228] h: accurately weigh the DM1 product according to the ratio of DM1: anti-CD79b antibody-SM (PEG) 24 = 1-5, and dissolve it in DMAC (dimethylacetamide) to prepare a 20 mg / ml DM1 / DMAC (dimethylacetamide) organic solution; add 0.1% to 1% V / V emulsifier (Tween 20 or Tween 80) to the anti-CD79b antibody-SM (PEG) 24 modified product solution prepared in step (4), and add 1-5% (volume ratio) of 20 mg / ml DM1 / DMAC (dimethylacetamide) solution reaction liquid, the reaction temperature is controlled at 15 to 30° C., and the reaction time is 1 h to 6 h or overnight.

[0229] (6) Antibody second step chemical coupling reaction solution purification

[0230] The reaction solution in step (5) is first purified by exchange with 20Mm sodium acetate buffer or sodium phosphate buffer cation (or anion) chromatography (Fractogel EMD COO-) at a linear flow rate of ≤300cm / h to remove small molecule drugs and drug aggregates, and then purified by ultrafiltration with 10 times the volume of 5-10mM sodium succinate buffer or phosphate buffer preparation buffer to collect the purified solution of anti-CD79b antibody-SM (PEG) 24-DM1 conjugated drug.

[0231] (7) Preparation of anti-CD79b antibody-SM(PEG)24-DM1 conjugate drug stock solution

[0232] The purified solutions of the anti-CD79b antibody-SM (PEG) 24-DM1 conjugate drug collected in groups g and h in step (6) were respectively added with a certain proportion of other formulation buffer components (preferably the formulation components and contents are 5-10 mM sodium succinate-2-10% sucrose-0.01-0.1% Tween 20 or Tween 80, pH 4.0-7.0) to prepare the anti-CD79b antibody-SM (PEG) 24-DM1 conjugate drug stock solution. The obtained drug mass is shown in Table 5:

[0233] Table 5 Comparison of the coupling efficiency and quality of the novel antibody, novel PEGylated linker and the anti-CD79b antibody-SM(PEG)24-DM1 coupling drug with 0.1% to 1% V / V of emulsifier (Tween 20) added in the second step chemical coupling reaction of the antibody:

[0234]

Claims

1. A method for preparing an antibody-cytotoxic small molecule drug conjugate, wherein: The method comprises preparing the antibody-cytotoxic small molecule drug conjugate drug by chemically modifying and connecting an antibody and an active cytotoxic small molecule chemical drug through a hydrophilic heterobifunctional non-cleavable linker; Wherein, the hydrophilic heterobifunctional non-cleavable linker has a structure as shown in the following formula (I): Wherein, L1 is selected from phenylene, or -(CH2) m -C(=O)-NHC2H4-(OC2H4) n -; R1 is selected from -SO3Na; m is 0, 1, 2 or 3; n is 1 to 30; The antibody is a humanized monoclonal antibody; the active cytotoxic small molecule chemical drug is maytansine alkaloids and their derivatives; The method comprises: (1) Preparation of antibody reaction buffer: The antibody is replaced by the antibody stock solution into the coupling reaction buffer system by ultrafiltration concentration or desalting chromatography, and then the coupling reaction buffer system is concentrated to an antibody concentration of 10 to 40 mg / ml to obtain the replaced antibody; the coupling reaction buffer system is a phosphate buffer with a pH value of 4.0-9.0; (2) Chemical modification of antibody reaction with linker: The hydrophilic heterobifunctional non-cleavable linker is added to the replaced antibody solution to carry out a chemical modification reaction to obtain a reaction solution containing a modified antibody; the reaction conditions for chemically modifying the antibody with the hydrophilic heterobifunctional non-cleavable linker include: a reaction temperature of 10°C-40°C, a reaction time of 0.5-5 hours, and a pH value of the reaction system solution of 4-9; the ratio of the amount of the antibody to the amount of the hydrophilic heterobifunctional non-cleavable linker is (2-10):1; and step (2) does not use an organic solvent; (3) Purification of antibody-linker modified products: Purifying the reaction solution containing the modified antibody obtained in step (2) by column chromatography or ultrafiltration; (4) Chemical coupling reaction: The purified modified antibody obtained in step (3), the active cytotoxic small molecule chemical drug and the emulsifier are mixed and then subjected to a chemical coupling reaction; the reaction conditions for the chemical coupling of the modified antibody and the active cytotoxic small molecule chemical drug include: a reaction temperature of 15° C. to 35° C., a reaction time of 0.5 to 6 hours, a pH value of the reaction system solution of 3 to 10; and a molar ratio of the active cytotoxic small molecule chemical drug to the modified antibody of (4 to 7): 1; Wherein, based on the total volume of the reaction solution of the chemical coupling reaction as 100%, the amount of the emulsifier is 0.1-1 v / v%; the emulsifier is selected from a combination of one or more of polysorbate 20, polysorbate 40, polysorbate 60 and polysorbate 80; (5) Cationic column chromatography purification: Purify the chemical coupling reaction solution obtained in step (4) by cation column chromatography and collect the eluate; (6) Ultrafiltration replacement and concentration: The eluate obtained in step (5) is concentrated by ultrafiltration using a preparation buffer, and the concentrate is collected to obtain the antibody-cytotoxic small molecule drug conjugate.

2. The preparation method according to claim 1, wherein The hydrophilic heterobifunctional non-cleavable linker has a structure as shown in the following formula (1) or (2):

3. The preparation method according to claim 1, wherein In step (3), the chromatography column is a G25 column chromatography.

4. The preparation method according to claim 1, wherein The amount of emulsifier used is 0.2-0.6 v / v%.

5. The preparation method according to claim 1, wherein Step (1) Concentrate the coupling reaction buffer system to an antibody concentration of 20 to 25 mg / ml.

6. The preparation method according to claim 1, wherein Step (3) flushing the chromatography column or ultrafiltration system with an equilibrium buffer; the equilibrium buffer is 10-30 mM sodium acetate or 20-100 mM phosphate buffer.

7. The preparation method according to claim 1, wherein The cationic chromatography in step (5) is G25-COO column chromatography; the eluent of the cationic chromatography is 5-30 mM sodium succinate buffer or 5-70 mM sodium phosphate buffer.

8. The preparation method according to claim 1, wherein The preparation buffer in step (6) is 5-30 mM sodium succinate buffer or 5-70 mM sodium phosphate buffer.

9. The preparation method according to claim 1, wherein The method further comprises adding other preparation components to the eluate obtained in step (6), wherein the other preparation components are a mixed solution of 5-30 mM sodium succinate buffer-sucrose-0.02% Tween 20, a mixed solution of 5-30 mM sodium succinate buffer-sucrose-0.02% Tween 80, a mixed solution of 5-30 mM sodium succinate buffer-trehalose-0.02% Tween 20, a mixed solution of 5-30 mM sodium succinate buffer - a mixed solution of trehalose-0.02% Tween 80, a mixed solution of 5-70 mM sodium phosphate buffer-sucrose-0.02% Tween 20, a mixed solution of 5-70 mM sodium phosphate buffer-sucrose-0.02% Tween 80, a mixed solution of 5-70 mM sodium phosphate buffer-trehalose-0.02% Tween 20, or a mixed solution of 5-70 mM sodium phosphate buffer-trehalose-0.02% Tween 80.

10. The preparation method according to claim 9, wherein: Other preparation components are a mixed solution of 5-10 mM sodium succinate-2-10% sucrose-0.01-0.1% Tween 20, or a mixed solution of 5-10 mM sodium succinate-2-10% sucrose-0.01-0.1% Tween 80, and the pH value of the mixed solution is 4.0-7.

0.

11. The preparation method according to claim 1, wherein: The reaction time for chemically modifying antibodies with hydrophilic heterobifunctional non-cleavable linkers is 60 min to 150 min.

12. The preparation method according to claim 1, wherein: The antibodies are anti-her2 antibody, anti-CD79b antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody and anti-EGFR antibody.

13. The preparation method according to claim 1, wherein The active cytotoxic small molecule chemical drug is selected from N 2 '-Deacetyl-N 2 '-(3-mercapto-1-oxopropyl) maytansine, N (2 ' ) -Deacetyl-N 2 -(4-mercapto-4-methyl-1-oxopentyl)-maytansine, and N 2’ -Deacetyl-N 2 A combination of one or more of '-(4-mercapto-1-oxopentyl)-maytansine or a MMAE / F composition series.

14. The preparation method according to claim 1, wherein: The phosphate buffer is sodium phosphate buffer, potassium phosphate buffer, Tris-HCl buffer or disodium hydrogen phosphate-citrate buffer.

15. An antibody-cytotoxic small molecule drug conjugate, wherein: The antibody-cytotoxic small molecule drug conjugate is prepared by the preparation method according to any one of claims 1 to 14.

16. Use of the antibody-cytotoxic small molecule drug conjugate according to claim 15 in the preparation of an anti-tumor drug, wherein the tumor is selected from breast cancer, colorectal cancer, gastric cancer or bladder cancer.

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