Anion column chromatography method for removing free small molecules in antibody-drug conjugate, and antibody-drug conjugate

Abstract

An anion column chromatography method for removing free small molecules in an antibody-drug conjugate, and the antibody-drug conjugate. A purification method comprises: using an anion column to purify an antibody-drug conjugate, wherein the antibody-drug conjugate contains negatively-charged free linker-payload molecules. The described purification method exhibits a binding capacity more than 50-fold higher than that of other currently available types of packing materials, and the operation is simple and time-saving and labor-saving are achieved.

Description

Anion column chromatography method for removing free small molecules in antibody conjugate drugs and antibody conjugate drugs TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular, the present application relates to an anion column chromatography method for removing free small molecules in antibody conjugate drugs and antibody conjugate drugs. BACKGROUND

[0002] Antibody conjugate drugs (ADC) are a kind of drugs in which monoclonal antibodies (mAb) are covalently linked to cytotoxic drugs through ADC cross-linking agents (linkers). Because mAb has good targeting ability and can sensitively identify healthy cells and tumor cells, it can bring highly active cytotoxic drugs into tumor cells to complete the killing. At present, 15 ADC drugs have been approved for marketing, and hundreds of ADC drugs are in different stages of clinical trials, so they have great development prospects and commercial value. However, because the development process of ADC involves both macromolecules and small molecules, there are great differences in many aspects, so the conjugate drug has many challenges such as great development difficulty, long research and development cycle, and high production cost.

[0003] ADC drugs are mainly administered by intravenous injection. ADC is similar to antibody, has the characteristics of low clearance rate and long half-life, and after experiencing plasma transport, binding to target points, cell internalization, small body-lysosome action, and release of cytotoxic drugs, the cytotoxic drugs kill tumor cells. Because the recognition ability of mAb to cancer cells and the efficiency of delivering cytotoxic drugs are limited, only 1-2% of cytotoxic drugs can really enter the cells to kill the cells, so the cytotoxic drugs generally have high toxicity (IC50 in nanomolar or picomolar level). Once the cytotoxic drugs are exposed in free form in the body, they will combine with albumin under the condition of the body, and along with the blood flow to all parts of the body, cause bleeding, infection, neuropathy and other adverse reactions, causing great harm to the human body. In ADC drugs, cytotoxic drugs usually exist in the form of free toxin linker (a compound formed by connecting cytotoxic drugs and linkers) or free toxin, and the removal of toxin linker is more common and more difficult in actual production, so the removal of free toxin linker is the top priority to ensure drug safety.

[0004] The most widely used method for removing free toxin linker at present is ultrafiltration and diafiltration (UF / DF), which plays a huge role in ADC small molecule toxin linker removal and other solution impurities. Although the UF / DF process can select a suitable membrane package to make the molecular weight cut-off much larger than the molecular weight of the toxin linker, the removal of some small molecules is not ideal. On the one hand, there is often non-specific adsorption between small molecules and proteins and membrane packages; on the other hand, since most small molecules are hydrophobic, they are prone to hydrophobic end aggregation and form nanoscale micelles in aqueous reactions, so their removal is not easy. In the removal of free small molecule toxin linker, hydrophobic column chromatography, cationic, affinity column chromatography and other methods have been reported and popularized to some extent, and the main principle is as shown in Figure 1: the ADC is adsorbed on the filler, the buffer is used to wash away the unbound small molecules and other impurities, and then the eluent is changed to make the ADC fall off from the filler to complete the purification. However, due to the low filler load, large sample processing difference and other problems, its development has been limited. There are also methods reported for removing toxin linker impurities in ADC by adding organic reagents and using molecular exclusion column chromatography, but they have a series of shortcomings such as complicated steps, long time consumption and damage to sample stability, which cannot meet the actual production needs well.

[0005] SUMMARY

[0006] One object of the present application is to provide a novel purification method that can efficiently remove free small molecules in antibody conjugate drugs, and the amount of antibody conjugate drugs treated is at least 50 times higher than the existing reported technology.

[0007] Another object of the present application is to provide an antibody conjugate drug prepared by the method.

[0008] To achieve the above object, on the one hand, the present application provides an anion column chromatography method for removing free small molecules in antibody conjugate drugs, wherein the method comprises: purifying the antibody conjugate drugs by using an anion column; and the antibody conjugate drugs contain free toxin linker molecules with negative charge.

[0009] The "toxin linker molecule" or "toxin linker" in the present application refers to a compound (linker-payload, LP) formed by connecting a cytotoxic drug with a linker in the preparation of an antibody conjugate drug, i.e. a "cytotoxic drug-linker" molecule, which can also be referred to as "small molecule" in the present application.

[0010] According to some specific embodiments of the present application, at least 3 kg of antibody conjugate drugs are treated per 1 L of filler.

[0011] According to some specific embodiments of the present application, 0-6 kg of antibody conjugate drugs are treated per 1 L of filler.

[0012] According to some specific embodiments of the present application, the method comprises formulating the antibody-drug conjugate and the buffer into a buffer mixed solution and flowing through an anion column for purification treatment; the pH value of the buffer mixed solution is 3-8.

[0013] According to some specific embodiments of the present application, the pH value of the buffer mixed solution is 5-7.

[0014] According to some specific embodiments of the present application, the pH value of the buffer mixed solution is 5, 5.5, 6.0, 6.5 or 7.0.

[0015] According to some specific embodiments of the present application, the buffer is selected from histidine buffer, acetate buffer, succinate buffer, phosphate buffer, citrate buffer, Tris buffer or glycine buffer, etc.; preferably, the buffer is histidine buffer or phosphate buffer.

[0016] According to some specific embodiments of the present application, the salt buffer can be potassium salt or sodium salt.

[0017] According to some specific embodiments of the present application, the molar concentration of the buffer is 10-50 mM.

[0018] According to some specific embodiments of the present application, the molar concentration of the buffer is 10-30 mM.

[0019] According to some specific embodiments of the present application, the molar concentration of the buffer is 20-30 mM.

[0020] According to some specific embodiments of the present application, the concentration of the antibody-drug conjugate in the buffer mixed solution containing the antibody-drug conjugate is 0.5-50 mg / mL.

[0021] The present application utilizes the adsorption of negative substances by anion fillers in an anion column to realize the separation of toxin linker (as shown in FIG. 2), for example, under weak acidic conditions, free toxin linker or other micellar by-products are negatively charged and can be adsorbed by positively charged anion fillers; the isoelectric point of antibodies / antibody-drug conjugates is generally above 8.0, which is positively charged under the weak acidic pH condition and cannot be adsorbed by the fillers, thus the purification can be completed by directly flowing through. Therefore, the existing anion fillers can generally achieve the effect of the present application.

[0022] According to some specific embodiments of the present application, the anion filler is composed of three parts: (1) a cross-linked network base frame; (2) a functional group fixed on the base frame; and (3) an ion with opposite charge to the functional group.

[0023] The cross-linked network matrix has the characteristics of porosity, hydrophilicity and good chemical stability.

[0024] The functional groups fixed on the matrix are charged groups, and quaternary ammonium salt or diethylaminoethyl is generally used as the functional group.

[0025] The ions with opposite charges to the functional groups can reversibly combine with the functional groups.

[0026] According to some specific embodiments of the present application, the anion filler can be an anion filler produced by Sartorius, Biorad, GE Healthcare (such as POROS series resins (D50, PI50, HQ50, XQ)), Sefine (such as Monomix series resins (Mab60-Q, HC60-DEAE Excel)), Nanomicro (such as NW Rose series resins (NW Rose DEAE FF, NW Rose Q FF)), and the like.

[0027] According to some specific embodiments of the present application, the anion filler in the anion column is selected from a strong anion filler or a weak anion filler.

[0028] According to some specific embodiments of the present application, the strong anion filler is selected from an anion filler with a quaternary ammonium salt structure.

[0029] According to some specific embodiments of the present application, the strong anion filler is selected from Capto Q of Sartorius Cytiva.

[0030] According to some specific embodiments of the present application, the weak anion filler is selected from an anion filler with a diethylaminoethyl structure.

[0031] According to some specific embodiments of the present application, the weak anion filler is selected from Diamond DEAE of Biorad.

[0032] According to some specific embodiments of the present application, 0.1 to 6 kg of antibody conjugated drugs are treated per 1 L of filler in the anion column.

[0033] According to some specific embodiments of the present application, 0.1 to 3 kg of antibody conjugated drugs are treated per 1 L of filler in the anion column.

[0034] According to some specific embodiments of the present application, the flow rate of the buffer solution containing the antibody conjugated drugs through the anion column is 0.1-10 CV / min.

[0035] According to some specific embodiments of the present application, the flow rate of the buffer solution containing the antibody conjugated drugs through the anion column is 1-5 CV / min.

[0036] According to some embodiments of the present application, the method further comprises, after the antibody-drug conjugate is purified by the anion column, flushing the anion column with a buffer.

[0037] According to some embodiments of the present application, the flow rate of the buffer during the flushing is 0.1-10 CV / min.

[0038] According to some embodiments of the present application, the flow rate of the buffer during the flushing is 1-5 CV / min.

[0039] It is understood that the buffer used during the flushing can be the same as or different from the buffer used in the buffer containing the antibody-drug conjugate.

[0040] According to some embodiments of the present application, the pH of the buffer mixture is 5-7, the buffer is selected from the group consisting of histidine buffer or phosphate buffer, and the anion filler in the anion column is selected from the group consisting of strong anion fillers.

[0041] The present application utilizes the electric charge of the toxin linker or the electric charge of the toxin linker after derivatization to achieve the adsorption of the toxin linker by the anion filler. The isoelectric point of the antibody and the antibody-drug conjugate is generally above 8.0, which is positively charged under the weak acid condition and cannot be adsorbed by the filler, so that the purification can be completed by directly flowing through. Therefore, the present application does not have special requirements for the antibody-drug conjugate itself.

[0042] Generally, if the toxin linker molecules in the obtained antibody-drug conjugate are negatively charged, the antibody-drug conjugate can be directly used in the method of the present application. For example, when the toxin linker structure contains one or more of sulfonic acid group, carboxylic acid group, nitric acid group and phosphoric acid group, the toxin linker is negatively charged. Alternatively, the toxin linker molecules can be negatively charged during the preparation of the antibody-drug conjugate. Both of the above-mentioned cases can purify the antibody-drug conjugate by the method of the present application.

[0043] Alternatively, if the free toxin linker in the obtained antibody-drug conjugate is not electrically charged, the free toxin linker can be made electrically charged by further derivatization:

[0044] That is, before the antibody-drug conjugate is purified by the anion column, the method further comprises a step of treating the antibody-drug conjugate with a derivatizing agent to make the free toxin linker molecules in the antibody-drug conjugate negatively charged (as shown in FIG. 3).

[0045] The derivatizing agent is a compound that can react with the toxin linker molecules and make the product after the reaction negatively charged.

[0046] According to some embodiments of the present application, the derivatizing agent is a compound containing a thiol group and a negatively charged group, or a compound containing a phosphorous group and a negatively charged group (a negatively charged phosphorous carboxylate salt).

[0047] According to some embodiments of the present application, the derivatizing agent is selected from the group consisting of one or more of acetylcysteine (NAC), thioethanesulfonate, thiophosphonate, thioperoxide, thio carboxylate, and phosphorous carboxylate; further, the derivatizing agent is selected from the group consisting of 2-mercaptoethanesulfonic acid sodium or N-acetylcysteine (NAC).

[0048] According to some embodiments of the present application, the step of treating the antibody conjugate with the derivatizing agent includes mixing the antibody conjugate with the derivatizing agent in a molar amount of 1-60 times of the antibody conjugate (based on one equivalent of the monoclonal antibody) to homogeneity (so that the free toxin linker in the antibody conjugate reacts with the derivatizing agent).

[0049] According to some embodiments of the present application, the step of treating the antibody conjugate with the derivatizing agent includes mixing the antibody conjugate with the derivatizing agent in a buffer to homogeneity.

[0050] According to some embodiments of the present application, the molar amount of the derivatizing agent is 2-60 times of the antibody conjugate.

[0051] According to some embodiments of the present application, the molar amount of the derivatizing agent is 4-60 times of the antibody conjugate.

[0052] According to some embodiments of the present application, the molar amount of the derivatizing agent is 5-40 times of the antibody conjugate.

[0053] According to some embodiments of the present application, the molar amount of the derivatizing agent is 10-40 times of the antibody conjugate.

[0054] According to some embodiments of the present application, the molar amount of the derivatizing agent is 10-20 times of the antibody conjugate.

[0055] According to some embodiments of the present application, the molar amount of the derivatizing agent is 10-15 times of the antibody conjugate.

[0056] According to some embodiments of the present application, the amount of the buffer is such that the concentration of the antibody conjugate in the resulting mixture is 0.5 mg / mL-50 mg / mL after the antibody conjugate is added to the buffer.

[0057] According to some specific embodiments of the present application, the step of treating the antibody conjugated drug with the derivatizing agent comprises mixing the derivatizing agent and the antibody conjugated drug uniformly at 0-37℃.

[0058] According to some specific embodiments of the present application, the step of treating the antibody conjugated drug with the derivatizing agent comprises mixing the derivatizing agent and the antibody conjugated drug uniformly at 15-30℃.

[0059] According to some specific embodiments of the present application, the step of treating the antibody conjugated drug with the derivatizing agent comprises mixing the derivatizing agent and the antibody conjugated drug uniformly at 20-30℃.

[0060] According to some specific embodiments of the present application, after the derivatizing agent and the antibody conjugated drug are mixed uniformly in the buffer, the reaction time of the free toxin linker contained in the derivatizing agent and the antibody conjugated drug is 0.25-24h.

[0061] According to some specific embodiments of the present application, the reaction time of the free toxin linker contained in the derivatizing agent and the antibody conjugated drug is 0.5-24h.

[0062] According to some specific embodiments of the present application, the reaction time of the free toxin linker contained in the derivatizing agent and the antibody conjugated drug is 0.5-1.5h.

[0063] According to some specific embodiments of the present application, the reaction time of the free toxin linker contained in the derivatizing agent and the antibody conjugated drug is 0.5-1h.

[0064] According to some specific embodiments of the present application, the molar mass of the derivatizing agent is 5-40 times of the antibody conjugated drug; the reaction time of the free toxin linker contained in the derivatizing agent and the antibody conjugated drug is 0.5-24h.

[0065] According to some specific embodiments of the present application, the molar mass of the derivatizing agent is 10-40 times of the antibody conjugated drug; the reaction time of the free toxin linker contained in the derivatizing agent and the antibody conjugated drug is 0.5-1.5h.

[0066] According to some specific embodiments of the present application, after the antibody conjugated drug is treated with the derivatizing agent, the step of adjusting the pH and / or concentrating the treatment solution can be further included, and then the anion column purification treatment is performed.

[0067] According to some specific embodiments of the present application, the pH adjustment is adjusting the pH of the solution to 3-8; or adjusting the pH to 5-7.

[0068] As described above, the antibody conjugate drug of the present application does not have a particular requirement, but according to some embodiments of the present application, the antibody conjugate drug of the present application can be prepared according to the following method:

[0069] The reducing agent is added to the reaction system of the antibody molecule, and the reaction is carried out at 0-37°C. After the reaction is completed, the toxin linker is added, and the reaction is carried out at 0-37°C.

[0070] According to some embodiments of the present application, the reducing agent is a compound containing a reducible disulfide bond of thiol or phosphorus.

[0071] According to some embodiments of the present application, the reducing agent is selected from one or a combination of two of dithiothreitol and tris(2-carboxyethyl)phosphine.

[0072] According to some embodiments of the present application, the molar amount of the reducing agent is 1-16 times the molar amount of the antibody molecule.

[0073] According to some embodiments of the present application, the molar amount of the reducing agent is 2-10 times the molar amount of the antibody molecule.

[0074] According to some embodiments of the present application, the molar amount of the reducing agent is 6-10 times the molar amount of the antibody molecule.

[0075] According to some embodiments of the present application, the molar amount of the toxin linker is 2-20 times the molar amount of the antibody molecule.

[0076] According to some embodiments of the present application, the molar amount of the toxin linker is 8-20 times the molar amount of the antibody molecule.

[0077] According to some embodiments of the present application, the molar amount of the toxin linker is 10-20 times the molar amount of the antibody molecule.

[0078] According to some embodiments of the present application, the reaction time of adding the reducing agent to the reaction system of the antibody molecule and carrying out the reaction at 0-37°C is 1-24h.

[0079] According to some embodiments of the present application, the reaction time of adding the reducing agent to the reaction system of the antibody molecule and carrying out the reaction at 0-37°C is 1-3h.

[0080] According to some embodiments of the present application, after the reducing agent is added to the reaction system of the antibody molecule, the reaction is carried out at 15-30°C.

[0081] According to some specific embodiments of the present application, the reaction is carried out at 20-30℃ after the reducing agent is added to the reaction system of the antibody molecule.

[0082] According to some specific embodiments of the present application, the reaction time is 1-24h at 0-37℃ after the toxin linker is added.

[0083] According to some specific embodiments of the present application, the reaction time is 1-3h at 0-37℃ after the toxin linker is added.

[0084] According to some specific embodiments of the present application, the reaction is carried out at 15-30℃ after the toxin linker is added.

[0085] According to some specific embodiments of the present application, the reaction is carried out at 20-30℃ after the toxin linker is added.

[0086] According to some specific embodiments of the present application, the reaction solvent in the reaction system is a buffer.

[0087] According to some specific embodiments of the present application, the concentration of the antibody molecule in the reaction system before the reducing agent is added is 5-20mg / mL.

[0088] According to some specific embodiments of the present application, the preparation of the antibody conjugated drug comprises:

[0089] An appropriate amount of monoclonal antibody is diluted to a concentration of 5-20mg / mL using a buffer, 1-16 equivalents of reducing agent are added to the antibody, and the reaction is carried out at 0-37℃ for 1-24h. 2-20 equivalents of toxin linker are added to the reaction system, and the reaction is carried out at 0-37℃ for 1-24h to obtain the antibody conjugated drug.

[0090] According to some specific embodiments of the present application, the preparation of the antibody conjugated drug comprises:

[0091] An appropriate amount of monoclonal antibody is diluted to a concentration of 5-20mg / mL using a buffer, 1-16 equivalents of reducing agent are added to the antibody, and the reaction is carried out at 0-37℃ for 1-24h. 2-20 equivalents of toxin linker are added to the reaction system, and the reaction is carried out at 0-37℃ for 1-24h to obtain the antibody conjugated drug.

[0092] According to some specific embodiments of the present application, the preparation of the antibody conjugated drug further comprises the step of adding a derivative agent in a molar amount of 1-60 equivalents of the antibody after the antibody conjugated drug is synthesized.

[0093] According to some embodiments of the present application, wherein the molar amount of the derivative agent added in the derivatization process is 2-20 times the molar amount of the antibody-drug conjugate when the antibody-drug conjugate is prepared by adding a reducing agent to the reaction system of the antibody molecule.

[0094] According to some embodiments of the present application, wherein the toxin linker structure in the antibody-drug conjugate of the present application has a group capable of reacting with a sulfhydryl group as the linker reacting with the antibody.

[0095] According to some embodiments of the present application, wherein the group capable of reacting with a sulfhydryl group in the antibody-drug conjugate of the present application is selected from one or more of a maleimide group, an α,β-unsaturated carbonyl group, a haloacetyl group, and a pyridyl disulfide group, a haloalkyl group, and a vinyl sulfone group.

[0096] According to some embodiments of the present application, wherein the α,β-unsaturated carbonyl group is an acrylate group.

[0097] According to some embodiments of the present application, wherein the cytotoxic drug in the structure of the antibody-drug conjugate is an anti-tumor drug.

[0098] According to some embodiments of the present application, wherein the cytotoxic drug in the structure of the antibody-drug conjugate is a DNA replication-related anti-tumor drug or a microtubulin formation-related anti-tumor drug.

[0099] According to some embodiments of the present application, wherein the DNA replication-related anti-tumor drug is a camptothecin or a pyrrolobenzodiazepine drug.

[0100] According to some embodiments of the present application, wherein the microtubulin formation-related anti-tumor drug is a dolastatin or a maytansinoid drug.

[0101] According to some embodiments of the present application, wherein the cytotoxic drug is an anti-tumor drug; preferably a DNA replication-related anti-tumor drug (preferably a camptothecin or a pyrrolobenzodiazepine drug), or a microtubulin formation-related anti-tumor drug (preferably a maytansinoid or a dolastatin drug); preferably the camptothecin drug is selected from Irinotecan, Exatecan, Dxd, or 7-ethyl-10-hydroxy camptothecin (SN38), the maytansinoid drug is selected from a maytansinoid thio derivative (preferably DM1 or DM4), the dolastatin is selected from an auristatin derivative (preferably MMAE or MMAF), and the pyrrolobenzodiazepine drug is selected from a PBD and a derivative thereof.

[0102] According to some embodiments of the present application, the toxin linker is LP1 (MC-GGFG-Dxd), LP2 (MC-VC-PAB-DM1) or LP3 (MC-VC-PAB-SN38).

[0103] The linker of LP1 is maleimide (i.e. MC).

[0104] GGFG of LP1 is a tetrapeptide: i.e. glycine-glycine-phenylalanine-glycine; the cytotoxic drug of LP2 is drubicin (i.e. Dxd).

[0105] The linker of LP2 is maleimide (i.e. MC).

[0106] VC-PAB of LP2 is Val-Cit-PAB, i.e. valine-citrulline-p-aminobenzyloxycarbonyl; the cytotoxic drug of LP2 is maytansine thio derivative (i.e. DM1).

[0107] The linker of LP3 is maleimide (i.e. MC).

[0108] VC-PAB of LP3 is Val-Cit-PAB, i.e. valine-citrulline-p-aminobenzyloxycarbonyl; the cytotoxic drug of LP1 is camptothecin derivative 7-ethyl-10-hydroxycamptothecin (i.e. SN38).

[0109] According to some embodiments of the present application, the structures of LP1, LP2 and LP3 are as follows, respectively:

[0110] The antibody in the antibody conjugate drug of the present application can be any antibody for preparing antibody conjugate drug, for example, can be trastuzumab, patitumumab or bevacizumab.

[0111] In another aspect, the present application also provides an antibody conjugate drug prepared by any method of the present application, wherein the content of free toxin linker in the antibody conjugate drug is less than or equal to 170 μg / mL.

[0112] According to some embodiments of the present application, the content of free toxin linker in the antibody conjugate drug is less than or equal to 0.1 μg / mL.

[0113] It can be understood that the embodiments of the present application can be combined arbitrarily without contradiction. Due to the limited space, the combinations of the embodiments of the present application are not described one by one.

[0114] In summary, the application provides an anion column chromatography method for removing free small molecules in antibody conjugated drugs and antibody conjugated drugs.

[0115] 1. Universality: Different types of derivatization reagents can make free toxin linkers negatively charged and suitable for subsequent purification; different sources of anion fillers have significant adsorption on different types of toxin linkers;

[0116] 2. Specificity: The anion filler has no adsorption effect on antibody conjugated molecules;

[0117] 3. High efficiency: The loading capacity is more than 50 times higher than that of other types of fillers, and the operation is simple, more time and labor saving. BRIEF DESCRIPTION OF DRAWINGS

[0118] Figure 1 is a schematic diagram of the existing cation filler purification technology.

[0119] Figure 2 is a schematic diagram of the purification technology principle of the application.

[0120] Figure 3 is a schematic diagram of the derivatization principle of the application.

[0121] Figure 4 is a schematic diagram of the coupling reaction and post-treatment process of Example 1.

[0122] Figure 5 is a concentration HPLC detection chart of the negatively charged free small molecule LP1 after UF / DF treatment after derivatization in Example 1.

[0123] Figure 6 is a concentration HPLC detection chart of the negatively charged free small molecule LP1 after anion filler purification treatment after derivatization in Example 1.

[0124] Figure 7 is a concentration HPLC detection chart of the negatively charged free small molecule LP2 after UF / DF treatment after derivatization in Example 1.

[0125] Figure 8 is a concentration HPLC detection chart of the negatively charged free small molecule LP2 after anion filler purification treatment after derivatization in Example 1.

[0126] Figure 9 is a concentration HPLC detection chart of the negatively charged free small molecule LP3 after UF / DF treatment after derivatization in Example 1.

[0127] Figure 10 is a concentration HPLC detection chart of the negatively charged free small molecule LP3 after anion filler purification treatment after derivatization in Example 1.

[0128] Figure 11 is a concentration HPLC detection chart of the negatively charged free small molecule LP4 after UF / DF treatment after derivatization in Example 1.

[0129] Figure 12 is a concentration HPLC detection chart of the negatively charged free small molecule LP4 after the anion filler purification treatment after derivatization of Example 1.

[0130] Figure 13 is a purification treatment schematic diagram of Example 2 (LP4).

[0131] Figure 14 is an HPLC detection result of two different filler treatments of Example 2 (LP4).

[0132] Figure 15 is an HPLC detection result of Example 4 (LP4).

[0133] Figure 16 is a standard curve result of Example 4 (LP4).

[0134] Figure 17 is an HPLC peak position chart of the small molecule LP4 after derivatization of Example 5. DETAILED DESCRIPTION

[0135] The technical solutions of the present application are described in detail below in combination with the drawings and examples, but the protection scope of the present application includes but is not limited to this.

[0136] Example 1

[0137] Adsorption of anion filler on four small molecules

[0138] Synthesis of ADC drugs:

[0139] Take 1 g of trastuzumab, dilute the monoclonal antibody to a concentration of 10 mg / mL using a buffer (10 mM pH 7.0 phosphate), add 6 times the amount of TCEP (tris (2-carboxyethyl) phosphine) to the antibody, place in a water bath at 25°C for 2 h, divide the reacted antibody into 4 equal parts, and add 10 times the amount of toxin linker LP1, LP2, LP3 and LP4 (LP3 and LP4 are the same small molecule, which is subsequently treated with different derivatization reagents) to each part, respectively, place in a constant temperature oscillator at 25°C for 2 h; After the reaction is completed, add 10 times the amount of derivatization reagent 2-mercaptoethanesulfonic acid sodium (ADC 1, 2, 3) or N-acetyl cysteine (NAC) (ADC 4), 25°C for 0.5 h and adjust the pH value to 6.0, adjust the concentration of the ADC solution to 10-15 mg / mL, and obtain the reaction solution ADC 1-1, ADC 2-1, ADC 3-1, ADC 4-1.

[0140] Anion column adsorption treatment of the reaction solution after derivatization:

[0141] The AKTA PURE 150 chromatography system and Capto Q 1 mL pre-packed column were used for adsorption treatment of the derivatized reaction solution. Half of the sample (ADC 1-1, ADC 2-1, ADC 3-1, ADC 4-1) was purified and the flow-through was collected, and the other half was purified by ultrafiltration as Comparative Example 1 (ADC 1-2, ADC 2-2, ADC 3-2, ADC 4-2) and the flow-through was collected, and then the flow-through was washed with a mobile phase of histidine buffer (20 mM, pH 6.0) at a flow rate of 1 CV / min. When the UV absorption at 280 nm decreased to 10 mAU, it was recorded as complete, i.e. the flow-through ADC 1-3, ADC 2-3, ADC 3-3, ADC 4-3 was obtained. The content of free small molecules in each was tested by RP HPLC, and Figure 4 is a schematic diagram of the coupling reaction and post-treatment process.

[0142] Determination of HPLC content:

[0143] The content of the charged toxin linker was tested by HPLC, with a detection wavelength of 370 nm (small molecule LP1, 3, 4) or 265 nm (small molecule LP2); mobile phase A was deionized water (containing 0.1% trifluoroacetic acid TFA), mobile phase B was acetonitrile (containing 0.1% trifluoroacetic acid TFA), and the gradient elution conditions were: start: 0 min, 95% A, 5% B; end: 15 min, 0% A, 100% B.

[0144] The HPLC detection chart of free LP1 after UF / DF treatment (ADC 1-2) is shown in Figure 5; and the detection chart after anion Capto Q filler purification treatment is shown in Figure 6 (ADC 1-3). The HPLC detection charts of free LP2 after UF / DF (ADC 2-2) and Q filler treatment (ADC 2-3) are shown in Figures 7 and 8, respectively. The HPLC detection charts of free LP3 after UF / DF (ADC 3-1) and Q filler treatment (ADC 3-3) are shown in Figures 9 and 10, respectively. The HPLC detection charts of free LP4 after UF / DF (ADC 4-1) and Q filler treatment (ADC 4-3) are shown in Figures 11 and 12, respectively.

[0145] As shown in Figure 5, the reaction solution treated by the UF / DF system of the ultrafiltration system cannot effectively remove small molecule impurities, and the impurity content is 4 μg / mL, and the characteristic peak of small molecule LP1 is near the retention time of 7.0 min. As shown in Figure 6, after Q filler treatment, the content of small molecules is lower than the detection limit, and the adsorption effect of Q filler is good.

[0146] As can be seen from FIG. 7, the reaction solution cannot effectively remove the charged small molecule impurities after UF / DF treatment, and the impurity content is 6 μg / mL, and the small molecule LP2 characteristic peak is near the retention time 7.2 min. As can be seen from FIG. 8, after Q filler treatment, the small molecule content is lower than the detection lower limit, and the Q filler adsorption effect is good.

[0147] As can be seen from FIG. 9, the reaction solution cannot effectively remove the charged small molecule impurities after UF / DF treatment, and the impurity content is 11 μg / mL, and the small molecule LP3 characteristic peak is near the retention time 9.0 min. As can be seen from FIG. 10, after Q filler treatment, the small molecule content is lower than the detection lower limit, and the Q filler adsorption effect is good.

[0148] As can be seen from FIG. 11, the reaction solution cannot effectively remove the charged small molecule impurities after UF / DF treatment, and the impurity content is 21 μg / mL, and the small molecule LP4 characteristic peak is near the retention time 9.2 min. As can be seen from FIG. 12, after Q filler treatment, the small molecule content is lower than the detection lower limit, and the Q filler adsorption effect is good.

[0149] The data before and after purification are shown in Table 1 below:

[0150] Table 1

[0151] Conclusion: By increasing the derivatization and using Q filler treatment steps, the adsorption of different types of small molecules can be effectively completed. The above operation can be used as a universal method for purifying free small molecules with maleimide linker or other derivatization reagents that can be derivatized with negative charged reagents.

[0152] Example 2

[0153] Adsorption of toxin linker by different anion columns

[0154] Using the ADC 4-1 crude product in Example 1 (see FIG. 4) as the sample (the phosphate buffer concentration is 10 mM, and the sample concentration of ADC 4-1 is adjusted to 10 mg / mL), different manufacturers' anion fillers (filler 1 is Sartobind Capto Q (strong anion filler); filler 2 is BIA Separations Diamond DEAE (weak anion filler)) are selected, and the column is packed with the same volume (1 mL). After completion, the ADC 4-1 sample is divided into two parts for loading, and the flow-through fluid after purification is collected. The free small molecule content is tested by RP-HPLC, and the process is shown in FIG. 13.

[0155] Anion column purification:

[0156] After loading, histidine buffer (20 mM, pH 6.0) is used for flushing, and the loading and flushing flow rates are both 1 CV / min.

[0157] Determination of HPLC content:

[0158] The content of the charged toxin linker was tested, with a detection wavelength of 370 nm; mobile phase A was deionized water (containing 0.1% TFA), and mobile phase B was acetonitrile (containing 0.1% TFA), with gradient elution conditions as follows: start: 0 min, 95% A, 5% B; end: 15 min, 0% A, 100% B.

[0159] As can be seen from the HPLC detection results in FIG. 14, both the strong anion filler 1 and the weak anion filler 2 have good adsorption effects on the same sample, and the content of the toxin linker (retention time: 9.2 min) is lower than the detection lower limit.

[0160] Example 3

[0161] Adsorption of anion column to ADC

[0162] The ADC samples (ADC 1-1, ADC 2-1, ADC 3-1, ADC 4-1, adjusted sample concentration to 10 mg / mL) in Example 1 (see FIG. 4) were detected using a UV-Vis ultraviolet spectrophotometer, wherein they were diluted to the same volume using a phosphate buffer (20 mM, pH 7.0), and phosphate was used as a blank control, with a detection wavelength of 280 nm. The results are shown in Table 2:

[0163] Table 2. Comparison of ultraviolet absorption values of each sample before and after AKTA anion filler treatment

[0164] Note: The differences between the samples ADC 1-3, ADC 3-3 and ADC 4-3 after anion chromatography treatment and the samples ADC 1-1, ADC 3-1 and ADC 4-1 before treatment are due to the fact that free LP1, LP3 and LP4 also have absorption at 280 nm, which can cause the overall sample absorption to be higher.

[0165] As can be seen from Table 2, there is no significant difference in the content of the ADC samples after anion filler treatment compared with ultrafiltration operation, proving that the anion filler has no adsorption to the ADC and will not cause loss of the sample.

[0166] Example 4

[0167] Adsorption loading test of anion filler to toxin linker

[0168] The toxin linker LP4 was selected for scale-up coupling experiments, and the reaction conditions were as follows: 3 g of trastuzumab was diluted to a concentration of 10 mg / mL using phosphate buffer (10 mM pH 7.0), 6 times the amount of TCEP was added to the antibody, and the reaction was stirred in a reaction bottle (25°C, 2 h); 10 times the amount of toxin linker LP4 was added, and the reaction was continued under the same conditions for 2 h; after the reaction was completed, 10 times the amount of derivatization reagent NAC was added and the reaction was continued under the same conditions for 0.5 h. Finally, the pH was adjusted to 6.0 and concentrated, and after completion, the volume V1 of the obtained retentate was recorded (105 mL), and the content of free small molecules in the retentate C1 was tested by HPLC (170 μg / mL).

[0169] The derivatized ADC solution was purified using an AKTA PURE 150 chromatography system and a Capto Q 1 mL pre-packed column, and the flow-through was collected, and then the anion filler was washed with a mobile phase, which was histidine buffer (20 mM, pH 6.0), and the loading and washing flow rates were both 1 CV / min. After loading was completed, the buffer was continued to be washed for 2-3 CV until the ultraviolet (UV) 280 nm absorption decreased to 10 mAU, which was recorded as the completion of the treatment. After the treatment was completed, the content of free small molecules in each tube was tested by RP HPLC, and the purified liquid that met the requirements was combined.

[0170] HPLC content determination: HPLC was used to test the content of the charged toxin linker, and the detection wavelength was 370 nm; the mobile phase A was deionized water containing 0.1% TFA, and the mobile phase B was acetonitrile containing 0.1% TFA, and the gradient elution conditions were as follows: start: 0 min, 95% A, 5% B; end: 15 min, 0% A, 100% B.

[0171] According to the HPLC test (FIG. 15) and the standard curve results (FIG. 16), the content of the toxin linker LP4 before anion chromatography treatment was 170 μg / mL, and the content of the toxin linker after treatment was all below the lower limit of 0.1 μg / mL, and no full loading or overloading occurred; according to the standard curve formula (calculated as 0.1 μg / mL after treatment), the data before and after the anion filler treatment showed that the amount of adsorbed toxin linker was about 17.8 mg, and according to the ADC mass calculation, 1 mL of Q filler could treat more than 3 g of ADC. Conversion: the anion filler loading was greater than 3,000 g / L (calculated according to the mass of ADC), which was much higher than the loading of the cationic filler 40-60 g / L and the hydrophobic filler 10-20 g / L. Therefore, the loading of the anion filler per liter relative to the mass of the ADC was 0.1-6 kg, preferably 0.1-3 kg.

[0172] Example 5 Effect of different derivatization conditions on derivatization efficiency

[0173] The toxin linker LP4 was selected for scale-up coupling experiments, and the reaction conditions were as follows: 5 g of trastuzumab was diluted to a concentration of 10 mg / mL using phosphate buffer (buffer 10 mM pH 7.0), 6 times the amount of TCEP was added to the antibody, and the reaction was stirred in a reaction bottle (25°C, 2 h); 10 times the amount of toxin linker LP4 was added, and the reaction was continued under the same conditions for 2 h; after the reaction was completed, the derivatization reagent NAC was added, and the reaction was continued under the same conditions, and Table 3 shows the comparison of derivatization conditions, wherein the derivatization reagent was set at 5, 10, 20, and 40 equivalents (calculated based on 1 equivalent of the antibody), and the reaction time was set at 15, 30, and 60 min, and the derivatization efficiency of derivatization was determined by HPLC integral area. The HPLC test method: detection wavelength: 370 nm; mobile phase A: deionized water (containing 0.1% TFA), mobile phase B: acetonitrile (containing 0.1% TFA), gradient elution conditions: start: 0 min, 95% A, 5% B; end: 15 min, 0% A, 100% B. Figure 17 shows the HPLC detection chart of the derivatization sample of No. 6, wherein the substance at 9.2 min is the negatively charged NAC-LP4 after derivatization, and the substance at 10.6 min is the uncharged LP4 after derivatization.

[0174] Table 3 Derivatization conditions of toxin linker

[0175] According to Table 3, it can be seen that when the amount of derivatization reagent is ≥10 equivalents, preferably 10-40 equivalents, and the reaction time is ≥30 min, preferably 30-60 min, the derivatization efficiency is basically stable. Since the derivatization reagent itself is negatively charged, it will be adsorbed by anion filler, affecting the adsorption effect of the toxin linker, therefore, the amount of derivatization reagent should be minimized. Considering time, cost, and chromatographic efficiency, the amount of derivatization reagent is preferably 10 equivalents, and the reaction time is preferably 30 min, which is the subsequent derivatization condition.

[0176] Example 6 Influence of pH value and filler type on adsorption effect

[0177] The toxin linker LP4 was selected for scale-up coupling experiments, and the reaction conditions were as follows: 5 g of trastuzumab was diluted to a concentration of 10 mg / mL using phosphate buffer (buffer 10 mM pH 7.0), 6 times the amount of TCEP was added to the antibody, and the reaction was stirred in a reaction bottle (25°C, 2 h); 10 times the amount of toxin linker LP4 was added, and the reaction was continued under the same conditions for 2 h; after the reaction was completed, the derivatization reagent NAC was added, and the reaction was continued, wherein the derivatization reagent was set at 10 equivalents (calculated based on 1 equivalent of the antibody), the reaction was continued at 25°C for 0.5 h, and the pH was adjusted to 6.0, thereby obtaining the reaction solution.

[0178] After completion, the anion column purification treatment was carried out according to the conditions in Table 4, i.e. the ADC reaction solution sample after derivatization was adjusted to pH 4, 5, 6, 7, and 8 in phosphate buffer, and Capto Q filler and Diamond DEAE filler were used for treatment according to the purification method of Example 1. Finally, HPLC was used to detect the free toxin linker, and the loss of ADC sample was detected by ultraviolet spectrophotometer, wherein if the difference in 280 nm absorption before and after treatment was greater than 30%, it was recorded as loss.

[0179] Table 4: Conditions for treating ADC crude product with anion filler

[0180] As can be seen from Table 4, within the pH range of 4-8, both strong anion filler Capto Q and weak anion filler Diamond DEAE can adsorb the derivatized LP4, and in general, Capto Q has better adsorption effect; at pH 4.0 and 8.0, the ADC sample has a loss, and there is a visible white flocculent material. At pH 5, 6 and 7, the adsorption effect is obvious. In summary, within the pH range of 5.0-7.0, both fillers have good adsorption effect on the free toxin linker, and especially, the adsorption effect on the free toxin linker is best under the condition of pH 6.0 and strong anion filler (No. 3).

[0181] Example 7: Application of different antibodies and derivatization reagents

[0182] Take 1 g of bevacizumab, dilute the concentration to 10 mg / mL using buffer (10 mM pH 7.0 phosphate), add 6 times the amount of TCEP to the antibody, and place it in a water bath at 25°C for 2 h. Add 10 times the amount of toxin linker LP4 to the reaction completed antibody, and place it in a constant temperature oscillator at 25°C for 2 h. After the reaction is completed, add 10 times the amount of derivatization reagent TCEP, react at 25°C for 0.5 h, and adjust the pH value to 6.0, to obtain reaction solution ADC 5-1.

[0183] Anion column adsorption treatment of the reaction solution after derivatization:

[0184] The AKTA PURE 150 chromatography system and Capto Q 1 mL pre-packed column were used to purify the ADC solution (ADC 5-1, sample concentration 10 mg / mL) after derivatization, and the mobile phase was used for washing, the mobile phase was histidine buffer (20 mM, pH 6.0), the sample loading and mobile phase flow rate were both 1 CV / min. The content of free small molecules was tested by RP HPLC of Example 1.

[0185] The experimental results: the content of free small molecules is <0.1 μg / mL, which shows that the anion column chromatography has a similar removal effect on the free toxin linker of the antibody conjugate drug obtained by coupling bevacizumab with LP4, which shows that the purification method is suitable for different types of antibodies.

[0186] Comparative Example 3

[0187] The cationic filler method described on pages 71-80 of Ion exchange chromatography was used, as follows:

[0188] Table 5 Performance parameters of cation exchange resin (containing ribonucleic acid binding capacity)

[0189] Table 6 Performance parameters of cation exchange resin (containing antibody binding capacity)

[0190] The principle is shown in Figure 1. The cationic filler adsorbs the ADC drug in the sample and does not adsorb the toxin linker, and then the ADC drug is eluted with a high salt solution. The defect of this method is that the ADC drug has a molecular weight significantly greater than the toxin linker, and the cationic filler has a low loading capacity for the ADC drug in the drug, 1 L of cationic filler can only adsorb 40-60 g of ADC drug, as shown in Tables 5-6, 1 ml of cationic filler (HiTrap SP HP) can adsorb 55 mg of ribonuclease, and 1 ml of cationic filler (HiTrap SP FF) can adsorb 50 mg of IgG antibody. At the same time, 1 L of anion filler of the present application can adsorb 3 kg of ADC sample, greatly increasing the cost of the filler. And the ADC drug needs to be eluted with a high salt, and it is reported that a high salt solution (0.15 M) can reduce the charge of the ADC, reduce the solubility of the ADC molecules in the solution, and increase the aggregation of the ADC, thereby causing loss, especially for high DAR value ADC molecules, which undoubtedly increases the process cost and instability.

[0191] Comparative Example 4

[0192] The ADC drug was purified according to CN115103691A "A pharmaceutical composition containing an antibody drug conjugate and its use" (see Example 1-52 ADC-32):

[0193] The intermediate I solution was prepared by stirring 5.0 g of trastuzumab stock solution (34.44 μmol, trastuzumab diluted in 20 mM histidine-hydrochloride buffer to a final antibody concentration of 15 mg / mL) with 34.64 mg of tris(2-carboxyethyl)phosphine hydrochloride (reducing agent TCEP, Sigma, 120.84 μmol) in a thermostatic water bath at 25 °C for 3 hours.

[0194] The shorter retention time compound 9-A (406.2 mg, 378.17 μmol) of compound 9 was dissolved in 9.98 mL of DMSO to form a DMSO solution of compound 9-A. The above intermediate I solution was pre-added with 23.42 mL of DMSO, and then the above DMSO solution of compound 9-A was added to the intermediate I solution pre-added with DMSO, and stirred at 25 °C in a water bath for 1 hour. The reaction was quenched with cysteine, and filtered. The reaction solution was subjected to ultrafiltration through a membrane bag (30 kd) and was exchanged with 10-fold and 16-fold volume of 20 mM histidine-hydrochloride buffer containing 10% (v / v) DMSO and 2.5 mM EDTA aqueous solution (pH = 6.0) and 10 mM histidine-hydrochloride buffer aqueous solution (pH = 5.5) to remove small molecules and residual solvents, to obtain an exemplary product ADC-32 of general formula FADC-4A.

[0195] The method for removing free small molecules described in Examples 1-52 of Comparative Example 3 above is a DMSO organic reagent method, which introduces an organic solvent in the purification process, which can easily affect the stability of the ADC molecules and cause environmental pollution, and requires a large amount of time. In order to save time and cost, the concentration of free toxin linker, which is difficult to remove, can only be barely qualified to meet the quality requirements, reaching <10 μg / mL (ADC concentration 10 mg / mL), which is much lower than <0.1 μg / mL (same ADC concentration) of the patent method.

[0196] Comparative Example 5

[0197] The ADC drug was purified according to CN105829346B "Anti-HER2 antibody-drug conjugate" (see the specification Examples 2 and Common Operation D):

[0198] Common Operation D: Purification of antibody-drug conjugate

[0199] The NAP-25 column was equilibrated with any one of commercially available phosphate buffered saline (PBS 7.4, Cat. No. 10010-023, Invitrogen), sodium phosphate buffer containing sodium chloride (137 mM) (10 mM, pH 6.0; referred to as PBS 6.0 in this specification), or acetic acid buffer containing sorbitol (5%) (10 mM, pH 5.5; referred to as ABS in this specification). The antibody-drug conjugate reaction aqueous solution (about 1.5 mL) was loaded into the NAP-25 column, and eluted with the amount of buffer as specified by the manufacturer, whereby the antibody fraction was isolated. The isolated fraction was again loaded into the NAP-25 column, and eluted with the buffer, and subjected to gel filtration purification, and the operation was repeated 2 to 3 times, whereby the antibody-drug conjugate was obtained, from which the unlinked drug linker, low molecular compounds (tris(2-carboxyethyl)phosphine hydrochloride (TCEP), N-acetyl-L-cysteine (NAC), dimethyl sulfoxide) were removed.

[0200] The purification method of Example 2 in the above Comparative Example 4 used SEC molecular exclusion chromatography, and the column was a NAP-25 column, and the packing requirements were: sample concentration < 1 mg / mL, and the loading amount was only 1 mg / mL. If the amount of purification was increased, the purification effect was significantly reduced, and it was not suitable for scale-up production, and the operation needed to be repeated 2 to 3 times. It can be seen that the operation method is complex, the impurity removal efficiency is not high, and the micellar complex formed by the aggregation of the toxin linker has a large molecular weight (> 10 KDa), which cannot meet the requirements of the NAP-25 column for the molecular weight of impurities, and therefore the separation effect is not good.

Claims

1. An anion column chromatography method for removing free small molecules in antibody conjugated drugs, wherein, The method comprises: purifying the antibody conjugated drug by using an anion column; removing the free toxin linker with negative charge contained in the antibody conjugated drug, wherein the antibody conjugated drug contains the free toxin linker molecule with negative charge; the toxin linker structure in the antibody conjugated drug has a group capable of reacting with thiol as a linker for reacting with the antibody, and the toxin linker structure in the antibody conjugated drug contains one selected from the following structures: a maleimide group, an α, β-unsaturated carbonyl group, a haloacetyl group, a pyridine dimercapto group, a haloalkyl group, or a vinyl sulfone group.

2. The method of claim 1, wherein, The method comprises: preparing a buffer mixed solution by mixing the antibody conjugated drug and a buffer, and flowing through an anion column for purification; the buffer mixed solution has a pH value of 3-8, and the buffer is selected from a histidine buffer, an acetate buffer, a succinate buffer, a phosphate buffer, a citrate buffer, a tris buffer, or a glycine buffer.

3. The method of claim 2, wherein, The buffer is selected from a histidine buffer or a phosphate buffer, and the buffer mixed solution has a pH value of 5-7.

4. The method of claim 1, wherein, The anion filler in the anion column is selected from a strong anion filler or a weak anion filler; 0.1-6 kg of the antibody conjugated drug is treated per 1 L of the anion column.

5. The method of claim 4, wherein, The anion filler in the anion column is selected from a quaternary ammonium salt structure anion filler or a diethylaminoethyl structure anion filler; 0.1-3 kg of the antibody conjugated drug is treated per 1 L of the anion column.

6. The method of claim 2, wherein, The flow rate of the buffer containing the antibody conjugated drug through the anion column is 0.1-10 CV / min.

7. The method according to any one of claims 1-6, wherein, The method further comprises a step of treating the antibody conjugated drug with a derivatizing agent before purifying the antibody conjugated drug by using an anion column, so that the free toxin linker molecule in the antibody conjugated drug is negatively charged. The derivatizing agent is selected from a compound containing a thiol group and a negative group, or a compound containing a phosphorus group and a negative group.

8. The method of claim 7, wherein, The derivatizing agent is selected from one or more of a combination of acetyl cysteine, thiol ethanesulfonate, thiol phosphate, thiol nitrate, thiol carboxylate, and phosphorus carboxylate.

9. The method of claim 8, wherein, The step of treating the antibody conjugated drug with the derivatizing agent comprises: uniformly mixing the derivatizing agent with the antibody conjugated drug, wherein the molar amount of the derivatizing agent is 1-60 times that of the antibody conjugated drug.

10. The method of claim 9, wherein, The molar amount of the derivatizing agent is 5-40 times that of the antibody conjugated drug; and the reaction time of the derivatizing agent with the free toxin linker contained in the antibody conjugated drug is 0.5-24 h.

11. The method according to any one of claims 1 to 6, wherein, The preparation process of the antibody conjugated drug comprises: adding a reducing agent to a reaction system of an antibody molecule, reacting at 0-37℃, adding a toxin linker molecule after the reaction is completed, and then reacting at 0-37℃, wherein the molar amount of the reducing agent is 1-16 times that of the antibody molecule. The reducing agent is one or more of a combination of dithiothreitol and tris(2-carboxyethyl)phosphine.

12. The method according to any one of claims 1-6, The toxin linker in the antibody conjugate drug also includes a cytotoxic drug, which is an antitumor drug selected from camptothecins, maytansinoids, dolastatins or pyrrolobenzodiazepines.

13. The method of claim 12, wherein, The camptothecins are selected from irinotecan, exatecan or deruxitecan or 7-ethyl-10-hydroxy camptothecin, the maytansinoids are selected from maytansine thio derivatives, the dolastatins are selected from auristatin derivatives, and the pyrrolobenzodiazepines are selected from PBD and derivatives thereof.

14. The method of claim 13, wherein the toxin linker comprises a cytotoxic drug and a linker structure, and the toxin linker is LP1, LP2 or LP3.

15. The antibody-drug conjugate prepared by the method of any one of claims 1 to 14, wherein, The free toxin linker content in the antibody conjugate drug is less than or equal to 0.1 μg / mL.

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