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

Abstract

The present invention provides an anion column chromatography method for removing free small molecules in an antibody-drug conjugate and an antibody-drug conjugate. The purification method comprises: using an anion column to purify the antibody-drug conjugate; the antibody-drug conjugate contains negatively charged free toxin linker molecules. The purification method of the present invention has a loading capacity more than 50 times higher than that of other types of fillers currently available, and is simple to operate, saving time and effort.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to an anion column chromatography method for removing free small molecules in an antibody-coupled drug and the antibody-coupled drug. Background Art

[0002] Antibody-drug conjugates (ADCs) are drugs in which a monoclonal antibody (mAb) is covalently linked to a cytotoxic drug via an ADC cross-linker (linker). Because mAbs have good targeting capabilities, they can sensitively identify healthy cells and tumor cells, and bring highly active cytotoxic drugs into tumor cells to kill them. Currently, 15 ADC drugs have been approved for marketing, and hundreds of ADC drugs have entered different stages of clinical trials, so they have huge development prospects and commercial value. However, since the development process of ADCs involves two fields, large molecules and small molecules, there are huge differences in many aspects, so conjugated drugs face many challenges such as high development difficulty, long R&D cycle, and high production costs.

[0003] ADC drugs are mainly administered by intravenous injection. ADC is similar to antibodies and has the characteristics of low clearance rate and long half-life. After undergoing plasma transport, binding to targets, cell internalization, body-lysosome action, and releasing cytotoxic drugs, cytotoxic drugs kill tumor cells. Due to the limited recognition ability of mAbs for cancer cells and the efficiency of delivering cytotoxic drugs, only 1-2% of cytotoxic drugs can actually enter cells to kill cells, so cytotoxic drugs generally have high toxicity (IC50 nanomolar or picomolar level). Once the cytotoxic drug is exposed to the body in a free form, it will bind to albumin under in vivo conditions, and flow to various parts of the body with blood, causing adverse reactions such as bleeding, infection, and neuropathy, causing great harm to the human body. In ADC drugs, cytotoxic drugs usually exist in the form of free toxin linkers (compounds formed by connecting cytotoxic drugs and linkers) or free toxins, and in actual production, the removal of toxin linkers is often more common and more difficult, so the removal of free toxin linkers is a top priority to ensure drug safety.

[0004] Currently, the most widely used method for removing free toxin linkers is ultrafiltration and diafiltration (UF / DF), which plays a huge role in the removal of ADC small molecule toxin linkers and other solution impurities. Although the UF / DF process can select suitable membrane packages so that the retention molecular weight can be 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, the hydrophobic ends are prone to aggregate in aqueous phase reactions to form nano-scale micelles, so their removal is not easy. In the removal of free small molecule toxin linkers, hydrophobic column chromatography, cationic column chromatography, affinity column chromatography and other methods have been reported in articles and have been promoted to a certain extent. The main principles are shown in the following. Figure 1 ADC is adsorbed on the filler, and a buffer solution is used to wash away impurities such as unbound small molecules. The eluent is then replaced to remove the ADC from the filler to complete the purification. However, due to various problems such as low filler loading capacity and large differences in processed samples, its development has been limited. There are also reported methods of removing toxin linker impurities in ADC by adding organic reagents and using molecular exclusion column chromatography, but these methods have a series of disadvantages such as complicated steps, long time consumption, and damage to sample stability, and cannot meet actual production needs well. Summary of the invention

[0005] One object of the present invention is to provide a novel purification method that can efficiently remove free small molecules in antibody-drug conjugates, and the amount of antibody-drug conjugates processed is at least 50 times higher than that of the existing reported technology.

[0006] Another object of the present invention is to provide an antibody-drug conjugate prepared by the method.

[0007] To achieve the above-mentioned purpose, on the one hand, the present invention provides an anion column chromatography method for removing free small molecules in antibody-drug conjugates, wherein the method comprises: purifying the antibody-drug conjugate using an anion column; the antibody-drug conjugate contains negatively charged free toxin linker molecules.

[0008] The "toxin linker molecule" or "toxin linker" described in the present invention refers to a compound (linker-payload, LP) formed by connecting a cytotoxic drug to a linker in the preparation of an antibody-drug conjugate, that is, a "cytotoxic drug-linker" molecule, which may also be referred to as a "small molecule" in the present invention.

[0009] According to some specific embodiments of the present invention, at least 3 kg of antibody-drug conjugates are processed per 1 L of filler.

[0010] According to some specific embodiments of the present invention, 0 to 6 kg of antibody-drug conjugates are processed per 1 L of filler.

[0011] According to some specific embodiments of the present invention, the method comprises preparing an antibody-drug conjugate and a buffer into a buffer mixed solution and passing the solution through an anion column for purification; the pH value of the buffer mixed solution is 3-8.

[0012] According to some specific embodiments of the present invention, the pH value of the buffer mixed solution is 5-7.

[0013] According to some specific embodiments of the present invention, the pH value of the buffer mixed solution is 5, 5.5, 6.0, 6.5 or 7.0.

[0014] According to some specific embodiments of the present invention, the buffer is selected from histidine buffer, acetate buffer, succinate buffer, phosphate buffer, citrate buffer, Tris buffer or glycine buffer, etc.; preferably, it is histidine buffer or phosphate buffer.

[0015] According to some specific embodiments of the present invention, the salt buffer may be a potassium salt or a sodium salt.

[0016] According to some specific embodiments of the present invention, the molar concentration of the buffer is 10-50 mM.

[0017] According to some specific embodiments of the present invention, the molar concentration of the buffer is 10-30 mM.

[0018] According to some specific embodiments of the present invention, the molar concentration of the buffer is 20-30 mM.

[0019] According to some specific embodiments of the present invention, the concentration of the antibody-drug conjugate in the buffered mixed solution containing the antibody-drug conjugate is 0.5-50 mg / mL.

[0020] The present invention utilizes the adsorption of negatively charged substances by the anion filler in the anion column to achieve the separation of toxin linkers (such as Figure 2 As shown, for example, under weakly acidic conditions, free toxin linkers or other micelle byproducts are negatively charged and can be adsorbed by positively charged anionic fillers; while the isoelectric point of antibodies / antibody-drug conjugates is generally above 8.0, and they are positively charged under the weakly acidic pH conditions and cannot be adsorbed by fillers, so they can be directly flowed through to complete purification. Therefore, existing anionic fillers can generally achieve the effects of the present invention.

[0021] According to some specific embodiments of the present invention, the anionic filler is composed of three parts: (1) a cross-linked network frame; (2) functional groups fixed on the frame; and (3) ions with opposite charges to the functional groups.

[0022] The cross-linked mesh frame is porous, hydrophilic and has good chemical stability;

[0023] The functional group fixed on the base frame is a charged group, and generally a quaternary ammonium salt or diethylaminoethyl is used as the functional group;

[0024] The ions with opposite charges to the functional groups can be reversibly combined with the functional groups.

[0025] According to some specific embodiments of the present invention, the anionic filler can be anionic fillers produced by manufacturers such as Situofan, Bogelon, Thermo Fisher (such as POROS series resins (D50, PI50, HQ50, XQ)), Saifen (such as Monomix series resins (Mab60-Q, HC60-DEAE Excel)), and Nanovita (such as NW Rose series resins (NW Rose DEAE FF, NW Rose Q FF)).

[0026] According to some specific embodiments of the present invention, the anion filler in the anion column is selected from a strong anion filler or a weak anion filler.

[0027] According to some specific embodiments of the present invention, the strong anion filler is selected from anion fillers having a quaternary ammonium salt structure.

[0028] According to some specific embodiments of the present invention, the strong anion filler is selected from Capto Q of Cytiva.

[0029] According to some specific embodiments of the present invention, the weak anion filler is selected from anion fillers having a diethylaminoethyl structure.

[0030] According to some specific embodiments of the present invention, the weak anionic filler is selected from DiamondDEAE of Boglund.

[0031] According to some specific embodiments of the present invention, each 1 L of filler in the anion column can process 0.1 to 6 kg of antibody-drug conjugates.

[0032] According to some specific embodiments of the present invention, each 1 L of filler in the anion column can process 0.1 to 3 kg of antibody-drug conjugates.

[0033] According to some specific embodiments of the present invention, the speed at which the buffer solution containing the antibody-drug conjugate flows through the anion column is 0.1-10 CV / min.

[0034] According to some specific embodiments of the present invention, the speed at which the buffer solution containing the antibody-drug conjugate flows through the anion column is 1-5 CV / min.

[0035] According to some specific embodiments of the present invention, the method further comprises purifying the antibody-drug conjugate with an anion column and then washing the anion column with a buffer.

[0036] According to some specific embodiments of the present invention, the flow rate of the buffer solution during flushing is 0.1-10CV / min.

[0037] According to some specific embodiments of the present invention, the flow rate of the buffer solution during flushing is 1-5CV / min.

[0038] It is understood that the buffer used for washing and the buffer used for the aforementioned buffer containing the antibody-drug conjugate may be the same or different.

[0039] According to some specific embodiments of the present invention, the pH value of the buffer mixed solution is 5-7, the buffer is selected from histidine buffer or phosphate buffer, and the anion filler in the anion column is selected from strong anion fillers.

[0040] The present invention utilizes the charge of the toxin linker, or the characteristics of the toxin linker being charged by derivatization, to achieve the adsorption of the toxin linker by the anionic filler, while the isoelectric point of antibodies and antibody-drug conjugates is generally above 8.0, and they are positively charged under the weakly acidic pH conditions and cannot be adsorbed by the filler, so they can be directly flowed through to complete the purification. Therefore, the present invention has no special requirements for the antibody-drug conjugate itself;

[0041] Generally speaking, if the toxin linker molecules in the obtained antibody-drug conjugates have a negative charge, they can be directly used in the method of the present invention. For example, when the toxin linker structure has one or more of sulfonic acid, carboxylic acid, nitrate and phosphate groups, the toxin linker can be made to have a negative charge; or, during the preparation of the antibody-drug conjugate, the toxin linker molecules can also be made to have a negative charge. In both cases, the antibody-drug conjugates can be purified using the method of the present invention.

[0042] Alternatively, if the free toxin linker in the obtained antibody-drug conjugate is not charged, the free toxin linker can be charged by further derivatization reaction:

[0043] That is, before the antibody-drug conjugate is purified by an anion column, the method further includes a step of treating the antibody-drug conjugate with a derivatizing agent so that the free toxin linker molecules in the antibody-drug conjugate are negatively charged (e.g. Figure 3 shown);

[0044] The derivatizing agent is a compound that can react with the toxin linker molecule and make the product after the reaction negatively charged.

[0045] According to some specific embodiments of the present invention, 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 (negatively charged phosphorous carboxylate).

[0046] According to some specific embodiments of the present invention, the derivatizing agent is selected from a combination of one or more of acetylcysteine ​​(NAC), mercaptoethanesulfonate, mercaptophosphate, mercaptonitrate, mercaptocarboxylate and phosphite carboxylate; further, the derivatizing agent is selected from sodium 2-mercaptoethanesulfonate or N-acetylcysteine ​​(NAC).

[0047] According to some specific embodiments of the present invention, the step of treating the antibody-drug conjugate with a derivatizing agent includes: mixing the antibody-drug conjugate with a molar amount of the derivatizing agent that is 1-60 times that of the antibody-drug conjugate (based on the monoclonal antibody as 1 equivalent) (so that the free toxin linker in the antibody-drug conjugate reacts with the derivatizing agent).

[0048] According to some specific embodiments of the present invention, the step of treating the antibody-drug conjugate with a derivatizing agent comprises: uniformly mixing the antibody-drug conjugate with a molar amount of the derivatizing agent that is 1-60 times that of the antibody-drug conjugate in a buffer.

[0049] According to some specific embodiments of the present invention, the molar amount of the derivatizing agent is 2-60 times that of the antibody-drug conjugate.

[0050] According to some specific embodiments of the present invention, the molar amount of the derivatizing agent is 4-60 times that of the antibody-drug conjugate.

[0051] According to some specific embodiments of the present invention, the molar amount of the derivatizing agent is 5-40 times that of the antibody-drug conjugate.

[0052] According to some specific embodiments of the present invention, the molar amount of the derivatizing agent is 10-40 times that of the antibody-drug conjugate.

[0053] According to some specific embodiments of the present invention, the molar amount of the derivatizing agent is 10-20 times that of the antibody-drug conjugate.

[0054] According to some specific embodiments of the present invention, the molar amount of the derivatizing agent is 10-15 times that of the antibody-drug conjugate.

[0055] According to some specific embodiments of the present invention, the amount of the buffer is such that after the antibody-drug conjugate is added to the buffer, the concentration of the antibody-drug conjugate in the obtained mixed solution is 0.5 mg / mL to 50 mg / mL.

[0056] According to some specific embodiments of the present invention, the step of treating the antibody-drug conjugate with a derivatizing agent comprises: the derivatizing agent and the antibody-drug conjugate are mixed uniformly at 0-37°C.

[0057] According to some specific embodiments of the present invention, the step of treating the antibody-drug conjugate with a derivatizing agent comprises: the derivatizing agent and the antibody-drug conjugate are mixed uniformly at 15-30°C.

[0058] According to some specific embodiments of the present invention, the step of treating the antibody-drug conjugate with a derivatizing agent comprises: the derivatizing agent and the antibody-drug conjugate are uniformly mixed at 20-30°C.

[0059] According to some specific embodiments of the present invention, after the derivatizing agent and the antibody-drug conjugate are evenly mixed in a buffer solution, the reaction time of the derivatizing agent and the free toxin linker contained in the antibody-drug conjugate is 0.25-24h.

[0060] According to some specific embodiments of the present invention, the reaction time between the derivatizing agent and the free toxin linker contained in the antibody-drug conjugate is 0.5-24h.

[0061] According to some specific embodiments of the present invention, the reaction time between the derivatizing agent and the free toxin linker contained in the antibody-drug conjugate is 0.5-1.5h.

[0062] According to some specific embodiments of the present invention, the reaction time between the derivatizing agent and the free toxin linker contained in the antibody-drug conjugate is 0.5-1h.

[0063] According to some specific embodiments of the present invention, the molar amount of the derivatizing agent is 5-40 times that of the antibody-drug conjugate; and the reaction time of the derivatizing agent and the free toxin linker contained in the antibody-drug conjugate is 0.5-24h.

[0064] According to some specific embodiments of the present invention, the molar amount of the derivatizing agent is 10-40 times that of the antibody-drug conjugate; and the reaction time of the derivatizing agent and the free toxin linker contained in the antibody-drug conjugate is 0.5-1.5h.

[0065] According to some specific embodiments of the present invention, after the antibody-drug conjugate is treated with a derivatizing agent, the steps of adjusting pH and / or concentrating the antibody-drug conjugate may be further included, and then the antibody-drug conjugate is purified using an anion column.

[0066] According to some specific embodiments of the present invention, adjusting the pH is to adjust the pH of the solution to 3-8; or to 5-7.

[0067] As mentioned above, the present invention has no special requirements for the antibody-drug conjugate itself, but according to some specific embodiments of the present invention, the antibody-drug conjugate of the present invention can be prepared according to the following method:

[0068] A 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, a toxin linker is added, and the reaction is carried out again at 0-37°C.

[0069] According to some specific embodiments of the present invention, the reducing agent is a compound containing a thiol group or a phosphorous group that can reduce disulfide bonds.

[0070] According to some specific embodiments of the present invention, the reducing agent is selected from one or a combination of two of dithiothreitol and tris(2-carboxyethyl)phosphine.

[0071] According to some specific embodiments of the present invention, the molar amount of the reducing agent is 1-16 times the molar amount of the antibody molecule.

[0072] According to some specific embodiments of the present invention, the molar amount of the reducing agent is 2-10 times the molar amount of the antibody molecule.

[0073] According to some specific embodiments of the present invention, the molar amount of the reducing agent is 6-10 times the molar amount of the antibody molecule.

[0074] According to some specific embodiments of the present invention, the molar amount of the toxin linker is 2-20 times the molar amount of the antibody molecule.

[0075] According to some specific embodiments of the present invention, the molar amount of the toxin linker is 8-20 times the molar amount of the antibody molecule.

[0076] According to some specific embodiments of the present invention, the molar amount of the toxin linker is 10-20 times the molar amount of the antibody molecule.

[0077] According to some specific embodiments of the present invention, a reducing agent is added to the reaction system of the antibody molecule, and the reaction is carried out at 0-37° C. for 1-24 h.

[0078] According to some specific embodiments of the present invention, a reducing agent is added to the reaction system of the antibody molecule, and the reaction is carried out at 0-37° C. for 1-3 h.

[0079] According to some specific embodiments of the present invention, after adding the reducing agent to the reaction system of the antibody molecule, the reaction is carried out at 15-30°C.

[0080] According to some specific embodiments of the present invention, after adding the reducing agent to the reaction system of the antibody molecule, the reaction is carried out at 20-30°C.

[0081] According to some specific embodiments of the present invention, after the toxin linker is added, the reaction time at 0-37° C. is 1-24 h.

[0082] According to some specific embodiments of the present invention, after the toxin linker is added, the reaction time at 0-37° C. is 1-3 h.

[0083] According to some specific embodiments of the present invention, after the toxin linker is added, the reaction is carried out at 15-30°C.

[0084] According to some specific embodiments of the present invention, the reaction is carried out at 20-30°C after the toxin linker is added.

[0085] According to some specific embodiments of the present invention, the reaction solvent in the reaction system is a buffer solution.

[0086] According to some specific embodiments of the present invention, before adding the reducing agent, the concentration of the antibody molecule in the reaction system is 5-20 mg / mL.

[0087] According to some specific embodiments of the present invention, the preparation process of the antibody-drug conjugate includes:

[0088] Take an appropriate amount of monoclonal antibody, dilute it to a concentration of 5-20 mg / mL with a buffer, add a reducing agent of 1-16 times the equivalent of the antibody, react at 0-37°C for 1-24 hours, add 2-20 times the equivalent of the toxin linker, respectively, and react at 0-37°C for 1-24 hours.

[0089] According to some specific embodiments of the present invention, the preparation process of the antibody-drug conjugate includes:

[0090] Take an appropriate amount of monoclonal antibody, dilute it to a concentration of 5-20 mg / mL with a buffer, add a reducing agent of 1-16 times the equivalent of the antibody, place it in a water bath or ice bath at 0-37°C to react for 1-24 hours, add 2-20 times the equivalent of the toxin linker, place it in a water bath or ice bath at 0-37°C to react for 1-24 hours.

[0091] According to some specific embodiments of the present invention, the preparation process of the antibody-drug conjugate further includes the step of adding a derivatizing agent with a molar amount of 1-60 equivalents of the antibody to react after the antibody-drug conjugate is synthesized.

[0092] According to some specific embodiments of the present invention, when the preparation process of the antibody-drug conjugate includes adding a reducing agent to the reaction system of the antibody molecule, the molar amount of the derivatizing agent added during the derivatization treatment is 2-20 times the molar amount of the antibody-drug conjugate.

[0093] According to some specific embodiments of the present invention, the toxin linker structure in the antibody-drug conjugate of the present invention has a group that can react with a thiol group as a linker that reacts with the antibody.

[0094] According to some specific embodiments of the present invention, the group capable of reacting with a thiol group is selected from one or more of a maleimide group, an α,β-unsaturated carbonyl group, a haloacetyl group and a pyridine dithiol group, a haloalkyl group, and a vinyl sulfone.

[0095] According to some specific embodiments of the present invention, the α,β-unsaturated carbonyl group is an acrylate group.

[0096] According to some specific embodiments of the present invention, the cytotoxic drug in the antibody-drug conjugate structure is an anti-tumor drug.

[0097] According to some specific embodiments of the present invention, the cytotoxic drug in the antibody-drug conjugate structure is a DNA replication-related anti-tumor drug or a microtubule formation-related anti-tumor drug.

[0098] According to some specific embodiments of the present invention, the DNA replication-related anti-tumor drug is a camptothecin or pyrrolobenzodiazepine drug.

[0099] According to some specific embodiments of the present invention, the microtubule protein formation-related anti-tumor drug is a dolastatin toxin or maytansine drug.

[0100] According to some specific embodiments of the present invention, the cytotoxic drug is an anti-tumor drug; preferably, it is an anti-tumor drug related to DNA replication (preferably camptothecin or pyrrolobenzodiazepine drugs), or an anti-tumor drug related to tubulin formation (preferably maytansine or dolastatin drugs); preferably, the camptothecin drug is selected from irinotecan, exatecan or derunotecan (Dxd) or 7-ethyl-10-hydroxycamptothecin (SN38), the maytansine drug is selected from maytansine thioderivatives (preferably DM1 or DM4), the dolastatin toxin is selected from auristatin derivatives (preferably MMAE or MMAF), and the pyrrolobenzodiazepine drug is selected from PBD and its derivatives.

[0101] According to some specific embodiments of the present invention, the toxin linker is LP1 (MC-GGFG-Dxd), LP2 (MC-VC-PAB-DM1) or LP3 (MC-VC-PAB-SN38).

[0102] The linker of LP1 is maleimide (ie MC).

[0103] The GGFG of LP1 is a tetrapeptide: glycine-glycine-phenylalanine-glycine; the cytotoxic drug of LP1 is deludecanoic acid (Dxd).

[0104] The linker of LP2 is maleimide (ie MC).

[0105] The VC-PAB of LP2 is Val-Cit-PAB, valine-citrulline-p-aminobenzyloxycarbonyl; the cytotoxic drug of LP2 is a thio derivative of maytansine (ie, DM1).

[0106] The linker of LP3 is maleimide (ie MC).

[0107] The VC-PAB of LP3 is Val-Cit-PAB, valine-citrulline-p-aminobenzyloxycarbonyl; the cytotoxic drug of LP3 is the camptothecin derivative 7-ethyl-10-hydroxycamptothecin (ie SN38).

[0108] According to some specific embodiments of the present invention, the structures of LP1, LP2 and LP3 are respectively as follows:

[0109]

[0110] The antibody in the antibody-drug conjugate of the present invention may be any antibody for preparing an antibody-drug conjugate, for example, trastuzumab, pertuzumab or bevacizumab.

[0111] On the other hand, the present invention also provides an antibody-drug conjugate prepared by any method of the present invention, wherein the content of free toxin linkers in the antibody-drug conjugate is less than or equal to 170 μg / mL.

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

[0113] It is understandable that, under the premise of no contradiction, the specific embodiments of the present invention can be arbitrarily combined. Due to space limitations, the combination of the specific embodiments of the present invention will not be described one by one.

[0114] In summary, the present invention provides an anion column chromatography method for removing free small molecules from an antibody-drug conjugate and an antibody-drug conjugate. The method of the present invention has the following advantages:

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

[0116] 2. Specificity: Anionic fillers have no adsorption effect on antibody-coupled molecules;

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

[0118] Figure 1 Schematic diagram of existing cationic filler purification technology.

[0119] Figure 2 It is a schematic diagram of the purification technology principle of the present invention.

[0120] Figure 3 Schematic diagram of the derivatization principle of the present invention.

[0121] Figure 4 Schematic diagram of the coupling reaction and post-processing process of Example 1.

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

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

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

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

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

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

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

[0129] Fig.12 This is a HPLC detection chart of the concentration of the negatively charged free small molecule LP4 after derivatization in Example 1 after purification by anionic filler.

[0130] Fig.13 Schematic diagram of the purification process of Example 2 (LP4).

[0131] Fig.14 These are the HPLC test results of two different filler treatments in Example 2 (LP4).

[0132] Fig.15 This is the HPLC test result of Example 4 (LP4).

[0133] Fig.16 This is the standard curve result of Example 4 (LP4).

[0134] Fig.17 This is the HPLC peak position diagram of the small molecule LP4 after derivatization in Example 5. DETAILED DESCRIPTION

[0135] The implementation process of the present invention and the beneficial effects produced are described in detail below through 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.

[0136] Example 1

[0137] Adsorption of four small molecules by anionic fillers

[0138] Synthesis of ADC drugs:

[0139] Take 1g of trastuzumab, dilute the monoclonal antibody to a concentration of 10mg / mL with a buffer solution (10mM pH 7.0 phosphate), add 6 times the equivalent of TCEP (tris(2-carboxyethyl)phosphine) of the antibody, place it in a water bath at 25°C for 2h, divide the antibody after the reaction into 4 parts, add 10 times the equivalent of the antibody toxin linkers LP1, LP2, LP3 and LP4 (LP3 and LP4 are the same small molecule, which are subsequently treated with different derivatization reagents. The above small molecules are all commercially available and come from Haoyuan Pharmaceutical), place it on a constant temperature oscillator at 25°C for 2h; after the reaction is completed, add 10 equivalents of the derivatization reagent sodium 2-mercaptoethane sulfonate (ADC 1, 2, 3) or N-acetylcysteine ​​(NAC) (ADC 4), react at 25°C for 0.5h and adjust the pH value to 6.0, and adjust the concentration of the ADC solution to 10-15mg / mL to obtain the reaction solution ADC 1-1, ADC 2-1, ADC 3-1, ADC 4-1.

[0140]

[0141]

[0142] Anion column adsorption treatment of the reaction solution after derivatization:

[0143] The reaction solution after derivatization was adsorbed using AKTA PURE 150 chromatography system and Capto Q 1mL prepacked column, wherein half of the samples (ADC 1-1, ADC 2-1, ADC 3-1, ADC 4-1) were purified and the flow-through was collected, and the other half were 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 mobile phase was used to rinse the anion column, and the mobile phase was histidine buffer (20mM, pH 6.0), and the sample loading and mobile phase flow rates were 1CV / min. When the UV absorption at 280nm dropped to 10mAU, it was marked as complete, that is, the flow-through ADC 1-3, ADC 2-3, ADC 3-3, ADC 4-3 were obtained, and the content of free small molecules therein was tested by RP HPLC, respectively. Figure 4 Schematic diagram of the coupling reaction and post-processing process.

[0144] HPLC content determination:

[0145] HPLC was used to test the content of charged toxin linkers, where the detection wavelength was 370 nm (small molecules 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 gradient elution conditions were: start: 0 min, 95% A, 5% B; end point: 15 min, 0% A, 100% B.

[0146] The HPLC detection chart of free LP1 concentration after UF / DF treatment (ADC 1-2) is as follows Figure 5 As shown; the detection diagram after purification by anionic CaptoQ filler is as shown Figure 6 The HPLC detection diagrams of free LP2 after UF / DF (ADC 2-2) and Q filler treatment (ADC 2-3) are shown in Figure 1. Figure 7 and Figure 8 The HPLC detection diagrams of free LP3 after UF / DF (ADC 3-1) and Q filler treatment (ADC 3-3) are shown in Figure 2. Fig. 9 and Fig.10 The HPLC detection diagrams of free LP4 after UF / DF (ADC 4-1) and Q filler treatment (ADC 4-3) are shown in Figure 2. Fig.11 and Fig.12 shown.

[0147] Depend on Figure 5 It can be seen that the reaction solution cannot effectively remove small molecule impurities after being treated by the UF / DF system. The impurity content is 4μg / mL, and the characteristic peak of small molecule LP1 is around the retention time of 7.0min. Figure 6 It can be seen that after being treated with Q filler, the content of small molecules is lower than the detection limit, and the adsorption effect of Q filler is good.

[0148] Depend on Figure 7 It can be seen that the reaction solution cannot effectively remove charged small molecule impurities after UF / DF treatment, the impurity content is 6μg / mL, and the small molecule LP2 characteristic peak is around the retention time of 7.2min. Figure 8 It can be seen that after being treated with Q filler, the content of small molecules is lower than the detection limit, and the adsorption effect of Q filler is good.

[0149] Depend on Fig. 9 It can be seen that the reaction solution cannot effectively remove charged small molecule impurities after UF / DF treatment, the impurity content is 11μg / mL, and the small molecule LP3 characteristic peak is around the retention time of 9.0min. Fig.10 It can be seen that after being treated with Q filler, the content of small molecules is lower than the detection limit, and the adsorption effect of Q filler is good.

[0150] Depend on Fig.11 It can be seen that the reaction solution cannot effectively remove charged small molecule impurities after UF / DF treatment, the impurity content is 21μg / mL, and the characteristic peak of small molecule LP4 is around the retention time of 9.2min. Fig.12 It can be seen that after being treated with Q filler, the content of small molecules is lower than the detection limit, and the adsorption effect of Q filler is good.

[0151] The data before and after purification are shown in Table 1 below:

[0152] Table 1

[0153]

[0154] Conclusion: By adding 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 to purify free small molecules containing maleimide linkers or other free small molecules that can be derivatized by negatively charged derivatization reagents.

[0155] Example 2

[0156] Adsorption of toxin linkers by different anion columns

[0157] Using the crude ADC 4-1 in Example 1 (see Figure 4 ) was used as the sample (the concentration of phosphate buffer was 10 mM, and the sample concentration of ADC4-1 was adjusted to 10 mg / mL), anion fillers from different manufacturers were selected (filler 1 was Capto Q (strong anion filler); filler 2 was Diamond DEAE (weak anion filler) from Bogelon, and the columns were loaded with the same volume (1 mL). After completion, the ADC 4-1 sample was divided into two portions for loading, and the purified flow-through was collected. The free small molecule content therein was tested by RP-HPLC. The process is shown in Fig.13 .

[0158] Anion column purification:

[0159] After loading, the sample was washed with histidine buffer (20 mM, pH 6.0), and the flow rate for loading and washing was 1 CV / min.

[0160] HPLC content determination:

[0161] The content of charged toxin linkers was tested, wherein the detection wavelength was 370 nm; mobile phase A was deionized water (containing 0.1% TFA), mobile phase B was acetonitrile (containing 0.1% TFA), and gradient elution conditions were: start: 0 min, 95% A, 5% B; end point: 15 min, 0% A, 100% B.

[0162] Depend on Fig.14The HPLC test results show that 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 below the detection limit.

[0163] Example 3

[0164] Adsorption of ADC on anion column

[0165] Take the ADC samples in Example 1 (ADC 1-1, ADC 2-1, ADC 3-1, ADC 4-1, adjust the sample concentration to 10 mg / mL) (see Figure 4 ) was detected using a UV-Vis spectrophotometer, wherein phosphate buffer (20 mM, pH 7.0) was used to dilute it to the same volume, and phosphate was used as a blank control, and the detection wavelength was 280 nm. The results are shown in Table 2:

[0166] Table 2. Comparison of UV absorption values ​​of each sample before and after AKTA anionic filler treatment

[0167]

[0168] Note: There are differences between samples ADC1-3, ADC 3-3 and ADC 4-3 after anion chromatography treatment and samples ADC1-1, ADC3-1, ADC4-1 before treatment. The reason is that free LP1, LP3, LP4 also absorb at 280nm, which will cause the overall sample absorption to be higher.

[0169] It can be seen from Table 2 that compared with the ultrafiltration operation, there is no significant difference in the ADC sample content after the anion filler treatment, which proves that the anion filler has no adsorption to ADC and will not cause sample loss.

[0170] Example 4

[0171] Adsorption capacity test of anionic fillers for toxin linkers

[0172] The toxin linker LP4 was selected for the amplified coupling experiment. The reaction conditions were as follows: 3g of trastuzumab was diluted to a concentration of 10mg / mL using phosphate buffer (10mM pH 7.0), 6 times the equivalent of TCEP was added to the antibody, and the reaction was stirred in a reaction bottle (25°C, 2h); 10 times the equivalent of toxin linker LP4 was added thereto, and the reaction was continued under the same conditions for 2h; after the reaction was completed, 10 times the equivalent of the derivatization reagent NAC was added and the reaction was continued under the same conditions for 0.5h. Finally, the pH was adjusted to 6.0 and concentrated. After completion, the volume of the retained solution V1 (105mL) was recorded, and the content of free small molecules in the retained solution C1 (170μg / mL) was tested using HPLC.

[0173] The derivatized ADC solution was purified and the flow-through was collected using the AKTA PURE 150 chromatography system and the Capto Q 1mL prepacked column, and then the anion filler was rinsed with the mobile phase, the mobile phase was histidine buffer (20mM, pH 6.0), and the loading and rinsing flow rates were 1CV / min. After the loading, 2-3CV buffer was continued to be rinsed until the UV280nm absorption dropped to 10mAU, which was considered complete. After the treatment, the content of free small molecules in each tube was tested using RP HPLC, and the purified solutions that met the test requirements were merged.

[0174] HPLC content determination: HPLC was used to test the content of charged toxin linkers, wherein the detection wavelength was 370 nm; mobile phase A was deionized water (containing 0.1% TFA), mobile phase B was acetonitrile (containing 0.1% TFA), and gradient elution conditions were: start: 0 min, 95% A, 5% B; end point: 15 min, 0% A, 100% B.

[0175] According to HPLC test ( Fig.15 ) and standard curve results ( Fig.16 ), the content of toxin linker LP4 before anion chromatography treatment was 170μg / mL, and the toxin linker content after treatment was lower than the lower limit of 0.1μg / mL, and there was no full load or overload phenomenon; according to the standard curve formula (calculated at 0.1μg / mL after treatment), the data before and after the anion filler treatment showed that: the amount of adsorbed toxin linkers was about 17.8mg, and according to the ADC mass calculation, 1mL Q filler can process more than 3g ADC. After conversion: the anion filler load is greater than 3,000g / L (calculated according to the ADC mass), which is much higher than the cationic filler load of 40-60g / L and the hydrophobic filler load of 10-20g / L. Therefore, the load of the anion filler per liter is 0.1-6kg relative to the mass of ADC, preferably 0.1-3kg.

[0176] Example 5 Effect of different derivatization conditions on derivatization efficiency

[0177] The toxin linker LP4 was selected for the amplified coupling experiment, 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 equivalent of TCEP of the antibody was added, and the reaction was stirred in a reaction bottle (25°C, 2h); 10 times the equivalent of the toxin linker LP4 was added thereto, and the reaction was continued for 2h under the same conditions; after the reaction was completed, the derivatization reagent NAC was added and the reaction was continued under the same conditions. Table 3 is a comparison of the derivatization conditions, in which the derivatization reagent was set at 5, 10, 20, and 40 equivalents (calculated based on the antibody equivalent as 1), the reaction time was set at 15, 30, and 60 min, and the derivatization efficiency was determined by HPLC integrated area. HPLC test method: detection wavelength: 370nm; mobile phase A is deionized water (containing 0.1% TFA), mobile phase B is acetonitrile (containing 0.1% TFA), gradient elution conditions: start: 0min, 95% A, 5% B; end point: 15min, 0% A, 100% B. Fig.17 This is the HPLC detection chart of the derivatized sample No. 6, where 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.

[0178] Table 3 Toxin linker derivatization conditions

[0179] serial number Derivatization Reagents Reaction time Derivatization efficiency 1 5 equivalent 15min 50％ 2 10 equivalent 15min 73％ 3 20 equivalent 15min 86％ 4 40 equivalent 15min 92％ 5 5 equivalent 30min 62％ 6 10 equivalent 30min 91％ 7 20 equivalent 30min 93％ 8 40 equivalent 30min 94％ 9 5 equivalent 60min 71％ 10 10 equivalent 60min 94％ 11 20 equivalent 60min 94％ 12 40 equivalent 60min 95％

[0180] According to Table 3, when the derivatization reagent dosage 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 agent itself is negatively charged, it will be adsorbed by the anionic filler and affect the adsorption effect on the toxin linker. Therefore, the amount of the derivatization agent should be minimized. Considering the time, cost and chromatography efficiency, the derivatization reagent dosage of 10 equivalents and the reaction time of 30 min are preferred as the subsequent derivatization conditions.

[0181] Example 6 Effect of pH value and filler type on adsorption effect

[0182] The toxin linker LP4 was selected for the amplified coupling experiment, 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 equivalent of TCEP to the antibody was added, and the reaction was stirred in a reaction bottle (25°C, 2h); 10 times the equivalent of the toxin linker LP4 was added thereto, and the reaction was continued under the same conditions for 2h; after the reaction was completed, the derivatization reagent NAC was added to continue the reaction, wherein the derivatization reagent was set at 10 equivalents (calculated based on the antibody equivalent as 1), the reaction was carried out at 25°C for 0.5h, and the pH was adjusted to 6.0 to obtain a reaction solution.

[0183] After completion, anion column purification treatment was performed according to the conditions in Table 4, that is, the pH of the derivatized ADC reaction solution sample was adjusted to 4, 5, 6, 7, and 8 in phosphate buffer, and treated with Capto Q filler and Diamond DEAE filler according to the purification method in Example 1. Finally, HPLC was used to detect free toxin linkers, and the loss of ADC samples was measured using a UV spectrophotometer, where a loss was recorded if the difference in 280nm absorption before and after treatment was greater than 30%.

[0184] Table 4 Conditions for treating crude ADC with anionic fillers

[0185]

[0186] As shown in Table 4, in the pH range of 4-8, both the strong anion filler Capto Q and the weak anion filler DiamondDEAE can adsorb the derivatized LP4. In general, Capto Q has a better adsorption effect. At pH 4.0 and 8.0, the ADC samples are lost and white flocculent substances visible to the naked eye are produced. At pH 5, 6 and 7, the adsorption effect is obvious. In summary, at pH = 5.0 to 7.0, the two fillers have a good adsorption effect on free toxin linkers, especially, preferably at pH = 6.0 and strong anion filler conditions (No. 3) The best adsorption effect on free toxin linkers.

[0187] Example 7 Application of different antibodies and derivatization reagents

[0188] Take 1 g of bevacizumab, dilute it to a concentration of 10 mg / mL using a buffer solution (10 mM pH 7.0 phosphate), add 6 times the equivalent of TCEP to the antibody, place it in a water bath at 25°C for 2 hours, add 10 times the equivalent of the antibody toxin linker LP4 to the antibody after the reaction, place it on a constant temperature oscillator at 25°C for 2 hours; after the reaction is completed, add 10 equivalents of the derivatization reagent TCEP, react at 25°C for 0.5 hours and adjust the pH value to 6.0 to obtain the reaction solution ADC 5-1.

[0189] Anion column adsorption treatment of the reaction solution after derivatization:

[0190] The derivatized ADC solution (ADC 5-1, sample concentration of 10 mg / mL) was purified using an AKTA PURE 150 chromatography system and a Capto Q 1 mL prepacked column, and washed with a mobile phase, the mobile phase being histidine buffer (20 mM, pH 6.0), and the sample loading and mobile phase flow rates were both 1 CV / min. The content of free small molecules therein was tested using the RP HPLC of Example 1.

[0191] Experimental results: The content of free small molecules was <0.1 μg / mL, indicating that anion column chromatography had a similar removal effect on the free toxin linkers of the antibody-drug conjugate obtained by conjugating bevacizumab to LP4, which shows that this purification method is suitable for different types of antibodies.

[0192] Comparative Example 3

[0193] The cationic filler method described on pages 71-80 of Ion exchange chromatography was used as follows:

[0194] Table 5 Performance parameters of cation exchange resin (including RNA binding capacity)

[0195]

[0196] Table 6 Performance parameters of cation exchange resin (including antibody binding capacity)

[0197]

[0198] The principle is shown in Figure 1 , the cationic filler adsorbs the ADC drug in the sample without adsorbing the toxin linker, and then elutes the ADC drug with a high salt solution. The defect of this method is that since the molecular weight of the ADC drug is significantly greater than the toxin linker, the cationic filler has a low loading capacity for the ADC drug in the drug. 1L of cationic filler can only adsorb 40-60g of ADC drug. As shown in Table 5-6, 1ml of cationic filler (HiTrap SP HP) can adsorb 55mg of ribonuclease, and 1ml of cationic filler (HiTrap SP FF) can adsorb 50mg of IgG antibody. At the same time, 1L of anionic filler in this application can adsorb 3kg of ADC sample, which greatly increases the cost of filler. And ADC drugs need to be eluted with high salt. It is reported that high salt solution (0.15M) will reduce the charge of ADC, reduce the solubility of ADC molecules in solution, and aggravate the aggregation of ADC, thereby causing losses, especially for ADC molecules with high DAR values. It is more serious, which will undoubtedly increase process costs and instability.

[0199] Comparative Example 4

[0200] According to CN115103691A "A pharmaceutical composition containing an antibody drug conjugate and its use", the ADC drug was purified (see Example 1-52 ADC-32):

[0201]

[0202] At 25°C, in a buffer (pH 5.6) containing 20 mM histidine-hydrochloric acid and 2.5 mM EDTA, 5.0 g of trastuzumab stock solution (34.44 μmol, 20 mM histidine-hydrochloric acid buffer diluted trastuzumab to a final antibody concentration of 15 mg / mL) and 34.64 mg of tris(2-carboxyethyl)phosphine hydrochloride (reducing agent TCEP, Sigma, 120.84 μmol) were stirred in a constant temperature water bath for 3 hours to generate an intermediate I solution.

[0203] Compound 9-A (406.2 mg, 378.17 μmol), which has a shorter retention time, was dissolved in 9.98 mL DMSO to generate a DMSO solution of compound 9-A. 23.42 mL DMSO was pre-added to the intermediate I solution, and the DMSO solution of compound 9-A was added to the intermediate I solution pre-added with DMSO, and the reaction was stirred in a water bath at 25°C for 1 hour, and cysteine ​​was added to quench the reaction, and the reaction was filtered. At 25°C, the reaction solution was passed through an ultrafiltration membrane (30 kd) and replaced with 20 mM histidine-hydrochloric acid and 2.5 mM EDTA buffer aqueous solution (pH = 6.0) containing 10% (v / v) DMSO, and 10 mM histidine-hydrochloric acid buffer aqueous solution (pH = 5.5) by 10 times and 16 times of equal volume ultrafiltration to remove small molecules and residual solvents, and obtain the exemplary product ADC-32 of the general formula of FADC-4A.

[0204] The method for removing free small molecules described in Examples 1-52 of Comparative Example 3 is a DMSO organic reagent method, which introduces an organic solvent in the purification process, which is easy to affect the stability of the ADC molecule and cause environmental pollution, and takes a lot of time. For the difficult-to-remove camptothecin toxin linkers, in order to save time and cost, the concentration of free toxin linkers can only barely meet the quality requirements, reaching <10μg / mL (ADC concentration 10mg / mL), which is much lower than the patent method's <0.1μg / mL (same ADC concentration).

[0205] Comparative Example 5

[0206] Purify the ADC drug according to CN105829346B "Anti-HER2 Antibody-Drug Conjugate" (see Example 2 and Common Operation D in the specification):

[0207] Common Procedure D: Purification of Antibody-Drug Conjugates

[0208] The NAP-25 column was equilibrated with any of the commercially available phosphate buffer (PBS7.4, Cat. No. 10010-023, Invitrogen), sodium phosphate buffer (10 mM, pH 6.0; referred to as PBS 6.0 in this specification) containing sodium chloride (137 mM), or acetic acid buffer (10 mM, pH 5.5; referred to as ABS in this specification) containing sorbitol (5%). The NAP-25 column was loaded with the antibody-drug conjugate reaction aqueous solution (about 1.5 mL), and eluted with the buffer in the amount specified by the manufacturer, thereby separating and obtaining the antibody fraction. The separated fraction was loaded again on the NAP-25 column, eluted with the buffer, and subjected to gel filtration purification operation, and the operation was repeated 2 to 3 times, thereby obtaining the antibody-drug conjugate from which the unconnected drug linker and low molecular weight compounds (tris (2-carboxyethyl) phosphine hydrochloride (TCEP), N-acetyl-L-cysteine ​​(NAC), dimethyl sulfoxide) were removed.

[0209] The purification method of Example 2 in the above Comparative Example 4 adopts SEC molecular exclusion chromatography, the column is a NAP-25 column, the filler requirements are: the loading concentration is less than 1 mg / mL, the loading capacity is only 1 mg / mL, if the amount of purification is increased, the purification effect is significantly reduced, it is not suitable for scale-up production, and the operation needs to be repeated 2-3 times. It can be seen that its operation method is complicated, the impurity removal efficiency is not high, and the micellar complex formed by the aggregation of toxin linkers has a large molecular weight (>10KDa), which cannot meet the NAP-25 column requirement for the molecular weight of impurities, so the separation effect is not good.

Claims

1. An anion column chromatography method for removing free small molecules in an antibody-drug conjugate, wherein the antibody-drug conjugate contains negatively charged free toxin linker molecules, wherein: The method comprises: purifying the antibody-drug conjugate by using an anion column; removing the negatively charged free toxin linker contained in the antibody-drug conjugate; the toxin linker structure in the antibody-drug conjugate has a group that can react with thiol as a linker that reacts with the antibody, The group capable of reacting with a thiol group is selected from a maleimide group; The toxin linker in the antibody-drug conjugate further comprises a cytotoxic drug, wherein the cytotoxic drug is an anti-tumor drug selected from camptothecins, maytansines, dolastatins or pyrrolobenzodiazepines; The camptothecin drug is selected from irinotecan, isitecan or derunotecan or 7-ethyl-10-hydroxycamptothecin, the maytansine drug is selected from maytansine thioderivatives, the dolastatin toxin is selected from auristatin derivatives, and the pyrrolobenzodiazepine drug is selected from PBD; the maytansine thioderivative is DM1 or DM4; the auristatin derivative is MMAE or MMAF; The method comprises preparing an antibody-drug conjugate and a buffer into a buffer mixed solution and passing the solution through an anion column for purification; the pH value of the buffer mixed solution is 5-7, and the buffer is selected from a histidine buffer, a phosphate buffer, a citrate buffer, a tris buffer or a glycine buffer; The anion filler in the anion column is selected from an anion filler with a quaternary ammonium salt structure or an anion filler with a diethylaminoethyl structure; The buffer containing the antibody-drug conjugate flows through the anion column at a rate of 0.1-10 CV / min; The method further comprises the step of treating the antibody-drug conjugate with a derivatizing agent before purifying the antibody-drug conjugate with an anion column, so that the free toxin linker molecules in the antibody-drug conjugate are negatively charged; the step of treating the antibody-drug conjugate with a derivatizing agent comprises: uniformly mixing the antibody-drug conjugate with a molar amount of 10-40 times that of the antibody-drug conjugate, and reacting the derivatizing agent with the free toxin linker contained in the antibody-drug conjugate for 0.5-24 hours; The derivatizing agent is selected from one of acetylcysteine, mercaptoethanesulfonate, mercaptophosphate, mercaptonitrate, mercaptocarboxylate and phosphorous carboxylate.

2. The method according to claim 1, wherein: Each 1L of anion column packing can process 0.1 to 6kg of antibody-drug conjugates.

3. The method according to claim 2, wherein: Each 1L of anion column packing can process 0.1 to 3kg of antibody-drug conjugates.

4. The method according to any one of claims 1 to 3, wherein: The preparation process of the antibody-drug conjugate includes: adding a reducing agent to the reaction system of the antibody molecule, reacting at 0-37°C, adding a toxin linker molecule after the reaction, and reacting again at 0-37°C, wherein the molar amount of the reducing agent is 1-16 times the molar amount of the antibody molecule; The reducing agent is a combination of one or more of dithiothreitol and tris(2-carboxyethyl)phosphine.

5. The method according to claim 4, wherein the toxin linker comprises a cytotoxic drug and a linker structure, and the toxin linker is LP1, LP2 or LP3; wherein, LP1 is MC-GGFG-Dxd, LP2 is MC-VC-PAB-DM1, and LP3 is MC-VC-PAB-SN38.

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