Purification method of antibody-drug conjugate based on anionic membrane and antibody-drug conjugate

Abstract

The present invention provides an anionic membrane-based purification method for antibody-drug conjugates and antibody-drug conjugates. The method comprises purifying the antibody-drug conjugates using an anionic membrane; the antibody-drug conjugates contain free toxin linkers. The purification method of the present invention has a good impurity removal effect, and is an antibody-drug conjugate flow-through mode with a high loading capacity (antibody-drug conjugate processing volume>10 kg / L), and the free toxin linkers after purification are below the detection limit.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to an anion membrane-based purification method for antibody-drug conjugates and the antibody-drug conjugates. Background Art

[0002] Antibody-drug conjugates (ADCs) are monoclonal antibodies (mAbs) covalently linked to cytotoxic drugs (toxin molecules) through ADC linkers. Because mAbs have good targeting capabilities, they can accurately identify and distinguish healthy cells from tumor cells, and bring highly active cytotoxic drugs (payloads) into cancer cells to kill them. As of the end of 2023, 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.

[0003] In order to increase the cell penetration of cytotoxic drugs and enhance the bystander effect, many ADCs choose to use small molecules with high hydrophobicity as cytotoxic drugs. 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 are generally highly toxic (IC50 nanomolar or picomolar level). Once the concentration of free toxins in ADC drugs is too high, serious adverse reactions will occur. At present, the most widely used cytotoxic drugs in clinical practice are camptothecins, such as irinotecan, exatecan, deruxtecan, etc. The small molecules of camptothecins are highly hydrophobic, especially after the linker is hydrophilic, the aggregation trend is more obvious, and it is easy to aggregate in aqueous solution to form nano-scale micelles. These toxin molecules or toxin linkers (LP) are often poorly removed by conventional ultrafiltration dialysis (UF / DF) methods, resulting in products that do not meet quality requirements.

[0004] To remove toxin linkers, the current widespread treatment method is to add organic solvents or inorganic salts to the UF / DF replacement fluid, which is not only not conducive to protein stability, but also cannot fundamentally solve the problem in most cases. Some data reported that adding column chromatography steps can effectively remove free toxin linkers, such as using cations (such as Figure 1As shown), hydrophobic column chromatography, but these methods are all combined with elution mode, the principle of which is to use non-covalent interaction to adsorb ADC molecules on the purification column, then use buffer to wash away impurities such as unbound small molecules, and finally replace the buffer to elute ADC. The process is complicated and the loading capacity is usually low; there are also reports on the use of molecular exclusion chromatography to purify toxin linkers, but it has serious problems such as low flow rate and difficulty in amplification, which will undoubtedly increase production costs and is difficult to promote and apply in actual production. Summary of the invention

[0005] The object of the present invention is to provide a method for purifying an antibody-drug conjugate in which conventional UF / DF purification cannot effectively remove free toxin linkers.

[0006] The principle of the present invention is: using a negatively charged derivatization reagent to carry out a derivatization reaction with a toxin linker to generate a negatively charged product, while under weakly acidic conditions, most antibody-drug conjugate molecules themselves are positively charged, and anionic membrane chromatography can be used to adsorb negatively charged or uncharged toxin linkers.

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

[0008] To achieve the above-mentioned purpose, on the one hand, the present invention provides a method for purifying antibody-drug conjugates based on anionic membranes, wherein the purification method comprises purifying the antibody-drug conjugates using anionic membranes to remove free toxin linkers contained in the antibody-drug conjugates, wherein the free toxin linkers include negatively charged or uncharged toxin linkers.

[0009] The toxin linker molecule described in the present invention refers to a compound (linker-payload, LP) formed by connecting a cytotoxic drug and a linker in the preparation of an antibody-drug conjugate, that is, a "cytotoxic drug-linker" molecule, hereinafter referred to as "toxin linker".

[0010] According to some specific embodiments of the present invention, at least 10 kg of antibody-drug conjugates are processed per 1 L of membrane.

[0011] According to some specific embodiments of the present invention, each 1 L of membrane can process 0.1 kg to 20 kg of antibody-drug conjugates.

[0012] According to some specific embodiments of the present invention, each 1 L of membrane can process 0.1 kg to 10 kg of antibody-drug conjugates.

[0013] According to some specific embodiments of the present invention, the method comprises passing a buffered mixed solution prepared by an antibody-drug conjugate and a buffer through an anionic membrane for purification; the pH value of the buffered mixed solution is 3-8.

[0014] According to some specific embodiments of the present invention, the pH value of the buffer mixed solution is 5-7.

[0015] According to some specific embodiments of the present invention, the pH value of the buffer mixed solution is 5.0, 5.5, 6.0, 6.5, or 7.0.

[0016] According to some specific embodiments of the present invention, the pH value of the buffer mixed solution is 5.5-6.5.

[0017] 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.

[0018] According to some specific embodiments of the present invention, the buffer is selected from histidine buffer, phosphate buffer and citrate buffer.

[0019] According to some specific embodiments of the present invention, the buffer is selected from histidine buffer.

[0020] According to some specific embodiments of the present invention, the molar concentration of the buffer is 10-50 mM.

[0021] According to some specific embodiments of the present invention, the molar concentration of the buffer is 10-30 mM.

[0022] According to some specific embodiments of the present invention, the concentration of the antibody-drug conjugate in the buffered mixed solution is 0.5 mg / mL to 50 mg / mL.

[0023] The present invention utilizes anionic membrane to adsorb negatively charged substances to achieve separation of toxin linkers (such as Figure 2 As shown in the figure, for example, under weakly acidic conditions, free toxin linkers or other micelle byproducts are negatively charged and can be adsorbed by positively charged anionic membranes; while the isoelectric point of antibodies / antibody-drug conjugates is generally above 8.0, and under this pH condition, they are positively charged and cannot be adsorbed by the membrane, so they can flow through directly to complete purification. Therefore, existing anion exchange membranes can generally achieve the effects of the present invention.

[0024] According to some specific embodiments of the present invention, the anion membrane is a polyethersulfone anion membrane, a polyacrylamide anion membrane or a regenerated cellulose (RC) anion membrane.

[0025] That is to say, the base frame of the anion membrane is made of polyethersulfone material, polyacrylamide material or regenerated cellulose material.

[0026] According to some specific embodiments of the present invention, the base frame of the anion membrane is made of polyethersulfone or polyacrylamide, which can further adsorb toxin linkers (such as Figure 2 as shown).

[0027] According to some specific embodiments of the present invention, the base frame of the anion membrane is made of polyethersulfone, which has the property of non-specific adsorption of lipophilic substances and can further adsorb toxin linkers (such as Figure 2 as shown).

[0028] According to some specific embodiments of the present invention, the anion membrane may be an anion membrane produced by the following companies: Cobite, Sartorius, Pall, or Merck Millipore.

[0029] For example, it can be: Purcise Q from Cobetter, Sartobind Q from Sartorius, Mustang Q XT from Pall, or Natrix Q from Merck Millipore.

[0030] The anionic membrane of the present invention can achieve the purpose of removing free charged and uncharged toxin linker impurities, and is expected to simplify the process route and reduce production costs.

[0031] According to some specific embodiments of the present invention, the speed at which the buffer solution containing the antibody-drug conjugate flows through the anionic membrane is 0.1-100 MV / min (MV: Membrane Volume).

[0032] According to some specific embodiments of the present invention, the speed at which the buffer solution containing the antibody-drug conjugate flows through the anionic membrane is 5-50 MV / min.

[0033] According to some specific embodiments of the present invention, the flow rate of the buffer solution containing the antibody-drug conjugate through the anionic membrane is 5-25 MV / min.

[0034] According to some specific embodiments of the present invention, the flow rate of the buffer solution containing the antibody-drug conjugate through the anionic membrane is 10-25 MV / min.

[0035] According to some specific embodiments of the present invention, the buffer is selected from histidine buffer, the pH value of the buffer mixture is 5-7, the speed at which the buffer containing the antibody-drug conjugate flows through the anion membrane is 10-25 MV / min, and the frame of the anion membrane is made of polyethersulfone.

[0036] The present invention utilizes the charge of the toxin linker, or the characteristics of the toxin linker being charged by derivatization, to achieve adsorption of the toxin linker by the anionic membrane, while the isoelectric point of antibodies and antibody-drug conjugates is generally above 8.0, and they are positively charged under this pH condition and cannot be adsorbed by the membrane, so they can flow through directly to complete purification. Therefore, the present invention has no special requirements for the antibody-drug conjugate itself;

[0037] Generally speaking, if the toxin linker molecules in the obtained antibody-drug conjugates are negatively charged, they can be directly used in the purification method of the present invention. For example, when the toxin linker structure contains one or more of sulfonic acid, carboxylic acid, nitric acid and phosphate groups, the toxin linker can be made negatively charged (such as nucleic acid drugs, etc.), and can be directly used in the purification method of the present invention; alternatively, the toxin linker molecules can also be made negatively charged during the preparation of the antibody-drug conjugates. In both cases, the antibody-drug conjugates can be purified using the purification method of the present invention;

[0038] Alternatively, the free toxin linker can be charged by further derivatization:

[0039] That is, before the antibody-drug conjugate is purified by an anionic membrane, the purification method further includes a step of derivatizing the antibody-drug conjugate with a derivatizing agent so that the free toxin linker in the antibody-drug conjugate is negatively charged (e.g. Figure 3 shown);

[0040] The derivatizing agent is a compound that can react with the toxin linker molecule and make the product after the reaction negatively charged.

[0041] The present invention can make the uncharged free toxin linker negatively charged through further derivatization reaction. The above-mentioned derivatization reaction can also be used to treat the antibody-drug conjugate whose free toxin linker is already negatively charged or part of the free toxin linker is negatively charged.

[0042] 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.

[0043] According to some specific embodiments of the present invention, the derivatizing agent is selected from one or more combinations of N-acetylcysteine ​​(NAC), 2-mercaptoethanesulfonate, thioglycolate and tris(2-carboxyethyl)phosphine (TCEP).

[0044] 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 (so that the free toxin linker in the antibody-drug conjugate reacts with the derivatizing agent).

[0045] 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 2-60 times that of the antibody-drug conjugate in a buffer.

[0046] According to some specific embodiments of the present invention, the step of treating the antibody-drug conjugate with a derivatizing agent comprises: mixing the derivatizing agent in a molar amount 4-60 times that of the antibody-drug conjugate and the antibody-drug conjugate evenly in a buffer.

[0047] 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 5-40 times that of the antibody-drug conjugate in a buffer.

[0048] According to some specific embodiments of the present invention, the step of treating the antibody-drug conjugate with a derivatizing agent comprises: mixing the derivatizing agent in a molar amount 8-30 times that of the antibody-drug conjugate and the antibody-drug conjugate evenly in a buffer.

[0049] 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 10-20 times that of the antibody-drug conjugate in a buffer.

[0050] 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 10-15 times that of the antibody-drug conjugate in a buffer.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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 0.5-2h.

[0056] 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 1-2 hours.

[0057] 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 1-1.5 h.

[0058] According to some specific embodiments of the present invention, after the antibody-drug conjugate is treated with a derivatizing agent, an ultrafiltration step is further included, and then a purification treatment is directly performed using an anionic membrane.

[0059] 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:

[0060] 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 sufficient amount of toxin linker is added, and the reaction is carried out again at 0-37°C.

[0061] 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:

[0062] A reducing agent is added to the reaction system of the antibody molecule, and the reaction is carried out at 4-37°C. After the reaction is completed, a sufficient amount of toxin linker is added, and the reaction is carried out again at 15-30°C.

[0063] According to some specific embodiments of the present invention, the reaction is carried out at 15-30°C after the reducing agent is added.

[0064] According to some specific embodiments of the present invention, the reaction is carried out at 20-30°C after the reducing agent is added.

[0065] According to some specific embodiments of the present invention, after the toxin linker is added, the reaction is carried out at 15-30°C.

[0066] According to some specific embodiments of the present invention, the reaction is carried out at 20-30°C after the toxin linker is added.

[0067] 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: adding a reducing agent to the reaction system of the antibody molecule, reacting at 0-37°C, adding a sufficient amount of toxin linker after the reaction, and then reacting at 0-37°C.

[0068] According to some specific embodiments of the present invention, the toxin linker is first dissolved in an organic solvent and then added to the reaction system; the organic solvent is selected from DMSO, DMA or DMF.

[0069] According to some specific embodiments of the present invention, the reaction solvent in the reaction system is a buffer solution.

[0070] According to some specific embodiments of the present invention, the amount of the buffer is such that after the antibody is added to the buffer, the concentration of the antibody in the reaction system is 5 mg / mL to 20 mg / mL.

[0071] 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 in antibodies.

[0072] According to some specific embodiments of the present invention, the reducing agent is selected from one or a combination of dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP).

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] According to some specific embodiments of the present invention, the reaction time after adding the reducing agent and the reaction time after adding the toxin linker are each independently 1-24 hours.

[0078] According to some specific embodiments of the present invention, the reaction time after adding the reducing agent and the reaction time after adding the toxin linker are each independently 1-3 hours.

[0079] According to some specific embodiments of the present invention, the preparation process of the antibody-drug conjugate includes:

[0080] Take an appropriate amount of monoclonal antibody, dilute it to a concentration of 5-20 mg / mL with buffer, add a metal chelator and mix thoroughly to make the final concentration in the range of 1-15 mM, add a reducing agent of 1-16 times the equivalent of the antibody, react at 0-37°C for 1-24 h, add 2-20 times the equivalent of toxin linker, respectively, and react at 0-37°C for 1-24 h.

[0081] According to some specific embodiments of the present invention, the preparation process of the antibody-drug conjugate includes the step of adding a metal chelator after adding the antibody.

[0082] According to some specific embodiments of the present invention, the metal chelator is selected from EDTA, DTPA, IDHA, EDDHA or HBED.

[0083] According to some specific embodiments of the present invention, the final concentration of the metal chelator is 1-15 mM.

[0084] According to some specific embodiments of the present invention, the preparation process of the antibody-drug conjugate further includes: after the toxin linker reaction is completed, ultrafiltration treatment is performed to obtain an ultrafiltration reaction liquid, and then the ultrafiltration reaction liquid is directly purified by anion membrane filtration.

[0085] 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.

[0086] According to some specific embodiments of the present invention, the preparation process of the antibody-drug conjugate includes:

[0087] Take an appropriate amount of monoclonal antibody, dilute it to a concentration of 5-20 mg / mL with a buffer, add a metal chelator and mix thoroughly to make the final concentration in the range of 1-15 mM, add a reducing agent 2-10 times the equivalent of the antibody, place it in a water bath or ice bath at 0-37°C for 1-3 h, add 2-20 times the equivalent of the toxin linker (dissolved in an appropriate amount of DMSO, DMA or DMF), place it in a water bath or ice bath at 15-30°C for 1-3 h.

[0088] According to some specific embodiments of the present invention, the ultrafiltration treatment of the present invention comprises using an ultrafiltration system to perform a fluid exchange of 4-16 times diafiltration volume (DV).

[0089] According to some specific embodiments of the present invention, the ultrafiltration treatment of the present invention comprises that the molecular weight cutoff of the ultrafiltration membrane package is 3kd-50kd.

[0090] According to some specific embodiments of the present invention, the ultrafiltration treatment of the present invention comprises that the molecular weight cutoff of the ultrafiltration membrane package is 20kd-30kd.

[0091] According to some specific embodiments of the present invention, the buffer used for ultrafiltration can be a conventional buffer, such as histidine-hydrochloric acid buffer (pH=6.0).

[0092] According to some specific embodiments of the present invention, the toxin linker molecule of the present invention has a group that can react with a sulfhydryl group to serve as a linker that reacts with an antibody.

[0093] According to some specific embodiments of the present invention, the toxin linker molecule of the present invention has a maleimide group, an α, β-unsaturated carbonyl group or a halogenated acetyl group, a pyridine dithiol group, a halogenated alkyl group, a vinyl sulfone or other groups that can react with a thiol group.

[0094] According to some specific embodiments of the present invention, the α, β-unsaturated carbonyl group is an acrylate group.

[0095] According to some specific embodiments of the present invention, the toxin linker in the antibody-drug conjugate structure further comprises a cytotoxic drug, and the cytotoxic drug is a camptothecin, maytansine, dolastatin or pyrrolobenzodiazepine drug.

[0096] The antibody described in the present invention can be any antibody used to prepare antibody-drug conjugates. According to some specific embodiments of the present invention, the antibody is an antibody containing a thiol group.

[0097] According to some specific embodiments of the present invention, the camptothecin drug is selected from irinotecan or exatecan or derunotecan (Dxd) or 7-ethyl-10-hydroxycamptothecin (SN38); the maytansine drug is selected from maytansine thioderivatives such as DM1 or DM4; the dolastatin toxin is selected from auristatin derivatives such as MMAE or MMAF; the pyrrolobenzodiazepine drug is selected from PBD and its derivatives.

[0098] According to some specific embodiments of the present invention, the toxin linker is LP1 (MC-VC-PAB-SN38) or LP2 (MC-GGFG-Dxd).

[0099] The linker of LP1 is maleimide (MC).

[0100] The VC-PAB of LP1 is Val-Cit-PAB, valine-citrulline-p-aminobenzyloxycarbonyl; the cytotoxic drug of LP1 is the camptothecin derivative 7-ethyl-10-hydroxycamptothecin (ie SN38).

[0101] The linker of LP2 is maleimide (MC).

[0102] The GGFG of LP2 is a tetrapeptide: glycine-glycine-phenylalanine-glycine; the cytotoxic drug of LP2 is deludecanoic acid (Dxd).

[0103] According to some specific embodiments of the present invention, the structures of LP1 and LP2 are respectively as follows:

[0104]

[0105] According to some specific embodiments of the present invention, the antibody is a monoclonal antibody.

[0106] According to some specific embodiments of the present invention, the antibody is trastuzumab, pertuzumab or bevacizumab.

[0107] According to some specific embodiments of the present invention, the antibody is trastuzumab.

[0108] On the other hand, the present invention also provides an antibody-drug conjugate prepared by any purification method of the present invention, wherein the content of free toxin linkers in the antibody-drug conjugate is less than or equal to 50 μg / mL.

[0109] 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.

[0110] 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.

[0111] In summary, the present invention provides a method for purifying antibody-drug conjugates based on anionic membranes. The purification method of the present invention has the following advantages:

[0112] 1. Impurity removal effect: Existing technologies: (1) Ultrafiltration dialysis cannot achieve the impurity removal effect; (2) Cationic and hydrophobic chromatography are combined elution modes with low loading capacity (antibody-drug conjugate processing volume 40-60 g / L); (3) Molecular exclusion chromatography has low impurity removal efficiency and requires multiple operations; (3) Organic reagent method has poor impurity removal effect and destroys product stability. The technology of the present invention: using derivatization + anion membrane chromatography, the impurity removal effect is good, and it is an antibody-drug conjugate flow-through mode with high loading capacity (antibody-drug conjugate processing volume>10 kg / L), and the free LP after purification is below the detection limit.

[0113] 2. Cost reasons: Existing technologies: (1) Cationic, hydrophobic, and molecular exclusion chromatography: require purification chromatography equipment, which is costly, has low loading capacity, and requires a large amount of fillers; (2) Organic reagents: require a large amount of organic reagents, pollute the environment, and are time-consuming and labor-intensive. The technology of the present invention: does not require complex equipment, has a high loading capacity, is conducive to reducing costs, saves more space, and is friendly to large-scale production.

[0114] 3. Efficiency reasons: Existing technologies: (1) Cationic / hydrophobic: the elution mode has long steps and takes a long time, the structural stability of the filler needs to be considered, and the flow rate is low; (2) Molecular exclusion: the operation is complicated and multiple operations are time-consuming; (3) Organic reagents: time-consuming and polluting the environment. The technology of the present invention: the elution mode has short steps and time, is easy to operate, has no unstable components, and can use high flow rates to remove impurities. BRIEF DESCRIPTION OF THE DRAWINGS

[0115] Figure 1 Schematic diagram of existing cationic filler purification technology.

[0116] Figure 2 Schematic diagram of anionic membrane treatment of ADC ultrafiltrate.

[0117] Figure 3 Schematic diagram of ADC coupling and post-processing of the present invention.

[0118] Figure 4 HPLC detection diagram (LP1) before and after adsorption of Example 1, peak 1 is charged NAC-LP1, and peak 2 is uncharged LP1.

[0119] Figure 5 HPLC detection diagram (LP2) before and after adsorption of Example 1, peak 1' is charged NAC-LP2, and peak 2' is uncharged LP2.

[0120] Figure 6 This is the UV absorption standard curve of different concentrations of the toxin linker in Example 1 (LP1).

[0121] Figure 7 This is the HPLC detection diagram before and after adsorption of different anion membranes in Example 2 (LP1).

[0122] Figure 8 This is the HPLC detection chart before adsorption of Example 3 (LP1+mAb1 / 2).

[0123] Fig. 9 This is the HPLC detection chart after adsorption of Example 3 (LP1+mAb1 / 2).

[0124] Fig.10 This is a HPLC detection chart of ADC purity after purification at different flow rates in Example 4.

[0125] Fig.11 This is the HPLC detection chart of the toxin linker content before anion membrane treatment in Example 4.

[0126] Fig.12 This is the HPLC detection chart of the toxin linker content at different flow rates in Example 4.

[0127] Fig.13 This is an HPLC detection diagram of the effect of the buffer components of Example 5 on the adsorption of anionic membranes.

[0128] Fig.14 This is the HPLC detection diagram before and after anion membrane adsorption in Example 6 (LP1). DETAILED DESCRIPTION

[0129] The following describes in detail the implementation process of the present invention and the beneficial effects produced by the specific embodiments, which is intended to help readers better understand the essence and characteristics of the present invention, and is not intended to limit the scope of implementation of the present invention.

[0130] Example 1

[0131] Antibody reduction: Take 4g of trastuzumab antibody (mAb1), calculate the amount of 20 mM phosphate buffer (PB buffer), pH 7.5 and 5 mM EDTA according to the concentration and dosage in parameter table 1, and add them to the reaction system in sequence. Use 1 M NaOH solution to adjust the pH of the reaction system to 7.5. Then add 20 mM TCEP and add it to the reaction system. Adjust the water bath to the required temperature for temperature control, and then perform the reduction reaction according to the required conditions.

[0132] Table 1. Reduction reaction parameters

[0133]

[0134] Coupling reaction: After the reduction is completed, the reduction solution is divided into two parts, and the toxin linkers LP1 and LP2 (dissolved in DMSO, DMSO final concentration 10 v / v%) are added to the two reaction systems respectively. The structures are as follows, and the addition amount is calculated according to Table 2. Adjust the water bath to the required temperature for temperature control, and then carry out the coupling reaction according to the required conditions.

[0135] Table 2. Coupling reaction parameters

[0136]

[0137] Derivatization reaction: The derivatization reagent NAC was dissolved in the reaction buffer to prepare a 20 mM derivatization master solution, and 10 times the equivalent of the derivatization reagent was added to the two coupling reactions respectively, and the reaction was stirred at 25°C for 1 h. The specific parameters are shown in Table 3.

[0138] Table 3. Derivatization reaction and dialysis parameters

[0139]

[0140] ADC ultrafiltration dialysis (UF / DF): After derivatization, the two ADC reaction solutions were ultrafiltration dialyzed using membrane packages (30kd) respectively, with the dialysis buffer being 20 mM His / His-HCl, pH 6.0, and the dialysis multiple being 10 DV (diafiltration volume). A small portion (1 mL) was taken as a control; the other portion was purified by anionic membrane chromatography as follows.

[0141] ADC anion membrane chromatography: 0.2 mL of Cobite Purcise TM The two ultrafiltration reaction solutions were purified by Q chromatography membrane. The buffer was 20 mM His / His·HCl, pH 6.0 to balance the anion membrane. The sample was loaded at a flow rate of 2 ml / min (i.e., 10 MV / min). 200 mL of the flow-through was collected (steps as Figure 3 as shown).

[0142] HPLC was used to test the content of toxin linkers, wherein the chromatographic column was Symmetry C18 Column, 100Å, 5 µm, 4.6 mm X 150 mm; the detection wavelength was 370 nm; the mobile phase A was deionized water (containing 0.1% trifluoroacetic acid TFA), the 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 point: 15 min, 0% A, 100% B. Figure 4 This is the HPLC detection diagram of No. 1 (LP1) before and after anion membrane adsorption, peak 1 shows the charged toxin linker NAC-LP1, and peak 2 is the uncharged LP1; Figure 5 This is the HPLC detection diagram of No. 2 (LP2) before and after anion membrane adsorption, peak 1 shows the charged toxin linker NAC-LP2, and peak 2 is the uncharged LP2; Figure 6 This is the UV absorption standard curve of different concentrations of LP1.

[0143] The results before and after purification using anionic membrane are summarized in Table 4:

[0144] Table 4. Comparison of free toxin content before and after anion membrane treatment

[0145]

[0146] The results are shown in Table 4. After derivatization, some toxin linkers of LP1 and LP2 still cannot be derivatized, and the derivatization efficiency is about 90%. Moreover, these toxin linkers cannot be completely removed by ultrafiltration. The final free drug residues are: charged toxin linker NAC-LP1: 21.3 ug / mL, toxin linker LP1: 2.1 ug / mL; charged toxin linker NAC-LP2: 4.1 ug / mL, toxin linker LP1: 0.4 ug / mL. After anion membrane treatment, the final free drug residues were all below the detection limit of 0.1 ug / mL. The results show that anion membrane chromatography has a significant effect on the removal of negatively charged and uncharged residual free toxin linkers.

[0147] Loading capacity calculation: calculated based on the mass of LP1 small molecules with a higher content before treatment: the adsorption capacity of the anion membrane for negatively charged toxin linkers is ≥ (21.3-0.1) ug / mL * 200 mL / 0.2 mL = 21.2 mg / mL, and the adsorption capacity for uncharged toxin linkers is ≥ (2.1-0.1) ug / mL * 200 mL / 0.2 mL = 2 mg / mL; calculated based on the ADC treatment mass: the processing capacity of the anion membrane is not less than 10,000 g / L, which is much higher than the current processing capacity of the cationic filler of 40-60 g / L. Similarly, based on the small molecular mass of LP2 toxin, the adsorption capacity of the anion membrane for negatively charged toxin linkers is ≥(4.1-0.1) ug / mL * 200 mL / 0.2 mL=4 mg / mL, and the adsorption capacity for uncharged toxin linkers is ≥(0.4-0.1) ug / mL *200 mL / 0.2 mL =0.3 mg / mL; calculated according to the ADC processing mass, the processing capacity of the anion membrane for ADC samples is not less than 10,000 g / L.

[0148] Example 2

[0149] Using 5 g of trastuzumab as the starting material, reduction, coupling, and derivatization were completed according to the parameters of Tables 1 to 3 in Example 1, and a portion of the derivatized solution was ultrafiltered. The ultrafiltrate was divided into 4 parts, and the ultrafiltrate was treated and purified using Q membranes from different manufacturers according to Example 1, wherein No. 1 was a sample not treated with a Q membrane, and No. 2-4 was a sample treated with a Q membrane from different manufacturers. The membrane materials No. 2-4 used the same charged ligand, but the base was different. After the treatment, HPLC and Nanodrop were used to test the free toxin linker (LP) content (charged LP and uncharged LP) and ADC product content.

[0150] Table 5. Comparison of content before and after treatment with different anion membranes

[0151]

[0152] It can be seen from Table 5 that the three anion exchange membranes have no obvious adsorption of ADC molecules and will not cause product loss; the three membranes can complete the efficient adsorption of charged LP1, and the charged LP1 concentration after adsorption is lower than the minimum detection limit of 0.1 ug / mL; PES and PAM also have good adsorption effects on uncharged LP1, and the LP1 content after adsorption is lower than the detection limit; after treatment with RC (NO.4) membrane, the uncharged LP1 concentration is 1.6 ug / ml, which is still slightly lower than the ultrafiltrate 2.4ug / ml in No. 1. It can be seen that the adsorption effect of RC on uncharged LP1 is slightly inferior to that of PES and PAM, but the adsorption effect on charged LP1 is comparable to that of PES and PAM membranes. Due to the high degree of reaction to LP molecules during the derivatization process, the proportion of charged LP1 in the overall LP1 is much greater than that of uncharged LP1, so the overall LP1 content after RC membrane purification can still meet the quality requirements. Figure 7 The changes of LP1 before and after adsorption using PES (NO.2), PAM (NO.3) and RC (NO.4) membranes were shown. The charged free toxin small molecule NAC-LP1 completely disappeared at 9.0-9.5 min peak in the three membrane materials, while the uncharged free toxin small molecule LP1 still existed at 10.5-11.0 min peak in the RC membrane.

[0153] Example 3

[0154] As shown in Table 6: Trastuzumab mAb1 and Pertuzumab mAb2 were used to perform reduction, coupling and derivatization experiments with toxin linker LP1 according to the method of Example 1. After the reaction was completed, a 30 Kd membrane package was used for 10 DV ultrafiltration, and the reaction was divided into two parts using Cobet Purcise TMThe two groups treated with Q membrane (refer to Example 1, No. 1 and 2) and not treated with Q membrane (No. 3 and 4) were tested for the total content of free toxin linkers and ADC content using HPLC and Nanodrop after completion. The detection method was the same as in Example 1.

[0155] Table 6. Summary of different antibody response information

[0156]

[0157] From the results in Table 6, it can be seen that after coupling with the same LP1 small molecule using different antibodies and undergoing the same ultrafiltration operation, the residues of the small molecules are basically the same (No. 3 VS No. 4); after being treated with the Q membrane, the concentrations of the free small molecules are significantly reduced, and the detection values ​​are lower than the minimum standard detection limit. Figure 8 and Fig. 9 The HPLC test chart of LP1 content before and after treatment with anion exchange membrane after coupling with different antibodies.

[0158] Therefore, for different combinations of antibodies (mAb1 and mAb2, connected to LP1) and toxin linkers (LP1 and LP2, connected to mAb1, Example 1), the anion exchange membrane can remove free toxin linkers (LP + NAC-LP) without affecting the ADC product. Therefore, this method can be extended to different combinations of antibodies and toxin linkers.

[0159] Example 4

[0160] Using a peristaltic pump and Cobetter Purcise TM The ultrafiltrate (obtained in Example 2) was filtered through an anionic membrane (volume 0.2 mL). Fig.10 The SEC-HPLC test chart of ADC at flow rates of 5 MV / min and 50 MV / min. The detection wavelength of SEC-HPLC is 280 nm; the mobile phase is 50 mM PB + 100 mM NaCl, pH 7.0, and the isocratic elution is 20 min. Fig.11 This is the HPLC detection chart of toxin linker content before anion membrane treatment. Fig.12 HPLC detection chart after filtration with flow rates from top to bottom: 5 MV / min, 10 MV / min, 25 MV / min and 50 MV / min (i.e., 1, 2, 5, 10 ml / min).

[0161] The HPLC test results show that: within the flow rate range of 5-50 MV / min, after the ADC molecules are treated with anion exchange membrane at different flow rates, there is no significant difference in the purity of the ADC molecules at different flow rates, all >95%, meeting the quality requirements; and within this flow rate range, the anion exchange membrane can efficiently adsorb charged and uncharged LP1, and the LP1 content after adsorption is lower than the minimum detection limit of 0.1 ug / mL, and its purification flow rate is not less than 50 MV / min, which is much higher than the flow rate of anion fillers on the market (Comparative Example 1), which can fully meet the requirements of actual production and improve production efficiency. Therefore, under different flow rate conditions, the anion membrane can efficiently complete the adsorption of the toxin linker LP1 without destroying the ADC molecules.

[0162] Since too high a flow rate will cause higher pressure, especially for liquids with high concentration and high viscosity, the manufacturer has limited the range of anion membrane flow rate to 0.2 mL Purcise TM For example, the recommended flow rate is 5-25MV / min. After comprehensive consideration, the purification flow rate is determined to be ≤25 MV / min.

[0163] Example 5

[0164] The reaction solution in Example 2 (derivation completed) was centrifuged and ultrafiltered with different buffers to replace the solution. After completion, 0.2 mL Purcise TM The samples were filtered through an anionic membrane at a flow rate of 25 MV / min. After the filtration, the contents of NAC-LP1 and LP1 were detected by HPLC. The specific test data are shown in Table 7, where No. 1 is the reaction solution that was only derivatized but not ultrafiltered or treated with anionic membrane. The other samples were ultrafiltered and treated with anionic membrane using buffer solutions of different components, pH values ​​and concentrations.

[0165] According to the HPLC test results, the use of 10-40 mM histidine in the pH range of 5.0-7.0 can effectively remove charged and uncharged toxin linker small molecules, and the small molecule content after treatment is lower than the minimum detection limit of 0.1 ug / mL. The use of phosphate and citrate buffers can also completely adsorb uncharged small molecules (LP1); but the adsorption capacity for charged small molecules (NAC-LP1) is limited, and the effect is not as ideal as that of histidine buffer, which may be due to the competition between phosphate and carboxylate with charged small molecules, reducing the adsorption effect. However, even so, after anion membrane purification treatment using phosphate and citrate buffers as buffers, the content of charged small molecules can still be significantly reduced, and the effect of the present invention is generally achieved. Fig.13The HPLC detection charts are before ultrafiltration and untreated (No. 1), ultrafiltration using histidine (No. 4), phosphate (No. 7) and citrate (No. 8) buffers and after passing through the membrane.

[0166] Table 7 Information of different samples

[0167]

[0168] Example 6

[0169] The experiment was carried out according to the reaction steps of Example 1. 100 mg of trastuzumab mAb1 was taken, and 8 times the equivalent of the monoclonal antibody was added to the reducing agent TCEP, and the reaction was carried out at 25°C for 2 h. After completion, the LP1 toxin linker was added, and the reaction was continued at 25°C for 2 h. Finally, 20 equivalents of the derivatization reagent TCEP were added to react for 1 h.

[0170] After the reaction is completed, ultrafiltration is first performed to replace the liquid, then an anion membrane is used for treatment, and finally HPLC is used to detect the small molecule content before and after the anion membrane treatment. Fig.14 It can be seen that the concentration of TCEP-LP1 before anion membrane treatment was 14 ug / mL, and the final residual amount of free drug TCEP-LP1 after membrane treatment was lower than the detection limit of 0.1 ug / mL.

[0171] Comparative Example 1

[0172] See also Figure 1 Schematic diagram of existing cationic filler purification technology. Its principle is to use non-covalent action to adsorb ADC molecules on the purification column, then use buffer to wash away impurities such as small molecules, and finally replace the buffer to elute ADC. The process is complicated and the load is usually low. Since too high a flow rate will destroy the spatial structure of the chromatography filler, there are generally restrictions on the flow rate. According to the information of Sitofan ion filler (as shown in Table 8 below), taking a 0.24 mL cationic filler column as an example, its maximum flow rate does not exceed 1 mL / min, which is much lower than the flow rate of Q membrane chromatography. Therefore, the anion membrane can use a higher flow rate for sample processing, which is beneficial to improve production efficiency.

[0173] Table 8

[0174]

[0175] Moreover, compared with column chromatography which requires expensive hardware equipment such as chromatography systems, purification columns, computers, and complex cleaning validation, anion exchange membranes only require peristaltic pumps and silicone tubes to complete subsequent purification processing, which is low-cost and easy to operate.

[0176] Comparative Example 2

[0177] 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):

[0178]

[0179] 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.

[0180] 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 then 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 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.

[0181] The above-mentioned method for removing free toxin linkers is to add DMSO organic reagent method, which introduces organic solvents in the purification process. The organic solvents are easy to affect the stability of ADC molecules and cause environmental pollution, and it takes a lot of time (total ultrafiltration volume 10+16=26 DV, >10 hours). 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 <10 μg / mL (ADC concentration 10 mg / mL), which is much lower than the patented method's <0.1 μg / mL (same ADC concentration).

[0182] Comparative Example 3

[0183] Purify the ADC drug according to CN105829346B "Anti-HER2 Antibody-Drug Conjugate" (see Example 2 and Common Operation D in the specification):

[0184] Common Procedure D: Purification of Antibody-Drug Conjugates

[0185] 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 PBS6.0 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-acetylcysteine ​​(NAC), dimethyl sulfoxide) were removed.

[0186] The above purification method uses SEC molecular exclusion chromatography to purify HER2 antibody-drug conjugates, and the column is NAP-25 column. If the purification amount is increased, the purification effect is significantly reduced, which is not suitable for scale-up production, and it needs to be repeated 2-3 times. It can be seen that the operation method is complicated and the impurity removal efficiency is not high. In addition, the micellar complex formed by the aggregation of toxin linkers has a large molecular weight (>10 KDa) and cannot meet the requirements of the NAP-25 column for the molecular weight of impurities, so the separation effect is not good.

Claims

1. A method for purifying an antibody-drug conjugate based on anionic membrane, wherein: The method comprises the steps of purifying the antibody-drug conjugate by using an anionic membrane to remove free toxin linkers contained in the antibody-drug conjugate, wherein the free toxin linkers include negatively charged or uncharged toxin linkers; the toxin linkers in the antibody-drug conjugate have a group that can react with a thiol group as a linker that reacts with the antibody, and the linker in the antibody-drug conjugate is a maleimide group; the toxin linker is LP1 or LP2; LP1 is MC-VC-PAB-SN38, and LP2 is MC-GGFG-Dxd; The method comprises passing a buffered mixed solution prepared by an antibody-drug conjugate and a buffer through an anionic membrane for purification; The pH value of the buffer mixed solution is 5-7, and the buffer is a histidine buffer; The anion membrane is a polyethersulfone anion membrane or a polyacrylamide anion membrane; The buffered mixed solution containing the antibody-drug conjugate flows through the anionic membrane at a rate of 5-50 MV / min; The purification method further comprises the step of treating the antibody-drug conjugate with a derivatizing agent before purifying the antibody-drug conjugate with an anionic membrane, so that the free toxin linker in the antibody-drug conjugate is negatively charged; The derivatizing agent is selected from one of N-acetylcysteine, 2-mercaptoethanesulfonate, thioglycolate and tris(2-carboxyethyl)phosphine; After the antibody-drug conjugate is treated with a derivatizing agent, an ultrafiltration step is also included, and then a purification treatment is directly performed using an anion membrane; The anionic membrane can process 0.1-20 kg of antibody-drug conjugate per 1 L of membrane; The step of treating the antibody-drug conjugate with a derivatizing agent comprises: uniformly mixing the derivatizing agent with a molar amount of 5-40 times that of the antibody-drug conjugate with the antibody-drug conjugate; The preparation process of the antibody-drug conjugate comprises: adding a reducing agent to the reaction system of the antibody molecule, reacting at 0-37° C., adding a sufficient amount of toxin linker to the reaction system after the reaction is completed, and then reacting 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 selected from one or a combination of tris(2-carboxyethyl)phosphine and dithiothreitol; The free toxin linker content in the antibody-drug conjugate is less than or equal to 0.1 μg / mL.

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