Method for preparing antibody-drug conjugate by using cyclodextrin compound

Abstract

Provided herein is a method for preparing an antibody-drug conjugate comprising: (a) treating an antibody with a reducing agent in the presence of transition metal ions; and (b) coupling a linker-drug intermediate to the reduced antibody obtained in step (a) in the presence of a macrocyclic compound.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a method for preparing an antibody-drug conjugate by using a macrocyclic compound. Background Art

[0002] Antibody-drug conjugates (ADCs) are obtained by connecting cytotoxic drugs to antibodies that bind to antigens expressed on the surface of target cells and can be internalized into cells, and include antibodies, linkers, and cytotoxic drugs. Among them, the antibody part accurately delivers the antibody-drug conjugate to the surface of target cells, and the linker part is responsible for releasing cytotoxic drugs in or on the surface of target cells, and maintaining stability in the blood circulation system, thereby promoting the accumulation of released cytotoxic drugs in target cells and killing target cells. Because of its excellent clinical characteristics in multiple indications, ADC has become a key treatment method in the field of oncology, and the market size is expected to continue to grow in the future.

[0003] In existing methods for preparing antibody-drug conjugates, a large amount of organic solvents (up to 20 vol%) are usually required to better dissolve the linker-drug in the reaction system. The introduction of a large amount of organic solvents will make subsequent purification difficult, for example: dissolving the semipermeable membrane, making the adsorption of proteins on the surface of the filler difficult, etc. In production, the reaction solution can be diluted to return the organic solvent level to the range that the purification equipment can tolerate, but this means that the amount of liquid that the purification equipment needs to handle will increase significantly, and the corresponding amount of waste liquid will increase by the same amount to meet the purification effect. In addition, when using some linkers-drugs for conjugation, a high content of organic solvents will usually result in a higher proportion of D6 in the resulting mixture, which has a greater impact on product quality.

[0004] Therefore, it is hoped to provide an improved method for preparing antibody-drug conjugates, which can reduce the amount of organic solvent used to improve production efficiency and improve the homogeneity of the antibody-drug conjugates required for production. Summary of the invention

[0005] Through long-term exploration and practice, the inventors have proposed a method for preparing antibody-drug conjugates, which can significantly reduce the amount of organic solvents used in the production process, improve the homogeneity of the antibody-drug conjugates required for production, and make the production process economical, efficient and environmentally friendly.

[0006] In one aspect, a method for preparing an antibody-drug conjugate is provided, comprising:

[0007] (a) treating the antibody with a reducing agent in the presence of a transition metal ion; and

[0008] (b) coupling the linker-drug intermediate to the reduced antibody obtained in step (a) in the presence of a macrocyclic compound.

[0009] In another aspect, provided herein is the use of a macrocycle for improving the homogeneity of a produced antibody-drug conjugate.

[0010] In another aspect, the present invention provides a use of a macrocyclic compound for improving production efficiency by reducing the amount of organic solvent used in the production process of an antibody-drug conjugate. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The present invention will be further described below in conjunction with the accompanying drawings, wherein these drawings are only for illustrating the embodiments of the present invention rather than for limiting the scope of the present invention.

[0012] Figure 1 The HIC spectra of the antibody drug conjugate Herceptin-Dxd (DAR=4) prepared at different organic solvent contents are shown. (a) is Comparative Example 1-1, the final organic solvent content is 20 vol%; (b) is Experimental Example 1-2, the final organic solvent content is 3.6 vol%.

[0013] Figure 2 HIC spectra of antibody drug conjugates (DAR=4) derived from different antibodies prepared at different organic solvent contents are shown. Sub-figures (a)-(b) are HIC spectra of antibody drug conjugate Daratumumab-Dxd, where (a) is Comparative Example 2-1, with a final organic solvent content of 20 vol%; (b) is Experimental Example 2-2, with a final organic solvent content of 3.6 vol%. Sub-figures (c)-(d) are HIC spectra of antibody drug conjugate Cetuximab-Dxd, where (c) is Comparative Example 2-3, with a final organic solvent content of 20 vol%; (d) is Experimental Example 2-4, with a final organic solvent content of 3.6 vol%.

[0014] Figure 3 HIC spectra of antibody drug conjugates (DAR=4) prepared with various linker-drug intermediates at different organic solvent contents are shown. Sub-figure (a) is the HIC spectrum of antibody drug conjugates (Experimental Example 3-3) prepared with linker-drug intermediate Herceptin-GGFG-Exatecan, which is prepared using the method of the present invention, and the final organic solvent content is 3.6 vol%. Sub-figures (b)-(c) are deconvoluted mass spectra of the prepared Herceptin-CL2A-SN38 (DAR value is about 4), wherein (b) is Comparative Example 3-1, and the final organic solvent content is 20 vol%; (c) is Experimental Example 3-2, and the final organic solvent content is 3.6 vol%.

[0015] Figure 4 The HIC spectra of the antibody drug conjugate Herceptin-Dxd (DAR=4) prepared by reducing the organic solvent content with various cyclodextrin compounds are shown. Sub-figures (a) and (b) correspond to the HIC spectra of the antibody drug conjugate Herceptin-Dxd (DAR=4) prepared by reducing the organic solvent content with hydroxypropyl-α-cyclodextrin (Experimental Example 4-1) and α-cyclodextrin (Experimental Example 4-2), respectively, wherein the final organic solvent content is 3.6 vol%. DETAILED DESCRIPTION

[0016] The meaning of scientific and technological terms in this application is consistent with the general understanding of those skilled in the art, unless otherwise specified. In this application, "one" or its combination with various quantifiers includes both singular and plural meanings, unless otherwise specified. In this application, for the same parameter or variable, when multiple numerical values, numerical ranges, or combinations thereof are given for description, it is equivalent to specifically revealing these numerical values, range ends, and numerical ranges formed by any combination thereof. In this application, any numerical value, whether or not it carries a modifier such as "about", covers an approximate range that can be understood by those skilled in the art, such as plus or minus 10%, 5%, etc. In this article, each "implementation method" refers to and covers the implementation methods of various methods and systems of this application equally. In this application, one or more technical features in any implementation method can be freely combined with one or more technical features in any one or more other implementation methods, and the implementation methods obtained thereby also belong to the content disclosed in this application.

[0017] ADC conjugation technologies can be roughly divided into two categories. One is the conjugation technology mediated by reactive natural amino acid residues in the antibody sequence (such as the side chain amino group of surface lysine and the thiol group after reduction of interchain disulfide bonds); the other is the introduction of reactive groups at specific sites of antibodies through chemical modification, genetic engineering technology or enzyme modification, and then conjugation of cytotoxic drugs to achieve specific site conjugation (such as engineered cysteine ​​site-specific insertion, non-natural amino acid site-specific insertion, enzyme-mediated and N-glycan-mediated conjugation technology).

[0018] Since the thiol group after the reduction of the interchain disulfide bond has extremely strong nucleophilicity, it becomes a special bioconjugation medium. However, when the linker-drug intermediate is conjugated to the thiol group, a high content of organic solvent is required in the reaction system to ensure the solubility of the linker-drug intermediate, which may affect the reactivity of the linker-drug intermediate and increase the difficulty of purifying the antibody-drug conjugate, resulting in a prolonged production process, low yield of the target product, and increased resource consumption, thus limiting the industrialization of ADC.

[0019] Through long-term exploration and experiments, the inventors have proposed an improved method for preparing antibody-drug conjugates, which can improve the homogeneity of the produced antibody-drug conjugates and significantly reduce the amount of organic solvents used in the production process, making the production process more environmentally friendly.

[0020] definition

[0021] As used herein, the term "antibody" refers to a Y-shaped tetrameric protein composed of four polypeptide chains, including two heavy (H) polypeptide chains and two light (L) polypeptide chains, which are held together by covalent disulfide bonds and non-covalent interactions.

[0022] Human light chains include a variable domain (VL) and a constant domain (CL), where the constant domain can be easily classified as either kappa or lambda based on amino acid sequence and locus. Each heavy chain includes one variable domain (VH) and one constant region, which in the case of IgG, IgA and IgD includes three domains named CH1, CH2 and CH3 (IgM and IgE have a fourth domain, CH4). In the IgG, IgA and IgD classes, the CH1 and CH2 domains are separated by a flexible hinge region, which is a proline and cysteine-rich segment with variable length (usually about 10 to about 60 amino acids in IgG). The variable domains in the light and heavy chains are joined to the constant domains by a "J" region of about 12 or more amino acids, and the heavy chain also has a "D" region, which has about 10 additional amino acids.

[0023] For the ADC of the present invention, the term "antibody" includes antibody fragments, such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments and scFv fragments. In addition, the term "antibody" extends to include functional equivalents, such as ligands and binding proteins that specifically recognize and bind to target molecules (such as antigens, such as tumor antigens), receptors or other surface molecules on target cells (such as disease-related cells, such as cancer cells or tumor cells), as long as these equivalent molecules have or can be modified to have functional groups that can react with the maleamide groups of the linker to covalently bind thereto. In some embodiments, the functional groups are thiol groups, such as those released by reduction of interchain disulfide bonds, and the antibody can thus be coupled to the linker portion via a sulfur-maleamide linkage.

[0024] Each class of antibodies further contains inter-chain and intra-chain disulfide bonds formed by paired cysteine ​​residues.

[0025] Herein, the term "interchain disulfide bond" refers to a disulfide between two heavy chains in an antibody (heavy chain-heavy chain disulfide bond) or a disulfide between a heavy chain and a light chain in an antibody (heavy chain-light chain disulfide bond).

[0026] The position and number of interchain disulfide bonds vary according to the immunoglobulin class and species. This article is not limited to any specific class or subclass of antibodies, and IgG1 immunoglobulins should be used for illustrative purposes only. Interchain disulfide bonds are located on the surface of immunoglobulins, are accessible to solvents, and are generally relatively easy to reduce. In human IgG1 isotypes, there are four interchain disulfide bonds, two between each heavy chain-light chain, and two between heavy chain-heavy chains. Chain association does not require interchain disulfide bonds. The IgG1 hinge region rich in cysteine ​​in the heavy chain is generally maintained as consisting of three parts: upper hinge (Ser-Cys-Asp-Lys-Thr-His-Thr), core hinge (Cys-Pro-Pro-Cys), and lower hinge (Pro-Ala-Glu-Leu-Leu-Gly-Gly). It will be understood by those skilled in the art that the IgG1 hinge region contains cysteine ​​in the heavy chain, and the heavy chain contains four interchain disulfide bonds, which provide structural flexibility that is conducive to Fab movement.

[0027] Herein, the terms "interchain sulfhydryl group" and "interchain thiol group" are used interchangeably and refer to sulfhydryl groups obtained by reducing the interchain disulfide bonds of an antibody.

[0028] As used herein, the term "drug" refers to an element, compound, agent, or molecular entity, including, for example, a drug, a therapeutic agent, or a pharmaceutical compound.

[0029] Herein, the term "linker" refers to a reactive molecule that connects a drug and an antibody. Specifically, the linker contains at least two reactive groups, one of which can be covalently bonded to the drug and the other can be covalently coupled to the antibody.

[0030] As used herein, the term "linker-drug intermediate" refers to a conjugate of "drug" and "linker".

[0031] As used herein, the terms "antibody-drug conjugate" and "ADC" are used interchangeably to refer to a composition comprising an antibody-drug conjugate bound by two drug linkers, an antibody-drug conjugate bound by four drug linkers, an antibody-drug conjugate bound by six drug linkers, an antibody-drug conjugate bound by eight drug linkers, and an antibody not bound by any drug linker, in any given ratio.

[0032] As used herein, the terms "DAR (drug:antibody ratio)" and "number of drug bound" are used interchangeably to refer to the number of drugs bound to a single antibody molecule in an antibody-drug conjugate.

[0033] Herein, the term "macrocyclic compound" refers to a cyclic organic compound with 12 or more ring members. Generally speaking, the macrocyclic compound used in the method described herein needs to include a hydrophobic limited cavity and a hydrophilic outer surface. Typically, the macrocyclic compound can include cyclodextrin compounds of different sizes, and chemically modified forms thereof.

[0034] In one aspect, the present invention provides a method for preparing an antibody-drug conjugate, comprising:

[0035] (a) treating the antibody with a reducing agent in the presence of a transition metal ion; and

[0036] (b) coupling the linker-drug intermediate to the reduced antibody obtained in step (a) in the presence of a macrocyclic compound.

[0037] 1. Antibody Reduction

[0038] Generally speaking, there is no particular limitation on the antibodies used herein, and those skilled in the art can select appropriate antibodies as needed.

[0039] In some embodiments, the antibody may have one or more of the following properties: the ability to recognize target cells, the ability to specifically bind to target cells via antigens (e.g., tumor-associated antigens (TAA)), viral antigens, or microbial antigens, the ability to enter target cells and internalize, and the ability to damage target cells. In some embodiments, the antibody has antibody-dependent cell-mediated phagocytosis (ADCP) activity or has in vivo anti-tumor, anti-viral, or anti-microbial activity.

[0040] In some embodiments, the antibody is an antitoxin, an antibacterial antibody, an antiviral antibody, or a cytotropic antibody.

[0041] In some embodiments, the antibody is selected from polyclonal antibodies, multiclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized and primatized antibodies, CDR-grafted antibodies, human antibodies, recombinantly produced antibodies, intracellular antibodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotypic antibodies, synthetic antibodies (including mutant proteins and variants thereof), immunospecific antibody fragments (e.g., Fd, Fab, F(ab')2, F(ab') fragments), single chain fragments (e.g., ScFv and ScFvFc); and derivatives thereof, including Fc fusions and other modifications, and any other immunoresponsive molecules. In some embodiments, the antibody is a monoclonal antibody or a polyclonal antibody. In other embodiments, the antibody is a monoclonal antibody (e.g., a human antibody or a humanized antibody). In yet other embodiments, the antibody is an IgG1 monoclonal antibody or an IgG4 monoclonal antibody.

[0042] In some embodiments, the antibody can be selected from any of IgA, IgD, IgE, IgG, IgM and IgY, such as IgG. In some embodiments, the antibody is selected from subclasses IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. In some embodiments, the antibody is selected from IgG1 and IgG4, such as IgG1. In some specific embodiments, the antibody is an IgG1 monoclonal antibody, such as but not limited to Herceptin (trastuzumab), Rituxan (rituximab), Daratumumab (daratumumab), Cetuximab (cetuximab).

[0043] The antibodies described herein can be produced according to methods known in the art.

[0044] The reduction of the antibody can be carried out by treating the antibody with a reducing agent in the presence of transition metal ions. This process can selectively reduce the disulfide bonds in the antibody structure (such as the interchain disulfide bonds in the Fab region) to sulfhydryl groups for coupling with the linker-drug.

[0045] In some embodiments, reduction of the antibody can be produced by reacting the antibody with a reducing agent at a temperature between -10°C and 37°C, for example, -10°C, -5°C, 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 37°C, or a temperature range formed by any two values ​​as endpoints.

[0046] In some embodiments, the amount of reducing agent is 0.04 mM-0.4 mM, such as 0.1 mM or 0.24 mM.

[0047] In some embodiments, the reducing agent can be tris (2-carboxyethyl) phosphine (TCEP) or a salt thereof, dithiothreitol (DTT) or 2-mercaptoethanol (2-ME), tris (3-hydroxypropyl) phosphine (THPP), 2-diphenylphosphinobenzenesulfonic acid (diPPBs), dithioerythritol (DTE), LiAlH4, Na2S2O3, KBH4 or hydrazine. In some specific embodiments, the reducing agent can be tris (2-carboxyethyl) phosphine or a salt thereof.

[0048] The reduction of the antibody can be carried out in a buffer system. In some embodiments, the available buffer system may include, for example, N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid buffer system (TAPS), N, N-bis (2-hydroxyethyl) glycine buffer system (Bicine), tris (hydroxymethylaminomethane) buffer system (Tris), N-tris (hydroxymethyl) methylglycine buffer system (Tricine), N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid buffer system (TES), 3- (N-morpholinyl) propanesulfonic acid buffer system (MOPS), piperazine-1,4-diethanesulfonic acid buffer system (PIPES), histidine buffer system, phosphate buffer system (PBS), borate buffer system (BBS), acetate buffer system (TAE), (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid) buffer system (HEPES) and combinations thereof. In some specific embodiments, the reduction of the antibody is carried out in a phosphate buffer system (such as PBS).

[0049] Transition metal ions are also used in the reduction of antibodies to produce selectivity in the reduction of disulfide bonds. In some embodiments, the transition metal ions are derived from metal elements of the first to third transition series, specifically, from metal elements in the second to fifth main groups. In some specific embodiments, the transition metal ions are derived from metal ions other than alkali metals. In some more specific embodiments, the transition metal ions are derived from first transition series metals, gallium, germanium, indium, tin, and bismuth.

[0050] In other embodiments, the transition metal ion may also be derived from a transition metal salt. The transition metal ion may include, but is not limited to, Zn 2+ 、Cd 2+ , Hg 2+ , Mn 2+ 、Ni 2+ , Fe 2+ , Fe 3+ and Cu 2+ .

[0051] In some embodiments, the concentration of the transition metal ion used for reduction of the antibody is 0.01 mM-0.2 mM.

[0052] The reduction of the antibody can last for 1 to 15 hours, for example, 1, 3, 5, 7, 9, 11, 13, 15 hours, or a range of time periods formed by using any two values ​​as endpoints.

[0053] Reduction of the antibody can be carried out at pH 5 to 9, such as pH 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or a pH range formed by any two values ​​thereof as endpoints.

[0054] 2. Conjugation of Antibodies to Linker-Drug Intermediates

[0055] The coupling of the antibody to the linker-drug intermediate is carried out by contacting the reduced antibody with the linker-drug intermediate solution. Usually, in order to make the linker-drug intermediate dissolve better, a large amount of organic solvent (concentration can be as high as 20vol%) is used to prepare the linker-drug intermediate solution, which in turn will cause problems in the subsequent purification process, such as semipermeable membrane dissolution, protein is difficult to adsorb to the surface of the filler, etc. Studies have found that the use of macrocyclic compounds to replace most organic solvents can not only greatly reduce the amount of organic solvents, save a lot of post-processing operations, but also improve the production efficiency of antibody-drug conjugates with the required DAR value and improve product quality.

[0056] Specifically, a linker-drug intermediate solution containing a macrocyclic compound is added to the reduced antibody so that they react with each other for coupling.

[0057] In some embodiments, the reduced antibody can be contacted (eg, mixed) with an excess of linker-drug intermediate for conjugation.

[0058] In some embodiments, the linker-drug intermediate is used at a concentration of no less than 4 times the antibody concentration. In some embodiments, the linker-drug intermediate can be used in an amount of 0.03mM-0.30mM, for example, 0.03, 0.06, 0.09, 0.12, 0.15, 0.18, 0.21, 0.24, 0.27, 0.30mM, or in an amount of a concentration range formed by any two values ​​as endpoints.

[0059] In some embodiments, macrocyclic compounds include, but are not limited to, porphyrins, crown ethers, pillararenes, and cyclodextrins and calixarenes.

[0060] In some specific embodiments, the macrocyclic compound is a cyclodextrin compound.

[0061] As used herein, "cyclodextrin" or "cyclodextrin-like compounds" are used interchangeably and refer to cyclic maltooligosaccharides having at least five pyranose glucopyranose units connected by α(1-4) bonds. Cyclodextrin molecules are characterized by a rigid, truncated conical molecular structure having a hollow interior, or pore, of a specific volume. "Cyclodextrin" or "cyclodextrin-like compounds" may also include cyclodextrin derivatives, or blends of one or more cyclodextrins. In some specific embodiments, the cyclodextrin-like compounds are selected from α-, β-, γ-cyclodextrins, and combinations thereof; wherein α-cyclodextrin has six glucose residues; β-cyclodextrin has seven glucose residues; and γ-cyclodextrin has eight glucose residues. More specifically, the cyclodextrin-like compounds are selected from α-cyclodextrin, hydroxypropyl-α-cyclodextrin, hydroxypropyl-β-cyclodextrin, β-cyclodextrin, hydroxypropyl-γ-cyclodextrin, and combinations thereof.

[0062] In some embodiments, the concentration of the macrocyclic compound (such as cyclodextrin compound) used in the coupling step (such as step (b)) can be 0.1-50 mM, for example, 0.1, 0.2, 0.24, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 mM, or a concentration range formed by any two values ​​as endpoints.

[0063] In some embodiments, suitable linkers are selected from cleavable linkers and non-cleavable linkers. Cleaving linkers are generally easy to cleave under intracellular conditions. Suitable cleavable linkers include, for example, peptide linkers that can be cleaved by intracellular proteases (e.g., lysosomal proteases or endosomal proteases). In exemplary embodiments, the linker can be a dipeptide linker, such as a valine-citrulline (val-cit) or a phenylalanine-lysine (phe-lys) linker. Other suitable cleavable linkers include disulfide bond linkers.

[0064] In some specific embodiments, the linker may include a group for attachment to a protein. For example, the linker may include an amino, hydroxyl, carboxyl or sulfhydryl reactive group (e.g., maleimide, haloacetamide (e.g., iodine, bromine or chloride), halogenated ester (e.g., iodine, bromine or chloride), halomethyl ketone (e.g., iodine, bromine or chloride), benzyl halide (e.g., iodine, bromine or chloride), vinyl sulfone and pyridine thio group).

[0065] The drug used herein is not particularly limited, as long as it is a compound having a desired effect (e.g., cytotoxicity, cell inhibition, immunosuppression or labeling effect) and also having a substituent or partial structure capable of binding to a linker structure. Herein, a drug linker comprising the drug and the linker portion may also be referred to as a "drug".

[0066] In some embodiments, the drug is selected from a cytotoxic or cytostatic agent, an immunosuppressant, a radioactive isotope, a toxin, a labeling agent, a fluorescent agent (e.g., an amine-derivatized fluorescent probe such as 5-dimethylaminonaphthalene-1-(N-(2-aminoethyl))sulfonamide-dansylethylenediamine, Oregon 488 cadaverine (cada verine) (e.g., catalog number O-10465, Molecular Probes), dansylcadaverine, N-(2-aminoethyl)-4-amino-3,6-dithio-1,8-naphthaleneimine, dipotassium salt (fluorescein ethylenediamine), rhodamine B ethylenediamine (e.g., catalog number L-2424, Molecular Probes), thiol-derivatized fluorescent probes such as FLL-cysteine ​​(e.g., catalog number B-20340, Molecular Probes)), and combinations thereof. In some exemplary embodiments, the drug can be a cytotoxic agent, for example, calimycin, bicamicin, PBD, DXd, SN38, MMAE, MMAF, maytansine derivatives (such as DM2 and DM4).

[0067] The linker-drug intermediate used herein is not particularly limited, as long as it is a compound capable of reacting with the interchain thiol of the antibody, thereby coupling the antibody to the linker-drug intermediate. In some embodiments, the compound that reacts with the interchain thiol of the antibody has an electrophilic group selected from the following group: (i) maleimide group, (ii) activated disulfide, (iii) active ester, such as NHS (N-hydroxysuccinimide) ester, HOBt (N-hydroxybenzotriazole) ester, haloformate, and acyl halide; (iv) alkyl and benzyl halides, such as haloacetamide; and (v) aldehydes, ketones, carboxyl groups. In some specific embodiments, the compound is an organic bromide, iodide, sulfone compound, and other olefins and alkynes with electron-deficient groups. However, the linker-drug intermediate used herein is not limited to the compounds described herein, and all types of drug linker intermediates can be applied to the method of the present invention, as long as they have a functional group for promoting the reaction with the interchain thiol of the antibody. The conjugation reaction can be terminated by inactivating the reactivity of the unreacted linker-drug intermediate with a sulfhydryl-containing reagent.

[0068] As such a thiol-containing reagent, for example, cysteine ​​or N-acetylcysteine ​​can be used. More specifically, the coupling reaction can be terminated by adding N-acetylcysteine ​​in an amount of 0.1 mM to 1.0 mM to the linker-drug intermediate used, for example, 0.1, 0.2, 0.24, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 mM or a concentration range formed by any two values ​​as endpoints.

[0069] The linker-drug intermediate can be initially dissolved in a solvent. Examples of solvents for the initial dissolution of the linker include organic solvents, such as acetone aqueous solution, ethanol aqueous solution, methanol aqueous solution, isopropanol aqueous solution, dimethyl sulfoxide aqueous solution, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA) and N-methyl-2-pyrrolidone (NMP). In some embodiments, the solvent is dimethyl sulfoxide (DMSO). Subsequently, a macrocyclic compound is added to the initially dissolved linker-drug intermediate to form a linker-drug intermediate solution in which the organic solvent content is not higher than 5 vol%.

[0070] In some embodiments, the molar ratio of the macrocyclic compound (such as a cyclodextrin compound) to the antibody is in the range of about 10:1-1000:1, for example, the molar ratio of the macrocyclic compound (such as a cyclodextrin compound) to the antibody is about 10:1, 60:1, 110:1, 160:1, 210:1, 260:1, 310:1, 360:1, 410:1, 460:1, 510:1, 560:1, 610:1, 660:1, 710:1, 760:1, 810:1, 860:1, 910:1, 960:1, 1000:1, or a range formed with any value as an endpoint. In some embodiments, the molar ratio of the macrocyclic compound (such as cyclodextrin compound) to the antibody is in the range of about 20:1-1000:2, more specifically in the range of about 30:1-1000:3.

[0071] The coupling of the antibody to the linker-drug intermediate can be carried out at a temperature of -10°C to 37°C, for example, -10°C, -5°C, 0°C, 5°C, 10°C, 12°C, 15°C, 20°C, 25°C, 30°C, 35°C, 37°C or a temperature range formed by any two values ​​as endpoints.

[0072] The coupling time of the antibody and the linker-drug intermediate can be 0.5 to 2 hours, for example 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 16, 1.7, 1.8, 1.9, 2.0 hours or a time range formed by any two values ​​as endpoints.

[0073] 3. Post-processing

[0074] In some embodiments, the method may further include: adding an effective amount of an oxidizing agent to reoxidize the unreacted thiol groups in step (b).

[0075] In some embodiments, the oxidant is selected from: 5,5'-dithiobis-2-nitrobenzoic acid, 4,4'-dithiodipyridine, 2,2'-dithiodipyridine, sodium tetrathionate, dehydroascorbic acid (DHAA), and combinations thereof. In some specific embodiments, the oxidant is DHAA. In some embodiments, the added concentration of the oxidant in the reaction solution is greater than the antibody concentration. In a specific embodiment, the oxidant concentration can be 0.08mM-0.8mM, for example 0.08mM, 0.16mM, 0.24mM, 0.32mM, 0.40mM, 0.48mM, 0.56mM, 0.64mM, 0.72mM, 0.8mM or a concentration range formed with any two values ​​as endpoints.

[0076] In some embodiments, the method may further comprise: purifying the antibody-drug conjugate.

[0077] In some embodiments, the method used to purify the antibody-drug conjugate in the purification step is selected from tangential flow filtration (TFF) (e.g., ultrafiltration diafiltration (UFDF)) and chromatography (e.g., size exclusion chromatography (SEC), hydrophobic chromatography (HIC), hydroxyapatite chromatography (HA) and membrane chromatography (MC)). In some specific embodiments, the antibody-drug conjugate is purified using a desalting column or size exclusion chromatography.

[0078] In a further embodiment, a chelating agent is used in the purification step to remove transition metal ions. In some embodiments, the chelating agent is selected from: aminocarboxylic acids, organic phosphonic acids, hydroxycarboxylic acids, amino acids, polycarboxylic acids, polyphosphates, and combinations thereof. In some specific embodiments, examples of chelating agents include ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA). Among them, ethylenediaminetetraacetic acid (EDTA) is preferred.

[0079] In some embodiments, the method may further comprise: recovering the antibody-drug conjugate.

[0080] In another aspect, the present invention also provides the use of macrocyclic compounds for improving the homogeneity of produced antibody-drug conjugates.

[0081] On the other hand, the present invention also provides the use of macrocyclic compounds for improving production efficiency by reducing the amount of organic solvent used in the production process of antibody-drug conjugates.

[0082] Example

[0083] The following examples are for illustration only and are not intended to limit the scope of the present invention.

[0084] Example 1: Effect of organic solvent content on drug distribution of antibody-drug conjugates

[0085] 1.1 Methods

[0086] Preparation of antibody-drug conjugate Herceptin-Dxd (DAR=4)

[0087] Comparative Example 1-1 was prepared according to the following method, and the specific steps are as follows:

[0088] (1) ZnCl2 (0.12 mM) and TCEP (0.1 mM) were added to a Herceptin solution (0.03 mM) in a phosphate buffer (pH 7, 20 mM), and the resulting reaction mixture was allowed to stand overnight at 12°C;

[0089] (2) MC-GGFG-Dxd (final concentration 0.3 mM; purchased from WuXi AppTec, DMSO was added to make the final organic solvent content 15 vol%, and the reaction was continued at 12°C for 1 hour;

[0090] (3) addition of N-acetylcysteine ​​(0.24 mM) to deplete excess MC-GGFG-Dxd;

[0091] (4) Add EDTA (0.24 mM) to capture Zn 2+ , and DHAA (0.24 mM) was added to oxidize excess thiol groups;

[0092] (5) The reaction mixture was purified using a desalting column (40K, 0.5 mL) (purchased from Thermo Fisher, product number 87766, batch number SJ251704).

[0093] Experimental Example 1-2 was prepared according to the following method. Compared with the method for preparing Comparative Example 1-1, the difference is that step (2) is changed to:

[0094] (2) MC-GGFG-Dxd (final content 0.12 mM; purchased from WuXi AppTec) in DMSO was introduced, hydroxypropyl-β-cyclodextrin (0.6 mM, purchased from ROQUETTE) was added, and the final organic solvent content was 3.6 vol%, and the reaction was continued at 12°C for 1 hour.

[0095] Homogeneity determination

[0096] The drug / antibody ratio (DAR) and product distribution of the resulting antibody-drug conjugates were analyzed using HIC-HPLC. The purification analysis of each component was performed by hydrophobic interaction chromatography (HIC) at a flow rate of 0.6 mL / min at ambient temperature on a Butyl-NPR HPLC column (purchased from TOSOH BIOSCIENCE, L x ID 3.5 cm x 4.6 mm, 2.5 μm). The injection volume was 50 μg, and solvent A was 1.5 M Na2SO4 and 50 mM Na3PO4 (pH 7). Solvent B was 25% v / v isopropanol and 75% v / v 50 mM Na3PO4 (pH 7). Substances with different DAR values ​​were eluted successively by sequential step gradients.

[0097] 1.2 Results

[0098] The results are shown in Table 1 and the corresponding diagrams are shown in Figure 1 .

[0099] Table 1

[0100]

[0101] As shown in Table 1, compared with Comparative Example 1-1, the average DAR value of Experimental Example 1-2 prepared by the method of the present invention is closer to 4, and the content of DAR4 component is higher, and the DAR6 component as the main by-product is greatly reduced. That is, when hydroxypropyl-β-cyclodextrin is added to reduce the content of organic solvents in the production process, the linker-drug intermediate can fully react with the antibody, and the antibody-drug conjugate prepared by the method of the present invention has better quality.

[0102] Example 2: Effect of organic solvent content on antibody-drug conjugates derived from different antibodies

[0103] In this example, an antibody-drug conjugate derived from Cetuximab and Daratumumab (both purchased from WuXi Biologics) was prepared.

[0104] 2.1 Methods

[0105] Preparation of antibody-drug conjugates (DAR=4) using Daratumumab and Cetuximab

[0106] Comparative Example 2-1 and Comparative Example 2-3 were prepared according to the following method, and the specific steps were as follows:

[0107] (1) ZnCl2 (0.12 mM) and TCEP (0.1 mM) were added to Daratumumab solution and Cetuximab solution (0.03 mM) in phosphate buffer (pH 7, 20 mM), respectively, and the resulting reaction mixture was allowed to stand at 12°C overnight;

[0108] (2) MC-GGFG-Dxd (final content of 0.3 mM; purchased from WuXi AppTec) in DMSO was introduced into Comparative Example 2-1 and Comparative Example 2-3, and DMSO was added to make the final content of the organic solvent 20 vol%, and the reaction was continued at 12° C. for 1 hour;

[0109] (3) addition of N-acetylcysteine ​​(0.24 mM) to deplete excess MC-GGFG-Dxd;

[0110] (4) Add EDTA (0.24 mM) to capture Zn 2+ , and DHAA (0.24 mM) was added to oxidize excess thiol groups;

[0111] (5) The reaction mixture was purified using a desalting column (40K, 0.5 mL) (purchased from Thermo Fisher, product number 87766, batch number SJ251704).

[0112] Experimental Examples 2-2 and 2-4 derived from Daratumumab and Cetuximab, respectively, were prepared as described herein, and compared with the method for preparing Comparative Examples 2-1 and 2-3, the difference was that step (2) was changed to:

[0113] (2) MC-GGFG-Dxd in DMSO (final content of 0.12 mM; purchased from WuXi AppTec) was introduced into Experimental Examples 2-2 and 2-4, respectively, and hydroxypropyl-β-cyclodextrin (10 mM, purchased from ROQUETTE) was added, with a final organic solvent content of 3.6 vol%, and the reaction was continued at 12°C for 1 hour.

[0114] Homogeneity analysis

[0115] The drug / antibody ratio (DAR) and product distribution of the resulting antibody-drug conjugates were analyzed using HIC-HPLC. The purification analysis of each component was performed by hydrophobic interaction chromatography (HIC) at a flow rate of 0.6 mL / min at ambient temperature on a Butyl-NPR HPLC column (purchased from TOSOH BIOSCIENCE, L x ID 3.5 cm x 4.6 mm, 2.5 μm). The injection volume was 50 μg, and solvent A was 1.5 M Na2SO4 and 50 mM Na3PO4 (pH 7). Solvent B was 25% v / v isopropanol and 75% v / v 50 mM Na3PO4 (pH 7). Substances with different DAR values ​​were eluted successively by sequential step gradients.

[0116] 2.2 Results

[0117] The results are shown in Table 2 and the corresponding diagrams are shown in Figure 2 .

[0118] Table 2

[0119]

[0120] As shown in Table 2, the environment in which hydroxypropyl-β-cyclodextrin is added to reduce the content of organic solvent is suitable for preparing antibody-drug conjugates derived from Daratumumab and Cetuximab. Compared with Comparative Examples 2-1 and 2-3, the average DAR values ​​of Experimental Examples 2-2 and 2-4 prepared by the method of the present invention are closer to the target DAR value of 4, and there are more DAR4 components.

[0121] Example 3: Effect of organic solvent content on linker-drug intermediates

[0122] In this example, the antibody-drug conjugate was prepared using camptothecin linker-drug intermediate MC-GGFG-Exatecan (purchased from MCE, catalog number HY-114233) and CL2A-SN38 (purchased from MCE, catalog number HY-128946).

[0123] 3.1 Methods

[0124] Preparation of antibody-drug conjugates Herceptin-SN38 / Herceptin- GGFG-Exatecan (DAR=4)

[0125] Comparative Example 3-1 was prepared according to the following method, and the specific steps are as follows:

[0126] (1) ZnCl2 (0.12 mM) and TCEP (0.1 mM) were added to a Herceptin solution (0.03 mM) in a phosphate buffer (pH 7, 20 mM), and the resulting reaction mixture was allowed to stand overnight at 12°C;

[0127] (2) CL2A-SN38 (final concentration 0.3 mM; purchased from MCE, structure:

[0128] ), adding DMSO to make the final organic solvent content 20 vol%, and continuing the reaction at 12° C. for 1 hour;

[0129] (3) addition of N-acetyl cysteine ​​(0.24 mM) to deplete excess MC-GGFG-CL2A-SN38;

[0130] (4) Add EDTA (0.24 mM) to capture Zn 2+ , and DHAA (0.24 mM) was added to oxidize excess thiol groups;

[0131] (5) The reaction mixture was purified using a desalting column (40K, 0.5 mL) (purchased from Thermo Fisher, product number 87766, batch number SJ251704).

[0132] Experimental Examples 3-2 and 3-3 were prepared according to the following method. Compared with the method for preparing Comparative Example 3-1, the difference is that for Experimental Example 3-3, CL2A-SN38 was replaced with MC-GGFG-Exatecan And for Experimental Examples 3-2 and 3-3, change step (2) to:

[0133] (2) MC-GGFG-Exatecan / CL2A-SN38 (final content 0.12 mM; purchased from MCE) in DMSO was introduced, hydroxypropyl-β-cyclodextrin (10 mM, purchased from ROQUETTE) was added, and the final organic solvent content was 3.6 vol%, and the reaction was continued at 12°C for 3 hours.

[0134] Homogeneity analysis

[0135] The drug / antibody ratio (DAR) and product distribution of the obtained antibody-drug conjugate were analyzed using HIC-HPLC. The purification analysis of each component was performed by hydrophobic interaction chromatography (HIC) at a flow rate of 0.6 mL / min at ambient temperature on an Agilent PLRP-S column (purchased from Agilent Technologies, Cat. No. PL1312-1502). The sample was injected after complete reduction with TCEP, the injection volume was 3 μg, and solvent A was an aqueous solution of 0.05% (v / v) TFA. Solvent B is an acetonitrile solution of 0.05% (v / v) TFA. Substances with different DAR values ​​were eluted successively by sequential step gradients. Due to the characteristics of the reduced mass spectrometry, only D4% and D6% and the average DAR values ​​calculated based on the heavy chain signal are displayed.

[0136] 3.2 Results

[0137] The results are shown in Table 3 and the corresponding diagrams are shown in Figure 3 middle.

[0138] Table 3

[0139]

[0140]

[0141] As shown in Table 3, the environment in which hydroxypropyl-β-cyclodextrin is added to reduce the organic solvent content is suitable for preparing antibody-drug conjugates with camptothecin linker-drug intermediates. Compared with Comparative Example 3-1, the average DAR value of Experimental Example 3-2 prepared by the method of the present invention is closer to the target DAR value of 4, the DAR4 component is more, and the DAR6 component as the main by-product is greatly reduced. Similarly, the average DAR value of Experimental Example 3-3 prepared by the method of the present invention is closer to the target DAR value of 4, and the amount of DAR4 component is significantly higher than the amount of DAR6 component.

[0142] Example 4: Universal application of cyclodextrin compounds in the preparation of antibody-drug conjugates

[0143] In this example, cyclodextrin compounds hydroxypropyl-α-cyclodextrin (purchased from Sigma, catalog number 390690) and α-cyclodextrin (purchased from Sigma, catalog number 778788) were used to explore the applicability of cyclodextrin compounds in reducing the content of organic solvents in the preparation of antibody-drug conjugates.

[0144] 4.1 Methods

[0145] Preparation of Antibody-Drug Conjugate Herceptin-Dxd (DAR=4) Using Cyclodextrin Compounds

[0146] Experimental Example 4-1 and Experimental Example 4-2 were prepared according to the following method. The specific steps are as follows:

[0147] (1) ZnCl2 (0.12 mM) and TCEP (0.1 mM) were added to a Herceptin solution (0.03 mM) in a phosphate buffer (pH 7, 20 mM), and the resulting reaction mixture was allowed to stand overnight at 12°C;

[0148] (2) MC-GGFG-Dxd (0.3 mM, purchased from MCE) in DMSO was introduced, and hydroxypropyl-α-cyclodextrin and α-cyclodextrin (10 mM, purchased from ROQUETTE) were added to Experimental Example 4-1 and Experimental Example 4-2, respectively, with a final organic solvent content of 3.6 vol%, and the reaction was continued at 12°C for 1 hour. ;

[0149] (3) addition of N-acetylcysteine ​​(0.24 mM) to deplete excess MC-GGFG-Dxd;

[0150] (4) Add EDTA (0.24 mM) to capture Zn 2+ , and DHAA (0.24 mM) was added to oxidize excess thiol groups;

[0151] (5) The reaction mixture was purified using a desalting column (40K, 0.5 mL) (purchased from Thermo Fisher, product number 87766, batch number SJ251704).

[0152] Homogeneity analysis

[0153] The drug / antibody ratio (DAR) and product distribution of the resulting antibody-drug conjugates were analyzed using HIC-HPLC. The purification analysis of each component was performed by hydrophobic interaction chromatography (HIC) at a flow rate of 0.6 mL / min at ambient temperature on a Butyl-NPR HPLC column (purchased from TOSOH BIOSCIENCE, L x ID 3.5 cm x 4.6 mm, 2.5 μm). The injection volume was 50 μg, and solvent A was 1.5 M Na2SO4 and 50 mM Na3PO4 (pH 7). Solvent B was 25% v / v isopropanol and 75% v / v 50 mM Na3PO4 (pH 7). Substances with different DAR values ​​were eluted successively by sequential step gradients.

[0154] 4.2 Results

[0155] The results are shown in Table 4 and the corresponding diagrams are shown in Figure 4 .

[0156] Table 4

[0157]

[0158] As shown in Table 4, the environment in which the cyclodextrin compound (hydroxypropyl-α-cyclodextrin and α-cyclodextrin, respectively, corresponding to Experimental Example 4-1 and Example 4-2) is added to reduce the organic solvent content is suitable for preparing antibody-drug conjugates. The average DAR values ​​of Experimental Example 4-1 and Experimental Example 4-2 prepared by the method of the present invention are closer to the target DAR value of 4, the DAR4 component is more, and the DAR6 component, which is the main by-product, is greatly reduced.

Claims

1. A method for preparing an antibody-drug conjugate, comprising: (a) treating the antibody with a reducing agent in the presence of a transition metal ion; and (b) coupling the linker-drug intermediate to the reduced antibody obtained in step (a) in the presence of a macrocyclic compound.

2. The method according to claim 1, wherein after step (b), the method further comprises: (c) adding an effective amount of an oxidant to reoxidize the unreacted thiol groups in step (b); more specifically, the oxidant is selected from the group consisting of 5,5'-dithiobis-2-nitrobenzoic acid, 4,4'-dithiodipyridine, 2,2'-dithiodipyridine, sodium tetrathionate, dehydroascorbic acid (DHAA), and combinations thereof; specifically, the oxidant is selected from DHAA.

3. The method of claim 1, wherein the antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, and a combination thereof.

4. The method according to claim 3, wherein the antibody is of IgG1 or IgG4 subtype; specifically, the antibody is selected from: trastuzumab, rituximab, daratumumab, cetuximab, and a combination thereof.

5. The method according to claim 1, wherein the transition metal ion is selected from the group consisting of: Zn 2+ 、Cd 2+ , Hg 2+ , Mn 2+ 、Ni 2+ , Fe 2+ , Fe 3+ , Cu 2+ , and combinations thereof; more specifically, selected from Zn 2+ .

6. The method according to claim 5, wherein step (a) is carried out in a buffer system; specifically, the buffer system is selected from the group consisting of N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid buffer system (TAPS), N,N-bis(2-hydroxyethyl)glycine buffer system (Bicine), tris(hydroxymethylaminomethane) buffer system (Tris), N-tris(hydroxymethyl)methylglycine buffer system (Tricine), N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid buffer system (TES), 3-(N-morpholino)propanesulfonic acid buffer system (MOPS), piperazine-1,4-diethanesulfonic acid buffer system (PIPES), histidine buffer system, phosphate buffer system (PBS), borate buffer system (BBS), acetate buffer system (TAE), (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer system (HEPES), and combinations thereof; more specifically, phosphate buffer system (PBS).

7. The method according to claim 1, wherein the reducing agent is selected from the group consisting of tris(2-carboxyethyl)phosphine (TCEP) or a salt thereof, dithiothreitol (DTT), 2-mercaptoethanol (2-ME), tris(3-hydroxypropyl)phosphine (THPP), 2-diphenylphosphinobenzenesulfonic acid (diPPBs), dithioerythritol (DTE), LiAlH4, Na2S2O3, KBH4, hydrazine, and combinations thereof; specifically, selected from tris(2-carboxyethyl)phosphine or a salt thereof.

8. The method according to claim 1, wherein the macrocyclic compound comprises: Porphyrin, crown ether, columnar hydrocarbon, cyclodextrin, calixarene and a combination thereof; specifically, the macrocyclic compound is a cyclodextrin compound; more specifically, the cyclodextrin compound is selected from α-cyclodextrin, hydroxypropyl-α-cyclodextrin, hydroxypropyl-β-cyclodextrin, β-cyclodextrin, hydroxypropyl-γ-cyclodextrin, and a combination thereof.

9. The method according to claim 8, wherein the molar ratio of the cyclodextrin compound to the antibody is about 10:1-1000:1, specifically about 20:1-1000:2, and more specifically about 30:1-1000:

3.

10. The method according to claim 1, wherein step (a) is carried out at a temperature range of -10°C to 37°C and a pH range of 5 to 9; further, step (b) is carried out at a temperature range of -10°C to 37°C for 0.5-2 hours.

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