Antibody drug conjugates and production thereof

Abstract

The present application provides antibody drug conjugates and methods of producing the same. The production method comprises: a first reduction reaction step in which an antibody is reduced in the presence of a transition metal ion; and one or more coupling steps wherein the reduced antibody is coupled to a linker-load to form an ADC; the product of the last coupling step is a final ADC product; wherein the method further comprises an optional second reduction reaction wherein the antibody moiety in the ADC is further reduced, and followed by a coupling step; and wherein the method further comprises an unmasking treatment to remove the antibody-incorporated transition metal, the unmasking treatment being synchronized with at least one of the coupling steps or with the second reduction step. The method is advantageous in that a multi-load ADC with a set drug loading pattern can be produced in a controllable manner.

Description

Technical Field

[0001] This invention relates to the field of antibody-drug conjugates, and more particularly to multi-loaded antibody-drug conjugates and their manufacturing methods. Background Technology

[0002] Antibody-drug conjugates (ADCs) are therapeutic drugs composed of a therapeutic agent, such as a cytotoxic drug, linked to a target-specific antibody. The therapeutic agent is guided by the antibody to the target site for release and effect. ADCs loaded with multiple drugs on different antibodies can circumvent ADC-related drug resistance and enhance therapeutic efficacy.

[0003] To expand the applications of ADCs, scientists are constantly exploring new multi-load ADC technologies. One strategy involves connecting two different loads to the same linker and then conjugating that linker to an antibody. For example, Levengood et al. designed a linker with two thiol groups, which were then blocked with S-(tert-butyl) or S-(isopropyl) disulfide. [1] Similarly, Yamazaki et al. developed a branched linker with azide and methyltetraazine functional groups. [2] Synaffix's "dual-head" ADC also employs this strategy, extending its "HydroSpace" technology to allow two different payloads to be connected to the same connector.

[0004] However, when two or more payloads are linked to a single linker, the linker may introduce highly hydrophobic groups (such as TCO or DBCO), resulting in a larger linker-payload size and even higher hydrophobicity. This can affect process development and CMC (such as free drug clearance), as well as the immunogenicity, stability, and efficacy of the ADC, especially when the payload itself is already highly hydrophobic. Introducing hydrophilic groups (e.g., in HydroSpace technology) can mitigate this problem, but large hydrophilic masking groups increase the hydrodynamic diameter of the antibody and may block antibody binding sites.

[0005] The second strategy involves linking two different linkers-loads to different sites on the antibody. Nilchan et al. engineered the protein to place a selenocysteine ​​residue at position 396 of the heavy chain and an additional cysteine ​​residue at position 114. [3] Recently, Boschanski developed a conjugation method that uses two enzymes to sequentially convert cysteine ​​in the sequence “CTPSR” or “CTAGR” to formylglycine, and then conjugates it with a linker-loaded reaction after each conversion. Sutro Biopharma also achieved dual-site labeling by introducing non-natural amino acids into antibody sequences.

[0006] This strategy avoids the problem of excessive hydrophobicity in the first strategy. However, to create the second type of coupling site, the antibody sequence usually needs to be engineered (e.g., engineered cysteine ​​residues), which may affect antibody activity and / or reduce monoclonal antibody yield. Alternatively, sequence-selective or chemoselective enzymes can be used for coupling, but these enzymes are difficult to remove, thus increasing the difficulty and cost of process development.

[0007] There is a need in the art for ADC manufacturing methods that can improve efficiency, operability, controllability, flexibility and / or scalability, especially for multi-load ADCs. Summary of the Invention

[0008] This application provides a method for producing an ADC having multiple connector-loads, preferably different connector-loads, more preferably connector-loads with different drug portions. An ADC product obtained by this method is also provided.

[0009] A first aspect of the present invention provides a method for producing antibody-drug conjugates, comprising:

[0010] The first reduction reaction step, wherein the antibody is reduced in the presence of transition metal ions; and

[0011] One or more coupling reaction steps, wherein a reduced antibody is coupled with a linker-loader to generate an antibody-drug conjugate;

[0012] The method further includes an optional second reduction reaction, wherein the antibody, as a component of the antibody-drug conjugate, is further reduced, and wherein the second reduction reaction is followed by a conjugation step; and

[0013] The method further includes a demasking process to remove transition metals bound to the antibody, the demasking process being performed simultaneously with at least one coupling step or with a second reduction step.

[0014] A second aspect of the present invention provides an antibody-drug conjugate product, wherein the purity of the target antibody-drug conjugate is at least 55 mol%; the target antibody-drug conjugate comprises four first linker-loaders connected to the Fab region and at least two second linker-loaders connected to the hinge region, wherein the first linker-loaders and the second linker-loaders are different or the same.

[0015] A third aspect of the present invention provides an antibody-drug conjugate product, wherein the purity of the target antibody-drug conjugate is at least 55 mol%; the target antibody-drug conjugate contains three or six first linker-loads and optionally one or two second linker-loads, wherein the first linker-loads are different from or the same as the second linker-loads.

[0016] A fourth aspect of the present invention provides an antibody-drug conjugate product, wherein the purity of the target antibody-drug conjugate is at least 55 mol%; the target antibody-drug conjugate contains two or four first linker-loaders linked to the antibody Fab region and at least one (e.g., one, two, three, and four) second linker-loader molecules linked to the antibody hinge region, and optionally may also contain one or two third linker-loaders linked to the antibody hinge region; wherein the first, second, and optional third linker-loaders are different or the same.

[0017] In some embodiments, at least one of the first, second, or third linker-loads is a thiol-bridge linker-load. In some embodiments, the antibody is a bispecific antibody, such as one of the WuXiBody structures, or a fusion protein, such as the VHH-Fc fusion protein.

[0018] A fifth aspect of the present invention provides an antibody-drug conjugate product prepared by the method of the present invention. Attached Figure Description

[0019] The following figures form part of the specification and are used to illustrate the invention, but are not intended to limit the scope of the invention in any way.

[0020] Figure 1 Overview of the Invention Strategy. Scheme 1-1: One embodiment of the present invention, producing a DAR6 type ADC; Scheme 1-2: One embodiment of the present invention, producing a 6+2 type multi-load ADC; Scheme 2: One embodiment of the present invention, producing a 4+4 type multi-load ADC; Scheme 3-1: One embodiment of the present invention, producing a 4+2 type multi-load ADC; and Scheme 3-2: One embodiment of the present invention, producing a 4+2+2 type multi-load ADC.

[0021] Figure 2 Variant implementations of scheme 1-1: (A): Implementation of producing DAR3 type ADC using thiol bridge linker-loader; (B): Implementation of producing DAR4 type ADC using VHH-Fc fusion protein; (C): Implementation of producing DAR4 type ADC using antibody with WuXiBody structure.

[0022] Figure 3 LC-MS characterization of a 4+4 dual-load mAb1-MMAE+MMAF product prepared according to one embodiment of the present invention.

[0023] Figure 4 LC-MS characterization of a 4+4 dual-load mAb1-MMAE+GGFG-DXd product prepared according to one embodiment of the present invention.

[0024] Figure 5 LC-MS characterization of a 4+4 dual-load mAb1-MMAF+MMAE product prepared according to one embodiment of the present invention.

[0025] Figure 6 LC-MS characterization of the 4+4 dual-load mAb1-MMAF+GGFG-DXd product prepared according to one embodiment of the present invention.

[0026] Figure 7 LC-MS characterization of a product containing 4+4 dual-load mAb1-MMAF+CL2A-SN38 prepared according to one embodiment of the present invention.

[0027] Figures 8A-8B The product containing the 4+4 dual-load mAb1-MMAE+DXd obtained in one embodiment of the present invention was characterized by AKTA pureHIC (hydrophobic interaction chromatography) and LC-MS.

[0028] Figure 9 LC-MS characterization of a 4+4 dual-load mAb2-MMAE+MMAF product prepared according to one embodiment of the present invention.

[0029] Figure 10 LC-MS characterization of a product containing a 4+4 dual-load mAb2-MMAE+GGFG-DXd obtained in one embodiment of the present invention.

[0030] Figure 11 LC-MS characterization of a product containing a 4+4 dual-load mAb2-MMAE+CL2A-SN38 obtained in one embodiment of the present invention.

[0031] Figure 12 LC-MS characterization of a product containing a 4+4 dual-load mAb2-MMAF+MMAE obtained in one embodiment of the present invention.

[0032] Figure 13 LC-MS characterization of a product containing a 4+4 dual-load mAb2-MMAF+CL2A-SN38 obtained in one embodiment of the present invention.

[0033] Figures 14A-14E Purity of the crude product obtained from process 2 in Example 1.

[0034] Figures 15A-15B Purity of the purified product obtained from process 3 in Example 1.

[0035] Figure 16 Example 2: LC-MS characterization of the product containing 4+2 dual-load mAb1-MMAF+MMAE obtained from process 1.

[0036] Figure 17 Example 2: LC-MS characterization of the 4+2 dual-load mAb2-MMAF+MMAE product obtained from process 1.

[0037] Figure 18 Purity of the crude product obtained from process 2 in Example 2.

[0038] Figure 19 Example 2: Purity of the purified product obtained from process 2.

[0039] Figure 20 LC-MS characterization of the 4+2+2 triple-load mAb1-DXd+MMAE+MMAF product obtained in Example 3.

[0040] Figures 21A-21B Example 4: HIC characterization of the DAR6-containing product obtained from Experiment 1.

[0041] Figure 22 Example 4: Purity of the product obtained in Experiment 1 before and after purification.

[0042] Figure 23 Example 4: HIC characterization of the product obtained in Experiment 2.

[0043] Figure 24 Example 4: HIC characterization of the product obtained in Experiment 3.

[0044] Figure 25 HIC characterization of the product obtained in Experiment 4 of Example 4.

[0045] Figure 26 HIC characterization of the product obtained in Experiment 5 of Example 4.

[0046] Figure 27 HIC characterization of the product obtained in Experiment 6 of Example 4.

[0047] Figure 28 HIC characterization of the product obtained in Experiment 7 of Example 4.

[0048] Figure 29 LC-MS characterization of a 6+2 dual-load mAb1-MMAE+Dxd product obtained in one embodiment of the present invention.

[0049] Figure 30 LC-MS characterization of a product containing a 6+2 dual-load mAb1-MMAE+MMAF obtained in one embodiment of the present invention.

[0050] Figure 31 LC-MS characterization of a 6+2 dual-load mAb2-MMAE+Dxd product obtained in one embodiment of the present invention.

[0051] Figure 32 LC-MS characterization of a product containing a 6+2 dual-load mAb2-MMAE+MMAF obtained in one embodiment of the present invention.

[0052] Figure 33 PLRP results of the conjugate obtained in Process 2 of Example 5 and PLRP results of the purified conjugate obtained in Process 2 of Example 5.

[0053] Figure 34 HIC characterization of the DAR3-containing product obtained in Example 6.

[0054] Figure 35 HIC characterization of the DAR4-containing product obtained in Example 7. Detailed Implementation

[0055] the term

[0056] In this document, the singular forms introduced by “a,” “an,” and “the,” etc., include the plural meaning, unless otherwise stated. Furthermore, the terms “a,” “a or more,” and “at least one” are used interchangeably herein.

[0057] In this document, unless otherwise stated, whether the terms “about,” “approximately,” or “roughly” precede a numerical value or range, they encompass reasonable approximations that would be understood by one of skill in the art, such as a range of ±10%, ±5%, ±3%, ±2%, ±1%, or ±0.5% of a specified value.

[0058] One or more features in one embodiment of this invention may be combined with one or more features in another embodiment without departing from the concept and idea of ​​the invention.

[0059] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All public and patent documents cited herein are incorporated by reference and are applicable for all purposes. References in this specification should be considered as indicating the level of expertise of those skilled in the art, but should not be construed as an admission that this invention is not entitled to precede such disclosures by prior invention.

[0060] Introduction

[0061] This application provides a method for producing an ADC having multiple linker-loaders, preferably different linker-loaders, more preferably linker-loaders with different drug moieties. The method also provides ADC products obtained by this invention. This invention achieves controlled loading of loads, preferably different loads, at specific cysteine ​​sites on an antibody via conventional cysteine ​​chemical coupling. The method of this invention requires no additional work, such as antibody engineering, the use of branched linkers that may pose a serious risk of hydrophilic masking groups, or additional enzymatic purification operations. The method of this invention can be used to design and produce ADC products with high purity target ADCs.

[0062] This invention draws upon transition metal-assisted coupling techniques to some extent, such as those described in WO2020164561. WO2020164561 discloses a transition metal-assisted coupling technique for producing highly homogeneous DAR4 (i.e., an ADC with a drug-to-resistance ratio of 4) ADC products, and this application incorporates the entire contents of WO2020164561 by reference. Compared to the traditional reduction-coupling method, the technique in WO2020164561 differs in that the two disulfide bonds in the hinge region are masked in the presence of transition metal ions (e.g., Zn(II)). Therefore, the ADC obtained after reduction coupling is primarily a DAR4-type ADC, with the loading being the free thiol groups released from the disulfide bonds in the Fab region.

[0063] This invention is based on the inventors’ discovery that disulfide bonds in the hinge region, which are masked and thus protected from reduction in transition metal-assisted coupling, can be selectively reduced under control to provide free thiol groups for subsequent coupling, thereby enabling the production of multi-load ADCs with a set drug loading configuration in a controllable manner.

[0064] Exemplary Implementation

[0065] 1. ADC Manufacturing Methods

[0066] In general, in a first aspect, a method for producing an antibody-drug conjugate is provided, comprising: a first reduction reaction step, wherein an antibody is reduced in the presence of a transition metal ion; and one or more coupling reaction steps, wherein the reduced antibody is coupled with a linker-load to generate an antibody-drug conjugate; wherein the method further comprises an optional second reduction reaction, wherein the antibody portion of the antibody-drug conjugate is further reduced, and wherein the second reduction reaction is followed by a coupling step; wherein the method further comprises a demasking treatment to remove the transition metal bound to the antibody, the treatment being performed simultaneously with at least one coupling step or the second reduction reaction step. The product of the final coupling step is an antibody-drug conjugate final product, and the method may optionally include a step of recovering the product of the final coupling reaction step as the antibody-drug conjugate final product.

[0067] This invention does not limit the antibodies that can be used; all antibodies intended for use in ADCs are acceptable. In some embodiments, antibodies containing interchain disulfide bonds in the Fab and hinge regions, like any IgG antibody, are suitable for this invention, especially antibodies with interchain disulfide bonds such as IgG1 and IgG4. In some embodiments, the antibody is an IgG1 or IgG4 antibody. In some embodiments, the antibody is a monoclonal antibody (mAb).

[0068] The term "antibody" broadly refers to a specific binding protein that can be used as the antibody portion in an ADC, especially a specific binding protein that has at least a hinge region with interchain disulfide bonds for coupling, such as the aforementioned IgG molecule. For example, for the purposes of this invention, in addition to the aforementioned IgG molecule, available antibody molecules also include engineered antibodies, such as bispecific or multispecific antibodies, such as antibodies with a WuXiBody structure (i.e., a multispecific antibody with an IgG-type structure in which the LC+CH1 segment of a pair of light-heavy chains (LC-HC) is replaced with a TCR constant region (e.g., Cα and Cβ)), and Fc fusion proteins, such as the VHH-Fc fusion. Specific examples of antibodies that can be used in this invention include, but are not limited to: trastuzumab, rituximab, cetuximab, adalimumab, alemtuzumab, atezolizumab, basiliximab, certolizumab, daclizumab, daratumumab, golimumab, ipilimumab, mepolizumab, necitumumab, obinutuzumab, and offatumumab.

[0069] In this document, the ordinal numbers "first," "second," and "third" preceding the connector-loads do not necessarily indicate that the connector-loads are different. In some embodiments, they represent connector-loads introduced in the first, second, and third coupling reactions, respectively. In some descriptions herein, they may also be used to represent three types of coupling sites, as detailed in the classification below. When connector-loads have different numbers (e.g., second connector-load and third connector-load) but are substantially the same, they may be grouped together and referred to by the smaller number (e.g., grouped together as the second connector-load).

[0070] In this document, the term "multi-load" as an adjective encompasses linker-loads with different linker structures, different drug components, or both, preferably with different drug components. In this document, "linker-load (LP)" refers to the load molecule itself capable of conjugating to an antibody without a linker. In this document, in context, "linker-load" can refer to the linker-load portion within an ADC that is a component of it, especially when subsequently described as "loaded / linked / bound to" an antibody, or it can refer to the corresponding complete linker-load molecule that forms the linker-load portion through conjugation with an antibody. In this document, "load" and "drug" are used interchangeably, both referring to therapeutic or diagnostic active reagents or components derived therefrom. Therefore, in context, a "multi-load" ADC includes ADCs containing different linker-loads (i.e., different in a broad sense) and ADCs containing different loads (i.e., different in a narrow sense). Available linker-loaded molecules include, but are not limited to: MC-vc-MMAE, MC-MMAF, MC-GGFG-DXd, MC-CL2A-SN38, mc-extecan, DBM-MMAF, DM21-glucose-DXd, YLlink-glucose-DXd, and MC-glucose-DXd. In some embodiments, thiol-bridged linkers-loaded molecules may be employed, such as those containing dibromomaleimide linkers, 4,5-dibromo-1,2-dihydropyrazine-3,6-dione linkers, or bismaleimide linkers.

[0071] The method of this invention enables the controlled production of multi-load ADCs with predetermined drug loading configurations, as well as high-purity ADC products of interest (hereinafter also referred to as "target products"). For example, the designed drug loading configurations include: "4+4", "4+2", "DAR4", "DAR6", "6+2", and "4+2+2", where each number represents the DAR value of the corresponding connector-load. In this document, as the context suggests, the DAR value refers to the theoretical value displayed or proven through measurement.

[0072] In this paper, "4+4 type" refers to an ADC with four first connector-load molecules connected to the Fab region and four second connector-load molecules connected to the hinge region; "4+2 type" refers to an ADC with four first connector-load molecules connected to the Fab region and two second connector-load molecules connected to the hinge region; "DAR4 type" refers to an ADC with two connector-load molecules connected to the Fab region and two of these connector-load molecules connected to the hinge region; "DAR6 type" refers to an ADC with four connector-load molecules connected to the Fab region and two of these connector-load molecules connected to the hinge region; "6+2 type" refers to an ADC with four first connector-load molecules connected to the Fab region, two first connector-load molecules connected to the first point of the hinge region, and two second connector-load molecules connected to the second point of the hinge region; "4+2+2 type" refers to an ADC with four first connector-load molecules connected to the Fab region, two second connector-load molecules connected to the first point of the hinge region, and two third connector-load molecules connected to the second point of the hinge region. Preferably, the first, second, and optional third connectors have different or identical loads; more preferably, the drug portions have different or identical loads. Even more preferably, the first, second, and third connectors have different loads; most preferably, the drug portions are different.

[0073] Specifically, in the first embodiment, the method of the present invention includes:

[0074] The first reduction reaction step, wherein the antibody is reduced in the presence of transition metal ions;

[0075] In the first coupling reaction step, the antibody is coupled to the first linker-loader to generate a first antibody-drug conjugate.

[0076] The second reduction reaction step is combined with a simultaneous demasking process, in which the antibody portion of the antibody-drug conjugate is further reduced; and

[0077] In the second coupling reaction step, the antibody portion of the first antibody-drug conjugate that has been further reduced is coupled to a second linker-loader to generate a second antibody-drug conjugate.

[0078] In some cases, the above method can produce a 4+4 type multi-load ADC, where four first connector-load molecules are loaded into the Fab region in the first coupling reaction step, and four second connector-load molecules are loaded into the hinge region in the second coupling reaction step. A schematic diagram of this process is shown in the figure. Figure 1 Option 2.

[0079] First reduction reaction in the presence of transition metal ions

[0080] In this embodiment of the method of the present invention, and in all embodiments thereafter, in the first reduction reaction, the antibody is partially reduced by a reducing agent in the presence of transition metal ions, thereby opening two interchain disulfide bonds (SS) in the Fab region. The partially reduced antibody has four free thiol groups released from the interchain disulfide bonds at cysteine ​​residues, for coupling with the linker-loader. The partial reduction in the presence of transition metal ions can be performed according to WO2020164561, the entire contents of which are incorporated herein by reference.

[0081] Specifically, this invention can utilize any ADC to produce a usable reducing agent, such as tris(2-carboxyethyl)phosphine (TCEP), diphenylarsine (DPAA), diPPB (2-diphenylphosphinebenzenesulfonic acid), 2-diphenylphosphinebenzenesulfonic acid (diPPBs), dithiothreitol (DTT), dithioerythritol (DTE), β-mercaptoethanol, LiAlH4, Na2S2O3, KBH4, and hydrazine. The reducing agent / mAb ratio can be 1-20, for example 2-16, 3-10, 4, 6, 8, 10, or 14. Transition metal ions usable in this invention include, but are not limited to, Zn. 2+ (Zn(II)), Mn 2+ (Mn(II)), Ni 2+ (Ni (II)), Fe 2+ (Fe(II)), Fe 3+ (Fe (III)), Ca 2+ (Ca (II)) and Cu 2+(Cu(II)). A mixture of transition metal ions may also be used. Transition metal ions can be added in the form of soluble salts, such as halides (e.g., ZnCl2, MnI2, CuBr2), sulfates (e.g., ZnSO4), nitrates (e.g., Zn(NO3)2), acetates (e.g., Zn(CH3COO)2), formates, and tetrafluoroborates. The ratio of ions to antibodies in the presence of these ions can be 0.5–50, for example 1–16, 2–8, or for example 4, 5, 6. The reaction can be carried out at temperatures of approximately 0–37°C (e.g., 2–25°C, 4°C, 12°C, 16°C, or 22°C) in a suitable buffer solution (e.g., pH approximately 6–8, such as approximately pH 6.5, pH 7.0, or pH 7.5; salt concentration approximately 20–80 mM, such as approximately 40 mM buffer) for 1–24 hours (e.g., approximately 12, 16, or 20 hours). Examples of reduction reaction buffers include, but are not limited to, PB, HEPES, MES, Tris, and MOPSO. Optionally, the reduction reaction buffer may contain additives such as Tween 20, benzyl alcohol, and trehalose as needed. The resulting reduction reaction solution contains a partially reducing antibody with four free thiol groups at the cysteine ​​site in its Fab region for coupling. This reduction reaction solution can be used directly for the next coupling step without removing the reducing agent, or a step to remove the reducing agent may be included as needed.

[0082] In some embodiments, the buffer for the first reduction reaction may be, for example, PB buffer (40 mM, pH 6.90) or HEPES buffer (40 mM, pH 7.00); the reducing agent / mAb ratio may be about 3–10, for example about 3.25 and about 10, and the reducing agent may be, for example, TCEP; and / or, the reaction may be carried out at 4–12°C for about 16–20 hours, for example, at about 4°C for about 16 hours or at about 12°C for about 20 hours.

[0083] First coupling reaction

[0084] The first coupling reaction can be performed according to standard practices in ADC production. Those skilled in the art know how to perform coupling based on the specific antibody, payload, or linker-load. For example, the linker-load / mAb ratio of the coupling reaction can be 6–20 (e.g., about 8, 10, 12, 14, or 16), and can be reacted in a buffer solution at a pH of about 4.0–8.0 at 0–37°C (e.g., 2–25°C, 4°C, 12°C, 16°C, or 22°C) for 1–4 hours (e.g., 1, 2, or 3 hours). In some embodiments, the first coupling reaction can be reacted at about 4–12°C for about 1–2 hours, for example, about 4°C for about 2 hours or about 12°C for about 1 hour. The first coupling reaction following the first reduction reaction produces an ADC with a first linker-load DAR value of 4, i.e., the first intermediate ADC.

[0085] Optionally, the resulting first coupling solution may be treated to remove any uncoupled linker-loads that may be present. This removal step is preferable if the linker-loads will be different in subsequent couplings. In some cases, removal can be achieved by incubation with a quencher, such as a cysteine ​​reagent, including cysteine ​​and its derivatives such as acetylcysteine ​​(NAC). Incubation can be performed under the same conditions as the coupling reaction for about 30–60 minutes (i.e., about 0.5–1 hour). In some embodiments, incubation can be performed at about 4–12°C for about 0.5 hours. The amount of quencher added can be in a ratio of 6–40 to the antibody, for example, 10, 12, 14, 16, or 21.

[0086] Optionally, the first intermediate ADC can be purified. Purification is preferred to remove present reaction reagents and enrich the ADC. In some cases, the intermediate ADC is purified or replaced with buffer using a desalting column. In some cases, the intermediate ADC is first removed before purification.

[0087] The second reduction reaction is combined with synchronous demasking.

[0088] This step includes (i) a second reduction reaction and (ii) a simultaneous demasking process. The term “second reduction (reaction)” refers only to the reduction reaction itself and does not include the simultaneous demasking process.

[0089] (i) Second reduction reaction

[0090] The second reduction reaction opens one or more remaining interchain disulfide bonds outside the Fab and masked regions as much as possible and as needed. In some embodiments, the transition metal ions used and applicable in the first reduction reaction are not present in the second reduction reaction. The second reduction reaction can be adapted. For example, the reducing agent / mAb ratio in the second reduction reaction can be about 2, 4, 6, or 8 lower than that in the first reduction reaction, such as about 6 or 8. Those skilled in the art can determine this ratio based on specific circumstances and practical needs. For example, the reducing agent / mAb ratio can be increased to 35 when using diPPBS. The temperature of the second reduction reaction can be higher than that of the first reduction reaction, such as about 4–37°C or room temperature (e.g., about 20–22°C) to 37°C, such as about 4°C, 12°C, 16°C, 22°C, or 37°C.

[0091] In some embodiments, the reducing agent for the second reduction reaction may be TCEP or DPAA, the reducing agent / mAb ratio may be about 6-8, and the reaction may be carried out at about 20-37°C (e.g., 22°C or 37°C) for about 4-16 hours, for example, about 16 hours at about 37°C or about 4 hours at about 22°C.

[0092] (ii) Synchronous demasking process

[0093] In this paper, the terms "joint" and "synchronous" in the context of demasking treatment both indicate that an operation or reaction occurs immediately after, simultaneously with, or overlaps with the demasking treatment, and depends on or utilizes the result of demasking. Therefore, in this step, the second reduction reaction can occur immediately after, simultaneously with, or overlap with the demasking treatment. The second reduction reaction utilizes the demasking result to produce a further reduced antibody with additional free cysteine ​​sites available for conjugation.

[0094] The demasking process of this invention stems from the inventors' discovery that some disulfide bonds in the hinge region (e.g., one of the SS bonds in the IgG1 and IgG4 hinge regions) that are masked in transition metal ion-assisted reduction reactions can be selectively opened by removing the transition metal ions chelated with thiol groups. The resulting free thiol groups can then be used for further coupling in a controlled manner. This has the advantage of enabling different couplings, thereby advancing the design and production of multi-load ADCs.

[0095] Demasking can be achieved through dechelation treatment. In this invention, demasking is performed using a chelating agent. In some cases, a second reduction reaction is carried out in the presence of a chelating agent. Examples of chelating agents include, but are not limited to, polyphosphates, aminocarboxylic acids, hydroxycarboxylic acids, polyamines, fatty amines, and o-phenanthroline derivatives, such as ethylenediamine, 2,2'-bipyridine, 1,10-o-phenanthroline, EDTA, EGTA, DTPA, etc. The chelating agent can be added in excess, for example, at a final concentration of 0.05–10 mM, such as 1–5 mM, 0.1 mM, 1 mM, 2 mM, 5 mM, and 8 mM.

[0096] Specifically, the second reduction reaction combined with simultaneous demasking can provide further reduced antibody (i.e., the antibody portion in the first ADC), which has free thiol groups at cysteine ​​sites masked in the presence of transition metal ions and at one or more other interchain SS bond sites. For example, with IgG1 or IgG4 antibodies, the second reduction reaction combined with simultaneous demasking opens two SS bonds in the hinge region, thereby obtaining four new coupling sites.

[0097] Second coupling reaction

[0098] Following the second reduction reaction and demasking treatment is the second coupling reaction, in which the second linker-loaded antibody is coupled to the further reduced antibody at a newly added coupling site to form a second ADC. For example, particularly with IgG1 and IgG4 antibodies, the second ADC can be a 4+4 type ADC, where the first "4" refers to the DAR value of the first linker-loaded molecule linked to the Fab region, and the second "4" refers to the DAR value of the second linker-loaded molecule linked to the hinge region. Preferably, the first and second loads in the resulting 4+4 type dual-loaded ADC are different.

[0099] The second coupling reaction can be the same as the first coupling reaction, except for some optional adjustments. For example, the linker-load / antibody ratio in the second coupling reaction can be lower than that in the first coupling reaction, corresponding to an excess of the stoichiometry or optional ratio of linker-load to antibody in the second coupling reaction, which can be, for example, 6-10. In some embodiments, the second coupling reaction can be carried out at around 4-22°C (e.g., around 4-12°C) for about 1-2 hours, for example, 2 hours at 4°C, 1 hour at 12°C, or 1 hour at 22°C.

[0100] Harvest

[0101] Optionally, the implementation may include a step of harvesting the product of the final coupling reaction step as the final antibody-drug conjugate product. A second ADC (i.e., a 4+4 multi-load ADC) may be harvested as the final ADC product. The final ADC product may be purified to enrich the target product. Various ADC purification techniques may be employed. Available purification techniques include, but are not limited to, filtration, ultrafiltration, dialysis, UF-DF (ultrafiltration-dialysis), desalting, buffer replacement, chromatography (such as HIC), or combinations thereof. In some cases, the final product is buffer-replaced using a desalting column. In some cases, the final product is further purified using chromatography, such as HIC-HPLC. The resulting product is an ADC product with high purity 4+4 multi-load ADC.

[0102] The above embodiments, especially the DAR type, are mainly for cases where the linker-load molecule reacts with the free thiol group on the antibody molecule in a 1:1 ratio (linker-load / free thiol group ratio). In some embodiments, the first and / or second linker-load molecule is a thiol-bridged linker-load capable of reacting with the free thiol group on the antibody molecule in a 1:2 ratio (linker-load / free thiol group ratio).

[0103] Therefore, when the first linker-loaded molecule in the first coupling reaction is a thiol-bridged linker-loaded molecule, an intermediate with two (2) first linker-loaded molecules loaded in the antibody Fab region is obtained (i.e., the aforementioned first antibody-drug conjugate); and / or, when the second linker-loaded molecule in the second coupling reaction is a thiol-bridged linker-loaded molecule, an intermediate with two (2) second linker-loaded molecules loaded in the antibody hinge region is obtained (i.e., the aforementioned second antibody-drug conjugate). Therefore, when the first or second coupling reaction or both reactions use thiol-bridged linker-loaded molecules, this process can produce 2s+4, 4+2s, or 2s+2s type multi-loaded ADCs, where the first number indicates the loading of the first linker-loaded portion of the antibody Fab region, and the second number indicates the loading of the second linker-loaded portion of the antibody hinge region. The superscript "s" indicates a thiol-bridged linker-loaded molecule.

[0104] In the second embodiment, the method of the present invention includes:

[0105] The first reduction reaction step involves the reduction of the antibody in the presence of transition metal ions;

[0106] In the first coupling reaction step, the antibody is coupled to a first linker-loader to form a first antibody-drug conjugate; and

[0107] The second coupling reaction step involves a combined synchronous demasking process, wherein the antibody portion of the first antibody-drug conjugate is coupled to a second linker-loader to generate a second antibody-drug conjugate.

[0108] This method can produce a 4+2 type multi-load ADC, in which four first linker-load molecules are loaded into the Fab region in the first coupling reaction step, and two second linker-load molecules are loaded into the hinge region in the second coupling reaction step. A schematic diagram of this process can be seen. Figure 1 Option 3-1.

[0109] The first coupling reaction following the first reduction reaction generates a first linker-loaded DAR4-type first intermediate ADC. Optionally, the intermediate ADC can be cleaned, purified, or both, as described in the first embodiment.

[0110] The second coupling reaction of joint synchronous demasking

[0111] This step includes a second coupling reaction and a simultaneous demasking process. The term "second coupling (reaction)" refers only to the coupling reaction itself and does not include the simultaneous demasking process.

[0112] In the second embodiment, there is no second reduction reaction before the second coupling reaction, and the second coupling reaction is followed by a simultaneous demasking process. In this embodiment, the antibody portion in the first antibody-drug conjugate is coupled with the second linker-loader to generate the second antibody-drug conjugate.

[0113] The simultaneous demasking process and the second coupling reaction are as described in the first embodiment. In some embodiments of the second embodiment, the second coupling reaction may be carried out at around 4-12°C for about 1-2 hours, for example, 4°C for 2 hours or 12°C for 1 hour.

[0114] The second coupling reaction can occur immediately after, simultaneously with, or overlap with the demasking process. In some cases, the second coupling reaction is carried out in the presence of a chelating agent. The second coupling reaction depends on demasking to provide additional free thiol groups for coupling. Therefore, the resulting second antibody-drug conjugate can be a 4+2 type multi-load ADC, wherein four first linker-load molecules are linked to the Fab region, and two second linker-load molecules are loaded at cysteine ​​sites in the hinge region provided by the demasking process. Preferably, the first and second loads are different in the resulting 4+2 type dual-load ADC.

[0115] Optionally, the implementation may include a step of harvesting the product of the final coupling reaction step as the final antibody-drug conjugate product. A second ADC (i.e., the 4+2 type multi-load ADC) may be harvested as the final ADC product. Optionally, the final ADC product may be purified as described in the first embodiment. The resulting product may be an ADC product with a high-purity 4+2 type multi-load ADC.

[0116] The above embodiments, especially the DAR type, are mainly for cases where the linker-load molecule reacts with the free thiol group on the antibody molecule in a 1:1 ratio (linker-load / free thiol group ratio). In some embodiments, the first and / or second linker-load molecule is a thiol-bridged linker-load that can react with the free thiol group on the antibody molecule in a 1:2 ratio (linker-load / free thiol group ratio). Therefore, when the first linker-load molecule in the first coupling reaction is a thiol-bridged linker-load molecule, an intermediate with two (2) first linker-load molecules in the antibody Fab region is obtained (i.e., the aforementioned first antibody-drug conjugate); and / or, when the second linker-load molecule in the second coupling reaction is a thiol-bridged linker-load molecule, an intermediate with one (1) second linker-load molecule in the antibody hinge region is obtained (i.e., the aforementioned second antibody-drug conjugate). Therefore, when the first or second coupling reaction, or both of these reactions, employ thiol-bridged linker-loaded molecules, this process can produce 2s+2, 4+1s, or 2s+1s type multi-loaded ADCs, where the first number represents the loading of the first linker-loaded portion of the antibody Fab region, and the second number represents the loading of the second linker-loaded portion of the antibody hinge region.

[0117] In the third embodiment, the method of the present invention includes:

[0118] The first reduction reaction step, wherein the antibody is reduced in the presence of transition metal ions;

[0119] In the first coupling reaction step, the antibody is coupled to the first linker-loader to generate a first antibody-drug conjugate.

[0120] The second coupling reaction step of the combined synchronous demasking process, wherein the antibody portion of the first antibody-drug conjugate is coupled with a second linker-loader to generate a second antibody-drug conjugate;

[0121] In the second reduction reaction step, the antibody portion of the second antibody-drug conjugate is further reduced; and

[0122] The third coupling reaction step involves coupling the antibody portion of the second antibody-drug conjugate with a third linker-loader to generate the third antibody-drug conjugate.

[0123] Compared to the second embodiment, this implementation further includes a second reduction reaction and a subsequent third coupling reaction after the combined simultaneous demasking second coupling reaction. A schematic diagram of this process is shown below. Figure 1 Option 3-2. Therefore, it is preferable to perform a purging process on the second intermediate ADC to remove residual uncoupled second linker-loads. Optionally, and preferably, the 4+2 type second intermediate ADC can be purified as described in the first embodiment.

[0124] The second reduction and third coupling reactions can be performed with reference to the second reduction and second coupling reactions in the first embodiment. The reducing agent in the second reduction reaction can be TCEP or DPAA, the reducing agent / mAb ratio can be about 6–10, and the reaction can be carried out at about 22°C–37°C for about 4–16 hours. In a preferred embodiment, the second reduction reaction can be carried out in the presence of a chelating agent such as EDTA to eliminate the risk of metal ion-mediated oxidation, excluding those available in the first reduction reaction. The third coupling reaction then loads additional linker-loads onto the remaining cysteine ​​sites of the SS bonds in the hinge region released by the second reduction reaction. For example, the resulting third ADC can be a 4+2+2 type triple-load ADC, wherein four first linker-load molecules are linked to the Fab region, two second linker-load molecules are linked to the demasked cysteine ​​sites in the hinge region, and two third linker-load molecules are linked to other cysteine ​​sites in the hinge region. Optionally, this embodiment may include a step of harvesting the product of the final coupling reaction step as the final antibody-drug conjugate product. A 4+2+2 type third ADC can be harvested as the ADC final product. Optionally, it can be purified as described in the first embodiment. The obtained product can be an ADC product with high purity 4+2+2 type multi-load ADC.

[0125] The above embodiments, especially the DAR type, are mainly for cases where the linker-load molecule reacts with the free thiol group on the antibody molecule in a 1:1 ratio (linker-load / free thiol group ratio). In some embodiments, the first and / or second linker-load molecule is a thiol-bridged linker-load capable of reacting with the free thiol group on the antibody molecule in a 1:2 ratio (linker-load / free thiol group ratio). Therefore, when the first linker-loaded molecule in the first coupling reaction is a thiol-bridged linker-loaded molecule, an intermediate with two (2) first linker-loaded molecules in the antibody Fab region is obtained (i.e., the aforementioned first antibody-drug conjugate); and / or, when the second linker-loaded molecule in the second coupling reaction is a thiol-bridged linker-loaded molecule, an intermediate with one (1) second linker-loaded molecule in the antibody hinge region is obtained (i.e., the aforementioned second ADC); and / or, when the third linker-loaded molecule in the third coupling reaction is a thiol-bridged linker-loaded molecule, an intermediate with one (1) third linker-loaded molecule in the antibody hinge region is obtained (i.e., the aforementioned third antibody-drug conjugate). Therefore, when one or more of the first, second, and third coupling reactions employ thiol-bridged linkers-loaded molecules, this method can produce 2s+2+2, 2s+2+1s, 2s+1s+2, 2s+1s+1s, 4+2+1s, 4+1s+2, or 4+1s+1s type multi-loaded ADCs, where the first number refers to the loading of the first linker-loaded portion of the antibody Fab region, the second number refers to the loading of the second linker-loaded portion of the antibody hinge region, and the third number refers to the loading of the third linker-loaded portion of the hinge region.

[0126] In the fourth embodiment, the method of the present invention includes:

[0127] The first reduction reaction step, wherein the antibody is reduced in the presence of transition metal ions; and

[0128] The first coupling reaction step of the combined synchronous demasking process, wherein the antibody is coupled with a first linker-loader to generate a first antibody-drug conjugate.

[0129] In some embodiments of the fourth implementation, in the first reduction reaction, the buffer solution may be, for example, PB (40 mM, pH 6.90), HEPES (20–80 mM, pH 6.8–8.0), Tris (40 mM, pH 7.0), MES (40 mM, pH 6.5), or MOPSO (40 mM, pH 7.0); the reducing agent / mAb ratio may be 2–14, for example 2.75, 3.00, 3.25, 3.75, 4, 8, or 14, and the reducing agent may be, for example, TCEP; and / or, the reaction may be carried out at about 4–22°C for about 16–20 hours, for example, 16 hours at 4°C, 20 hours at 12°C, 16 hours at 16°C, or 16 hours at 22°C.

[0130] The key feature of this process is the combination of the first coupling reaction step and the simultaneous demasking process. This step includes the first coupling reaction and the simultaneous demasking process. The term "first coupling (reaction)" refers only to the coupling reaction itself and does not include the simultaneous demasking process.

[0131] A schematic diagram of this method's process can be seen. Figure 1 Scheme 1-1. In this method, the reduced reaction solution formed by the first reduction reaction can be directly used for the first coupling reaction in conjunction with simultaneous demasking without removing the reducing agent. Both the first coupling reaction and the simultaneous demasking treatment are as described in the first embodiment. In some cases, the first coupling reaction is carried out in the presence of a chelating agent. Utilizing the demasking effect, the first coupling reaction generates a DAR6-type ADC, in which four first linker-loaded molecules are attached to the Fab region, and two first linker-loaded molecules are loaded into the hinge region to demask the released cysteine ​​sites.

[0132] In some embodiments of the fourth embodiment, during the demasking process, the chelating agent may be added in excess, for example, at a final concentration of 2-5 mM, and the chelating agent may be, for example, EDTA; and / or, the first coupling reaction may be carried out at about 4-22°C for about 1-2 hours, for example, at about 4°C for about 2 hours, at about 12°C for about 1 hour, at about 16°C for about 1 hour, or at about 22°C for about 1 hour. Optionally, the resulting first coupling solution may be treated to remove any residual uncoupled linker-loads that may be present. In some embodiments of the fourth embodiment, a quencher such as NAC may be added at a ratio of 10-16 to the antibody, and / or the reaction may be carried out at about 4-22°C (e.g., about 4°C, 12°C, 16°C, or 22°C) for 0.5 hours.

[0133] Optionally, the implementation may include a step of harvesting the product of the final coupling reaction step as the final antibody-drug conjugate product. The first DAR6-type ADC can be harvested as the final ADC product, and optionally, it can be purified as described in the first embodiment. The advantage of this method is that it can produce ADC products with high-purity DAR6-type ADCs.

[0134] The above-described embodiments, especially the DAR type, primarily address the scenario where the linker-loador molecule reacts with the free thiol group on the antibody molecule at a 1:1 ratio (linker-loador / free thiol group ratio). In some embodiments, the linker-loador molecule is a thiol-bridged linker-loador molecule, which can couple with the free thiol group at a 1:2 ratio (linker-loador / free thiol group ratio). Therefore, as... Figure 2 As shown in Figure (A), when thiol-bridged linker-loaded molecules are used as linkers-loaded molecules in the coupling reaction, this method can generate DAR3-type ADCs with two linker-loaded molecules in the antibody Fab region and one linker-loaded molecule in the hinge region.

[0135] The above-described embodiments, especially the DAR type, are primarily IgG antibodies with two interchain disulfide bonds in the two Fab regions and two interchain disulfide bonds in the hinge region. In some embodiments, the antibody can be an engineered antibody with more or fewer disulfide bonds in the Fab region, thereby obtaining more DAR types.

[0136] For example, Figure 2 In the embodiment shown in Figure (B), the antibody is a VHH-Fc protein. The linker-loaded molecule reacts with the free thiol group for coupling at a 1:1 ratio (linker-loaded / free thiol group ratio). Therefore, the target product of this embodiment is a DAR4-type ADC with two linker-loaded molecules loaded in the Fab region and two linker-loaded molecules loaded in the hinge region. Accordingly, when the first or second coupling reaction, or both reactions, use thiol-bridged linker-loaded molecules, this process can produce multi-loaded ADCs of the 1s+2, 2+1s, or 1s+1s type, where the first number represents the loading of the first linker-loaded portion in the Fab region, and the second number represents the loading of the second linker-loaded portion in the hinge region.

[0137] Figure 2In the embodiment shown in Figure (C), the antibody is a bispecific antibody with a WuXiBody structure, in which the LC+CH1 segment of one of the light-heavy chain pairs (LC-HC) is replaced with a TCR constant region (such as Cα and Cβ). In this embodiment, the target product is a DAR4-type ADC, where the Fab region of the other LC-HC pair carries two linker-loaded molecules, and the hinge region carries two linker-loaded molecules. Therefore, when the first or second coupling reaction, or both reactions, employ thiol-bridged linker-loaded molecules, this process can produce multi-loaded ADCs of the 1s+2, 2+1s, or 1s+1s type, where the first number indicates the loading of the first linker-loaded portion in the Fab region, and the second number indicates the loading of the second linker-loaded portion in the antibody hinge region.

[0138] In the fifth embodiment, the method of the present invention includes:

[0139] The first reduction reaction step, wherein the antibody is reduced in the presence of transition metal ions;

[0140] The first coupling reaction step of the combined synchronous demasking process, wherein the antibody is coupled with the first linker-loader to generate the first antibody-drug conjugate;

[0141] In the second reduction reaction step, the antibody portion of the first antibody-drug conjugate is further reduced; and

[0142] The second coupling reaction step involves coupling the antibody portion of the first antibody-drug conjugate with a second linker-loader to generate a second antibody-drug conjugate.

[0143] Compared to the fourth embodiment, this method further includes a second reduction reaction and a subsequent second coupling reaction after the first coupling reaction for simultaneous demasking. A schematic diagram of this method's process is shown below. Figure 1 Schemes 1-2. Therefore, it is preferable to purge the first solution to remove residual uncoupled first linker-loads, as described in the first embodiment. Optionally, and preferably, the DAR6-type first intermediate ADC can be purified, as described in the first embodiment.

[0144] The second reduction reaction and the second coupling reaction are both as described in the first embodiment. In some embodiments of the fifth embodiment, the reducing agent for the second reduction reaction may be TCEP or DPAA, the reducing agent / mAb ratio may be about 6-10, and the reaction may be carried out at about 22°C-37°C for about 4-16 hours.

[0145] In a preferred embodiment, the second reduction reaction can be carried out in the presence of a chelating agent such as EDTA to eliminate the risk of metal ion-mediated oxidation, excluding those available in the first reduction reaction. The second coupling reaction then loads additional linker-loads onto the remaining cysteine ​​sites of the SS bonds in the hinge region released by the second reduction reaction. For example, the resulting second ADC can be a 6+2 type dual-load ADC, wherein four first linker-load molecules are linked to the Fab region, two first linker-load molecules are linked to the demasked cysteine ​​sites in the hinge region, and two second linker-load molecules are linked to other cysteine ​​sites in the hinge region. Optionally, this embodiment includes a step of harvesting the product of the final coupling reaction step as the final antibody-drug conjugate product. The 6+2 type second ADC can be harvested as the final ADC product, and optionally, it can be purified as described in the first embodiment. The resulting product can be an ADC product with a high-purity 6+2 type multi-load ADC.

[0146] The above-described embodiments, especially the DAR type, primarily address the scenario where the linker-loador molecule reacts with the free thiol group on the antibody molecule at a 1:1 ratio (linker-loador / free thiol group ratio). In some embodiments, the first and / or second linker-loador molecules are thiol-bridged linker-loadors capable of reacting with the free thiol group at a 1:2 ratio (linker-loador / free thiol group ratio). Therefore, when the first linker-loador molecule in the first coupling reaction is a thiol-bridged linker-loador molecule, an intermediate is obtained with two first linker-loador molecules in the antibody Fab region and one first linker-loador molecule in the hinge region (i.e., the aforementioned first antibody-drug conjugate); and / or, when the second linker-loador molecule in the second coupling reaction is a thiol-bridged linker-loador molecule, an intermediate is obtained with one second linker-loador molecule in the antibody hinge region (i.e., the aforementioned second ADC). Therefore, when the first or second coupling reaction, or both of these reactions, employ thiol-bridged linker-loaded molecules, this process can produce multi-loaded ADCs of the 3s+2, 6+1s, or 3s+1s type, where the first number indicates the loading of the first linker-loaded portion of the antibody Fab region, and the second number indicates the loading of the second linker-loaded portion of the antibody hinge region.

[0147] 2. Antibody-drug conjugates

[0148] In a second aspect of this disclosure, this application provides antibody-drug conjugate (ADC) products containing high-purity target products, with a purity of at least 55 mol%, at least 60 mol%, at least 65 mol%, at least 68 mol%, at least 70 mol%, at least 75 mol%, at least 80 mol%, at least 85 mol%, at least 87 mol%, at least 88 mol%, at least 90 mol%, at least 93 mol%, at least 95 mol%, at least 97 mol%, at least 98 mol%, or at least 99 mol%.

[0149] In one implementation, the target product, i.e., the target ADC, contains four first connector-load molecules connected to the Fab region and four second connector-load molecules connected to the hinge region. Specifically, the target product can be a DAR6-type ADC, a 4+4-type dual-load ADC, a 4+2-type dual-load ADC, a 6+2-type dual-load ADC, or a 4+2+2-type triple-load ADC.

[0150] In another embodiment, the target product, i.e. the target ADC, contains three or six first connector-load molecules and optionally one or two second connector-load molecules, wherein the first and second connector-load molecules are the same or different.

[0151] In another embodiment, the target product, i.e. the target ADC, contains two or four first linker-loaders linked to the antibody Fab region and at least one (e.g., one, two, three, or four) second linker-loaders linked to the antibody hinge region, and optionally contains one or two third linker-loaders linked to the antibody hinge region; wherein the first, second, and third linker-loaders are the same as each other, not all the same, or different from each other.

[0152] In another embodiment, the ADC product is manufactured using the method of the present invention described above.

[0153] In this article, "antibody-drug conjugate product" refers to a collection of ADC molecules, wherein the drug loading patterns of the ADC molecules are either consistent (i.e., homogeneous) or inconsistent (i.e., heterogeneous). In this article, the "purity" of an ADC product refers to the percentage of the target product in the ADC collection. Purity can be determined using methods known in the art, such as hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC), hydrophobic interaction chromatography-high performance liquid chromatography (PLRP-HPLC), and liquid chromatography-mass spectrometry (LC-MS).

[0154] In some embodiments, the target ADC is a 4+4 dual-load ADC, containing four first connector-load molecules connected to the Fab region and four second connector-load molecules connected to the hinge region, wherein the first connector-load molecules and the second connector-load molecules are different. In one embodiment, the ADC product of the present invention contains a 4+4 dual-load ADC with a purity of at least 68 mol%, at least 72 mol%, at least 73 mol%, at least 74 mol%, at least 76 mol%, at least 77 mol%, at least 88 mol%, at least 93 mol%, at least 95 mol%, at least 97 mol%, at least 98 mol%, or at least 99 mol%.

[0155] In some implementations, the target ADC is 2 s +4, 4+2 s or 2 s +2 s The term "multi-load ADC" refers to a multi-load ADC where the first number indicates the loading of the first linker-load portion of the antibody Fab region, and the second number indicates the loading of the second linker-load portion of the antibody hinge region. One or both of the first and second linker-load portions are formed by thiol-bridged linker-load molecules. In this paper, the linker-load portion formed by linker-load molecules in the ADC refers to the portion of the original linker-load molecule that has been incorporated into the ADC and become part of it. The superscript "s" indicates a thiol-bridged linker-load.

[0156] In some embodiments, the target ADC is a 4+2 type dual-load ADC, which contains four first connector-load molecules connected to the Fab region and two second connector-load molecules connected to the hinge region, wherein the first connector-load molecules and the second connector-load molecules are different. In one embodiment, the ADC product of the present invention contains a 4+2 type dual-load ADC with a purity of at least 61 mol%, at least 62 mol%, at least 64 mol%, at least 69 mol%, at least 76 mol%, at least 84 mol%, at least 87 mol%, or at least 88 mol%.

[0157] In some implementations, the target ADC is 2 s +2, 4+1 s or 2 s +1 s A multi-load ADC, wherein the first number represents the loading of the first linker-load portion of the antibody Fab region, and the second number represents the loading of the second linker-load portion of the antibody hinge region, wherein one or both of the first and second linker-loads are formed by thiol-bridged linker-load molecules.

[0158] In some embodiments, the target ADC is a 4+2+2 type triple-load ADC, which contains four first connector-load molecules connected to the Fab region, two second connector-load molecules connected to the hinge region, and two third connector-load molecules connected to the hinge region, wherein the first, second, and third connector-load molecules are all different. In one embodiment, the ADC product of the present invention contains a 4+2+2 type triple-load ADC with a purity of at least 60 mol%, at least 70 mol%, and at least 90 mol%.

[0159] Therefore, the target ADC is 2 s +2+2、2 s +2+1 s 2 s +1 s +2、2 s +1 s +1 s 4+2+1 s 4+1 s +2 or 4+1 s +1 s A multi-load ADC, wherein the first number refers to the load of the first linker-load portion of the antibody Fab region, the second number refers to the load of the second linker-load portion of the antibody hinge region, and the third number refers to the load of the third linker-load portion of the hinge region, and wherein one, two, or three of the first, second, and third linker-load portions are formed by thiol-bridged linker-load molecules.

[0160] In one embodiment, the target ADC is a DAR6-type ADC, which contains four linker-load molecules connected to the Fab region and two linker-load molecules connected to the hinge region. In another embodiment, the ADC product of this invention contains a DAR6-type ADC with a purity of at least 74 mol%, at least 75 mol%, at least 76 mol%, at least 77 mol%, at least 78 mol%, at least 79 mol%, at least 81 mol%, and at least 90 mol%.

[0161] In some embodiments, the target ADC is a DAR3-type ADC, wherein the antibody Fab region carries two linker-loaders and the hinge region carries one linker-loader, wherein the linker-loader portion is formed by thiol-bridged linker-loaders. In some embodiments, the ADC product of the present invention contains at least 60 mol% or at least 70 mol% of the DAR3 product in purity.

[0162] In some embodiments, the target ADC is a DAR4 product containing VHH-Fc protein, comprising two linker-loaded molecules loaded in the Fab region of the VHH-Fc protein and two linker-loaded molecules loaded in the hinge region, wherein the linker-loaded portion is formed by reacting a linker-loaded molecule with a free thiol-donating coupling group at a linker-load / free thiol ratio of 1:1. In some embodiments, the ADC product of the present invention contains at least 60 mol%, at least 65 mol%, at least 70 mol%, or at least 75 mol% of DAR4 product with a purity of at least 60 mol%, at least 65 mol%, at least 70 mol%, or at least 75 mol%.

[0163] In some embodiments, the target ADC is a DAR4 product containing a WuXiBody structure bispecific antibody, comprising two linker-loaded molecules loaded in the Fab region of one of the LC-HC pairs and two linker-loaded molecules loaded in the hinge region. The WuXiBody structure is characterized in that the LC+CH1 segment of one pair of paired light-heavy chains (LC-HC) is replaced with a TCR constant region (e.g., Cα, Cβ), while retaining the Fab region of the other LC-HC pair. Furthermore, the linker-loaded portion is formed by linker-loaded molecules reacting with free thiol-donating molecules at a linker-load / free thiol ratio of 1:1. In some embodiments, the ADC product of the present invention contains at least 60 mol%, at least 65 mol%, at least 70 mol%, or at least 75 mol% of the DAR4 product with a purity of at least 60 mol%, at least 65 mol%, at least 70 mol%, or at least 75 mol%.

[0164] In some implementations, the target ADC is 1 s +2, 2+1 s Or 1 s +1 s A multi-load ADC of type, wherein the first number represents the loading of the first linker-load portion of the antibody Fab region, and the second number represents the loading of the second linker-load portion of the antibody hinge region, wherein one or both of the first and second linker-loads are formed by thiol bridge linker-load molecules.

[0165] In some embodiments, the target ADC is a 6+2 type dual-load ADC, which contains four first connector-load molecules connected to the Fab region, two first connector-load molecules connected to the hinge region, and two second connector-load molecules connected to the hinge region, wherein the first connector-load molecules and the second connector-load molecules are different. In one embodiment, the ADC product of the present invention contains a 6+2 type dual-load ADC with a purity of at least 60 mol%, at least 64 mol%, at least 65 mol%, at least 66 mol%, at least 67 mol%, or at least 74 mol%.

[0166] In some implementations, the target ADC is 3 s +2, 6+1 s Or 3s +1 s A multi-load ADC, wherein the first number represents the load of a first linker-load in the antibody Fab region and hinge region, and the second number represents the load of a second linker-load in the antibody hinge region, wherein one or both of the first and second linker-loads are formed by thiol-bridged linker-load molecules.

[0167] This application also provides an ADC product produced by the method of the present invention, containing the target product of the DAR type described above, and optionally having the purity described above.

[0168] Example

[0169] The following examples are for illustrative purposes only and do not constitute a limitation on the scope of the present invention.

[0170] The antibodies used in the examples are as follows:

[0171] mAb 1 = Trastuzumab, mAb 2 = Rituximab, mAb 3 = Cetuximab, mAb 4 = Adalimumab, mAb 5 = Alemtuzumab, mAb 6 = Atezolizumab, mAb 7 = Basiliximab, mAb 8 = Certolizumab, mAb 9 = Daclizumab, mAb 10 = Daratumumab, mAb 11 = Golimumab, mAb 12 = Ipilimumab, mAb 13 = Mepolizumab, mAb 14 = Necitumumab, mAb 15 = Obinutuzumab, mAb 16 = Ofatumumab, mAb 17 = Anti-claudin 18.2 Fc fusion protein (VHH-FC) (cAb17), mAb 18 = W329001-U5T5.E17R-57.uIgG1, i.e., cAb18 (WuXiBody).

[0172] Example 1: Production of a 4+4 Dual-Load ADC

[0173] This embodiment describes... Figure 1 An exemplary process of Scheme 2.

[0174] Process 1

[0175] In PB buffer (40 mM, pH 6.90), 5 mg / mL mAb1 (trastuzumab, light chain as shown in SEQ ID NO:1, heavy chain as shown in SEQ ID NO:2, WuXi Biologics) was reduced with TCEP (C4706-2G, Sigma-Aldrich) in the presence of ZnCl2 (14422-500G, Sigma-Aldrich) (Zn(II) / mAb molar ratio = 10) at 4°C for 16 h. The reduction reaction solution containing the reducing antibody was used directly for the next coupling step without removing the TCEP.

[0176] A DMA (dimethylacetamide, ARK2190-1L, SAFC) solution (10 mg / mL) containing linker-loading 1 was added to the reduction reaction solution and mixed. The linker-loading / mAb (LP / mAb) molar ratio was 12.0, and the first coupling reaction was carried out. The coupling reaction was carried out at 4 °C for 2 hours to obtain mAb1 intermediate ADC with four linker-loading 1 molecules coupled together.

[0177] Acetylcysteine ​​(NAC) (A9165-100G, Sigma-Aldrich) (10mM stock solution prepared with ddH2O, NAC / mAb molar ratio = 16) was added to the first coupling reaction solution containing intermediate ADC, and the reaction was carried out at 4°C for 0.5 hours to remove excess linker-loading.

[0178] Next, the intermediate was purified by centrifugation and desalting column (40 kDa, 0.5 mL, REF: 87766, batch number SJ251704, manufacturer: Thermofisher) with PB buffer (40 mM, pH 6.90).

[0179] Concentrated EDTA (EDTA∙2Na, 10009719, SCR) (50 mM stock solution prepared with ddH2O) was added to a final concentration of 5 mM in PB buffer (40 mM, pH 6.90) containing the purified intermediate ADC. TCEP (TCEP / mAb molar ratio = 6) was then added to the EDTA-treated intermediate to initiate a second reduction reaction at 37°C for 16 hours. The resulting second reduction reaction solution containing the further reduced antibody could be used directly for the next conjugation step without removing TCEP.

[0180] Add the DMA solution of linker-load 2 (total DMA 10% v / v, linker-load / mAb molar ratio = 10) to the second reduction reaction solution, and then couple the reaction at 4°C for 2 hours.

[0181] The coupling product was transferred to preservation solution (20 mM histidine-acetic acid buffer, pH 5.5) using a centrifugal desalting column (40 kDa, 0.5 mL, REF: 87766, batch number SJ251704, manufacturer: Thermofisher). The identification results of the crude product are shown in Table 1-1 and... Figures 3 to 7 .

[0182] Table 1-1: PLRP-HPLC purity of 4+4 dual-loaded molecules in crude product

[0183]

[0184] The crude product was further purified using an AKTA pure 25M system for preparative-grade HIC purification. All chromatographic steps were performed at room temperature. A ProPac™ HIC-10, 7.8 x 75 mm column (Thermo Scientific) was equilibrated with 5 column volumes of buffer A (0.7 M (NH₄)₂SO₄, 25 mM PB, pH 6.5). To prepare a sample for column loading, 1 mL of the crude product (3.5 mg / mL) containing the 4+4 dual-loaded ADC mAb₁-4MMAE-4GGFG-DXd was mixed with an equal volume of buffer A. After loading, the column was washed with buffer A to a baseline of A280 nm. Elution was then performed using a 0-100% gradient of buffer B (25 mM phosphate buffer, 25% isopropanol, pH 6.5) in buffer A, followed by 3 CV of 100% buffer B, collecting the eluent at 280 nm, 248 nm, and 370 nm. The collected target components were subjected to buffer exchange via a centrifugal desalting column (40 kDa, 0.5 mL, serial number: 87766, batch number: SJ251704, manufacturer: Thermo Fisher) to replace the buffer with PB buffer (40 mM PB, pH 6.90; Na2HPO4 24.4 mM, NaH2PO4 15.6 mM).

[0185] The purity of the HIC purified product was determined by HIC-HPLC and LC-MS, and the results are shown in Tables 1-2 and 2-3. Figures 8A-8B The purified ADC was characterized by LC-MS (Agilent 1290 Infinity II; QTOF: Agilent 6530 LC\Q-TOF).

[0186] Table 1-2: Purity of 4+4 dual-loaded molecules in HIC purified products

[0187]

[0188] The above process was repeated by replacing mAb1 with mAb2 (rituximab, light chain as shown in SEQ ID NO:3, heavy chain as shown in SEQ ID NO:4). The characterization of the resulting products is shown in Tables 1-3 and 1-4. Figures 9 to 13 .

[0189] Table 1-3: PLRP-HPLC purity of 4+4 dual-loaded molecules in crude product

[0190]

[0191] The crude product was purified using preparative-grade HIC as described above.

[0192] Process 2

[0193] In HEPES buffer (40 mM, pH 6.90), 5 mg / mL mAb1 / mAb2 / mAb3 was reduced with TCEP (C4706-2G, Sigma-Aldrich) in the presence of ZnCl2 (14422-500G, Sigma-Aldrich), with a Zn(II) / mAb molar ratio of 4 and a TCEP / mAb molar ratio of 3.25, at 12°C for 22 hours. The reduction reaction solution containing the reducing antibody was used directly for the next coupling step without removing the TCEP.

[0194] A DMA (dimethylacetamide, ARK2190-1L, SAFC) solution (10 mg / mL) containing linker-loading 1 was added to the reduction reaction solution and mixed. The linker-loading / mAb (LP / mAb) molar ratio was 12.0, and the first coupling reaction was carried out. The coupling reaction was carried out at 12 °C for 1 hour to obtain mAb1 intermediate ADC with four linker-loading 1 molecules coupled together.

[0195] Acetylcysteine ​​(NAC) (A9165-100G, Sigma-Aldrich) (10mM stock solution prepared with ddH2O, NAC / mAb molar ratio = 16) was added to the first coupling reaction solution containing intermediate ADC, and the reaction was carried out at 12℃ for 0.5 hours to remove excess linker-loading.

[0196] Next, the intermediate was purified by centrifugation and desalting column (40 kDa, 0.5 mL, REF: 87766, batch number SJ251704, manufacturer: Thermofisher) with PB buffer (40 mM, pH 6.90).

[0197] Concentrated EDTA (EDTA∙2Na, 10009719, SCR) (50 mM stock solution prepared with ddH2O) was added to HEPES buffer (40 mM, pH 6.90) containing the purified intermediate to a final concentration of 5 mM. TCEP (TCEP / mAb molar ratio = 6) was then added to the EDTA-treated intermediate to initiate a second reduction reaction at 22°C for 16 hours. The resulting second reduction reaction solution containing the further reducing antibody was used directly for the next conjugation step without the need to remove TCEP.

[0198] Add the DMA solution of linker-load 2 (total DMA 10% v / v, linker-load / mAb molar ratio = 10) to the second reduction reaction solution, and then couple the reaction at 12°C for 1 hour.

[0199] The coupling product was transferred to storage buffer (20 mM histidine-acetic acid buffer, pH 5.5) using a centrifugal desalting column (40 kDa, 0.5 mL, REF: 87766, batch number SJ251704, manufacturer: Thermofisher). The identification results of the crude product are shown in Tables 1-4 and... Figures 14A to 14E .

[0200] Table 1-4: PLRP-HPLC purity of 4+4 dual-loaded molecules in crude product

[0201]

[0202] Process 3

[0203] In PB buffer (40 mM, pH 6.90), 5 mg / mL mAb1 was reduced with TCEP in the presence of ZnCl2, with a Zn(II) / mAb molar ratio of 4 and a TCEP / mAb molar ratio of 3.25, and the reaction was carried out at 12°C for 20 hours. The reduction reaction solution containing the reducing antibody was used directly for the next coupling step without removing the TCEP.

[0204] A DMA solution (10 mg / mL) containing linker-load 1 was added to the reduction reaction solution and mixed. The molar ratio of linker-load / mAb (LP / mAb) was 12.0, and the first coupling reaction was carried out. The coupling reaction was carried out at 12 °C for 1 hour to obtain mAb1 intermediate ADC with four linker-load 1 molecules coupled together.

[0205] NAC is added to the first coupling reaction solution containing the intermediate ADC to remove excess linker-load, as described in process 1.

[0206] The crude intermediate was then purified using a preparative-grade HIC system on an AKTA pure 25M system. All chromatographic steps were performed at room temperature. A 1 mL HiTrap ButylHP column (Cytiva) (Thermo Scientific) was equilibrated with more than 5 column volumes of buffer A (0.7 M (NH4)2SO4 and 25 mM PB, pH 6.5). To prepare a sample for column loading, 1 mL of the crude intermediate (3.5 mg / mL) containing the four-loaded ADC (mAb1-4MMAE) was mixed with an equal volume of buffer A. After loading, the column was washed with buffer A to a baseline of A280 nm. Elution was then performed using a 0-100% gradient of buffer B (25 mM phosphate buffer, 25% isopropanol, pH 6.5) in buffer A at 50 column volumes. The eluent was then collected at 280 nm, 248 nm, and 370 nm using 3 CV of 100% buffer B. The coupling product was replaced with PB (PB 40mM, pH 6.90; Na2HPO4 24.4mM, NaH2PO4 15.6mM) using a centrifugal desalting column (40 kDa, 0.5 mL, REF: 87766, batch number SJ251704, manufacturer: Thermo Fisher) using a centrifugal desalting column.

[0207] Concentrated EDTA (50 mM stock solution prepared with ddH2O) was added to PB buffer (40 mM, pH 6.90) containing the purified intermediate to a final concentration of 5 mM. TCEP (TCEP / mAb molar ratio = 6) was then added to the EDTA-treated intermediate to initiate a second reduction reaction at 22°C for 16 hours. The resulting second reduction reaction solution containing the further reducing antibody was used directly for the next conjugation step without the need to remove TCEP.

[0208] Add the DMA solution of linker-load 2 (total DMA 10% v / v, linker-load / mAb molar ratio = 10) to the second reduction reaction solution, and then couple the reaction at 22°C for 1 hour.

[0209] The coupling product was transferred to storage buffer as described in Process 1. The resulting crude product was purified by HIC as described in Process 1. The purity of the purified HIC product was determined by PLRP HPLC, and the results are shown in Tables 1-5. Figure 15A and 15B .

[0210] Table 1-5: Purity of 4+4 dual-loaded molecules in the purified product

[0211]

[0212] Example 2: Production of a 4+2 Dual-Load ADC

[0213] This embodiment describes... Figure 1 An exemplary process of scheme 3-1.

[0214] Process 1

[0215] In PB buffer (40 mM, pH 6.90), 5 mg / mL antibody was reduced with TCEP (C4706-2G, Sigma-Aldrich) in the presence of ZnCl2 (14422-500G, Sigma-Aldrich), with a Zn(II) / mAb molar ratio of 4 and a TCEP / mAb molar ratio of 10, at 4°C for 16 hours. The reduction reaction solution containing the reduced antibody was used directly for the next coupling step without removing the TCEP.

[0216] A DMA (dimethylacetamide, ARK2190-1L, SAFC) solution (10 mg / mL) containing linker-loading 1 was added to the reduction reaction solution and mixed. The linker-loading / mAb (LP / mAb) molar ratio was 12.0, and the first coupling reaction was carried out. The coupling reaction was carried out at 4°C for 2 hours to obtain the mAb1 intermediate ADC with 4 molecules of linker-loading 1 coupled together.

[0217] Acetylcysteine ​​(NAC) (acetylcysteine, A9165-100G, Sigma-Aldrich) (10mM stock solution prepared with ddH2O, NAC / mAb molar ratio = 16) was added to the first coupling reaction solution containing intermediate ADC, and the reaction was carried out at 4°C for 0.5 hours to remove excess linker-loading.

[0218] Next, the intermediate was purified by centrifugation and desalting column (40 kDa, 0.5 mL, REF: 87766, batch number SJ251704, manufacturer: Thermofisher) with PB buffer (40 mM, pH 6.90).

[0219] Add concentrated EDTA (EDTA∙2Na, 10009719, SCR) (50 mM stock solution prepared with ddH2O) to the eluent to a final concentration of 5 mM. Add the DMA solution of linker-loador 2 (total DMA 10% v / v) to the EDTA-treated eluent and mix, with a linker-loador / mAb molar ratio of 10. Then, perform a coupling reaction at 4°C for 2 hours.

[0220] The coupling product was transferred to storage buffer (20 mM histidine-acetic acid buffer, pH 5.5) using a centrifugal desalting column (40 kDa, 0.5 mL, REF: 87766, batch number SJ251704, manufacturer: Thermofisher). The identification results of the crude product are shown in Table 2-1 and... Figure 16 and 17.

[0221] Table 2-1: Purity of 4+2 double-loaded molecules in crude product

[0222]

[0223] The crude product was purified using preparative-grade HIC as described above.

[0224] Process 2

[0225] In HEPES buffer (40 mM, pH 7), 5 mg / mL mAb1 was reduced with TCEP in the presence of ZnCl2, with a Zn(II) / mAb molar ratio of 4 and a TCEP / mAb molar ratio of 3.25, and the reaction was carried out at 12°C for 20 hours. The reduction reaction solution containing the reducing antibody was used directly for the next coupling step without removing the TCEP.

[0226] A DMA (dimethylacetamide, ARK2190-1L, SAFC) solution (10 mg / mL) containing linker-loading 1 was added to the reduction reaction solution and mixed. The linker-loading / mAb (LP / mAb) molar ratio was 12.0, and the first coupling reaction was carried out. The coupling reaction was carried out at 12 °C for 1 hour to obtain mAb1 intermediate coupled with 4 linker-loading 1 molecules.

[0227] NAC (10 mM stock solution prepared with ddH2O, NAC / mAb molar ratio = 16) was added to the first coupling reaction solution containing intermediate ADC, and the reaction was carried out at 12°C for 0.5 hours to remove excess linker-loading.

[0228] Next, the intermediate was purified by HEPES (40 mM, pH 7) using a centrifugal desalting column (40 kDa, 0.5 mL, REF: 87766, batch number SJ251704, manufacturer: Thermofisher).

[0229] Add concentrated EDTA to the eluent to a final concentration of 2 mM. Add a DMA solution of linker-loador 2 (total DMA 10% v / v) to the EDTA-treated eluent, with a linker-loador / mAb molar ratio of 10, and couple the reaction at 12°C for 1 hour.

[0230] The coupling product was transferred to storage buffer (20 mM histidine-acetic acid buffer, pH 5.5) using a centrifugal desalting column (40 kDa, 0.5 mL, REF: 87766, batch number SJ251704, manufacturer: Thermofisher). The identification results of the crude product are shown in Table 2-2 and... Figure 18The crude product was purified using preparative-grade HIC as described in Example 1. The purified ADC was characterized by LC-MS (Agilent 1290 Infinity II; QTOF: Agilent 6530 LC\Q-TOF). The results are shown in Tables 2-2 and 2-3. Figure 19 .

[0231] Table 2-2: Purity of Target Molecules under 4+2 Dual Loading

[0232]

[0233] Example 3: Production of a 4+2+2 Triple Load ADC

[0234] This embodiment describes... Figure 1 An exemplary process of scheme 3-2.

[0235] In HEPES buffer (40 mM, pH 6.90), 5 mg / mL mAb1 (trastuzumab, same as above) was reduced with TCEP (C4706-2G, Sigma-Aldrich) in the presence of ZnCl2 (14422-500G, Sigma-Aldrich), with a Zn(II) / mAb molar ratio of 4 and a TCEP / mAb molar ratio of 3.25, at 12°C for 20 hours. The reduction reaction solution containing the reducing antibody was used directly for the next coupling step without removing the TCEP.

[0236] A DMA (dimethylacetamide, ARK2190-1L, SAFC) solution (10 mg / mL) containing linker-loading 1 was added to the reduction reaction solution and mixed, with a linker-loading / mAb (LP / mAb) molar ratio of 12.0, to carry out the first coupling reaction. The coupling reaction was carried out at 12℃ for 1 hour to obtain the first intermediate, which is an mAb coupled with 4 linker-loading 1 molecules.

[0237] Acetylcysteine ​​(NAC) (acetylcysteine, A9165-100G, Sigma-Aldrich) (10mM stock solution prepared with ddH2O, NAC / mAb molar ratio = 16) was added to the first coupling reaction solution containing intermediate ADC, and the reaction was carried out at 12℃ for 1 hour to remove excess linker-loading.

[0238] Next, the intermediate DAR4 ADC was purified with HEPES buffer (40 mM, pH 6.90) using a centrifugal desalting column (40 kDa, 0.5 mL, REF: 87766, batch number SJ251704, manufacturer: Thermofisher).

[0239] Add concentrated EDTA (EDTA∙2Na, 10009719, SCR) (50 mM stock solution prepared with ddH2O) to the eluent to a final concentration of 5 mM. Add a DMA solution of linker-loador 2 (total DMA 10% v / v) to the EDTA-treated eluent, with a linker-loador / mAb molar ratio of 10, and perform a coupling reaction at 12°C for 1 hour.

[0240] Acetylcysteine ​​(NAC) (10 mM stock solution prepared with ddH2O, NAC / mAb molar ratio = 16) was added to the second coupling reaction solution, and the reaction was carried out at 12°C for 0.5 hours to remove excess linker-loading.

[0241] The second intermediate of the 4+2 dual-load ADC was purified using a centrifugal desalting column (40 kDa, 0.5 mL, REF: 87766, Lot #SJ251704, Thermo Fisher) with HEPES buffer (40 mM, pH 6.90).

[0242] In HEPES buffer (40 mM, pH 7.0), the second intermediate ADC product was reduced with DPAA at a DPAA / mAb molar ratio of 6. The second reduction reaction was carried out at 22°C for 16 hours. The resulting second reduction reaction solution containing the further reducing antibody was used directly for the next coupling step without the need to remove the DPAA.

[0243] Add a DMA solution of linker-load 3 (total DMA 10% v / v, linker-load / mAb molar ratio = 10) to the second reduction reaction solution to obtain the third coupling solution, and incubate the third coupling solution at 22°C for 2 hours.

[0244] The product of the third coupling reaction was transferred to storage buffer (20 mM histidine-acetic acid buffer, pH 5.5) using a centrifugal desalting column (40 kDa, 0.5 mL, REF: 87766, batch number SJ251704, manufacturer: Thermofisher). The crude product was identified as described in Example 1, and the results are shown in Table 3-1 and... Figure 20 .

[0245] Table 3-1: Purity of 4+2+2 Triple Load ADC

[0246]

[0247] Example 4: Production of DAR6 ADC

[0248] This embodiment describes... Figure 1 An exemplary process of Scheme 1-1.

[0249] Experiment 1

[0250] In PB buffer (40 mM, pH 6.90) or HEPES buffer (40 mM, pH 7), in the presence of ZnCl2 (14422-500G, Sigma-Aldrich), 5 mg / mL antibody was reduced with TCEP (C4706-2G, Sigma-Aldrich) at a Zn(II) / mAb molar ratio of 4. The TCEP / mAb molar ratio, reaction temperature, and time are described below. The reduction reaction solution containing the reduced antibody was used directly for the next coupling step without removing the TCEP.

[0251] A DMA solution containing the linker-loading agent (10 mg / mL, i.e., a linker-loading agent / mAb molar ratio of LP / mAb = 12.0) and EDTA (final concentration shown below) were added to the reduction reaction solution and mixed to carry out the coupling reaction. The coupling reaction was carried out at 4°C for 2 hours to obtain the DAR6 ADC product.

[0252] Acetylcysteine ​​(NAC) (acetylcysteine, A9165-100G, Sigma-Aldrich) (10mM stock solution prepared with ddH2O) was added to the first coupling reaction solution containing the ADC product to remove excess linker-loading. The NAC / mAb molar ratio, reaction temperature and time are described below.

[0253] The product was desalted using a centrifugal desalting column (40 kDa, 0.5 mL, REF: 87766, batch number SJ251704, manufacturer: Thermofisher) and the buffer was changed to storage buffer (20 mM histidine-acetic acid buffer, pH 5.5). The identification results of the crude product are shown in Table 4-1 and... Figure 21A , 21B .

[0254] Table 4-1: Production and Characterization of DAR6 ADC Products

[0255]

[0256] like Figure 22 As shown, the purity of the target product can be further purified and improved. The production method of mAb1-MMAE is the same as that of mAb4-MMAE described above. The crude product is then subjected to preparative-grade HIC purification as described in Example 1.

[0257] Experiment 2

[0258] The reduction reaction steps in this experiment used various buffers. Specifically, in PB buffer (40 mM, pH 6.90) or other buffers listed below, 5 mg / mL mAb1 was reduced with TCEP in the presence of ZnCl2, with a Zn(II) / mAb molar ratio of 4, a TCEP / mAb molar ratio of 8 (PB only) or 3.25, and reacted at 4°C for 16 hours (PB only) or 12°C for 20 hours. The reduction reaction solution containing the reducing antibody was used directly in the next coupling step without removing the TCEP.

[0259] Add the linker-loading DMA solution (10 mg / mL) and EDTA (5 mM final concentration (PB only) or 2 mM (other buffers)) to the reduction reaction solution and mix. The linker-loading / mAb (LP / mAb) molar ratio is 12.0, and the coupling reaction is carried out. The coupling reaction is carried out at 4°C for 2 hours to obtain the DAR6 ADC product.

[0260] Add acetylcysteine ​​(NAC) (acetylcysteine, A9165-100G, Sigma-Aldrich) (10mM stock solution prepared with ddH2O, NAC / mAb molar ratio = 16) to the first coupling reaction containing the ADC product, and react at 4°C (PB only) or 12°C (other buffers) for 0.5 hours to remove excess linker-loading.

[0261] Next, the product was transferred to the storage buffer as described in Experiment 1. The identification results of the crude product are shown in Table 4-2 and... Figure 23 .

[0262] Table 4-2: Difference Test of Reduction Reaction Buffer

[0263]

[0264] Experiment 3

[0265] This experiment tested different salt concentrations in the reduction reaction buffer. Specifically, in HEPES buffers with different salt concentrations at pH 7, 5 mg / mL mAb1 was reduced with TCEP in the presence of ZnCl2, with a Zn(II) / mAb molar ratio of 4 and a TCEP / mAb molar ratio of 3.25, and the reaction was carried out at 12°C for 20 hours. The reduction reaction solution containing the reducing antibody was used directly for the next coupling step without removing the TCEP.

[0266] A DMA solution containing the linker and load (mc-vc-PAB-MMAE, 10 mg / mL, i.e., linker-load / mAb molar ratio = 12.0) and EDTA (final concentration 2 mM) were added to the reduction reaction solution and mixed to carry out the coupling reaction. The coupling reaction was carried out at 12℃ for 1 hour to obtain the DAR6 ADC product.

[0267] Acetylcysteine ​​(NAC) (10 mM stock solution prepared with ddH2O, NAC / mAb molar ratio = 16) was added to the first coupling reaction solution containing the ADC product, and the reaction was carried out at 12°C for 0.5 hours to remove excess linker-loading.

[0268] Next, the product was transferred to the storage buffer as described in Experiment 1. The identification results of the crude product are shown in Table 4-3 and... Figure 24 .

[0269] Table 4-3: Test of salt concentration in reduction reaction buffer

[0270]

[0271] Experiment 4

[0272] This experiment tested different mAb concentrations. Specifically, in HEPES buffer (40 mM, pH 7), in the presence of ZnCl2, a specific concentration of mAb1 was reduced with TCEP, with a Zn(II) / mAb molar ratio of 4 and a TCEP / mAb molar ratio of 3.25, and the reaction was carried out at 12°C for 20 hours. The reduction reaction solution containing the reducing antibody was used directly in the next coupling step without the need to remove the TCEP.

[0273] A DMA solution containing the linker and load (mc-vc-PAB-MMAE, 10 mg / mL, i.e., linker-load / mAb molar ratio = 12.0) and EDTA (final concentration 2 mM) were added to the reduction reaction solution and mixed to carry out the coupling reaction. The coupling reaction was carried out at 12℃ for 1 hour to obtain the DAR6 ADC product.

[0274] Acetylcysteine ​​(NAC) (10 mM stock solution prepared with ddH2O, NAC / mAb molar ratio = 16) was added to the first coupling reaction solution containing the ADC product, and the reaction was carried out at 12°C for 0.5 hours to remove excess linker-loading.

[0275] Next, the product was transferred to the storage buffer as described in Experiment 1. The identification results of the crude product are shown in Table 4-4 and... Figure 25 .

[0276] Table 4-4: mAb Concentration Difference Test

[0277]

[0278] Experiment 5

[0279] In this experiment, experiments were conducted at different temperatures and with different TCEP equivalents (i.e., the TCEP / mAb ratio). Specifically, in PB buffer (40 mM, pH 6.9) or HEPES buffer (40 mM, pH 7), 5 mg / mL mAb1 was reduced with TCEP in the presence of ZnCl2, with a Zn(II) / mAb molar ratio of 4. The TCEP / mAb molar ratio was as indicated in the ADC name. The reaction temperature and time are described below. The HEPES reduction reaction solution containing the reducing antibody was used directly for the next coupling step without the need for TCEP removal. The reduction reaction solution containing the reducing antibody in PB buffer was subjected to TCEP removal treatment. Specifically, the reduction reaction solution was replaced with storage buffer (20 mM histidine-acetic acid buffer, pH 5.5) using a centrifugal desalting column (40 kDa, 0.5 mL, REF: 87766, batch number SJ251704, manufacturer: Thermo Fisher).

[0280] A DMA solution containing the linker and load (mc-vc-PAB-MMAE, 10 mg / mL, i.e., linker-load / mAb molar ratio = 12.0) and EDTA were added to the reduction reaction solution and mixed to carry out the coupling reaction. The final concentration of EDTA is described below. The temperature and time of the coupling reaction are as described below, and the reaction yielded the DAR6 ADC product.

[0281] Acetylcysteine ​​(NAC) (10 mM stock solution prepared with ddH2O, NAC / mAb molar ratio = 16) was added to the first coupling reaction solution containing the ADC product, and the reaction was carried out at 12°C for 0.5 hours to remove excess linker-loading.

[0282] Next, the product was transferred to the storage buffer as described in Experiment 1. The identification and results of the crude product are shown in Tables 4-5 and 5. Figure 26 .

[0283] Table 4-5: Experiments at different reduction reaction temperatures and TCEP equivalents

[0284]

[0285] Experiment 6

[0286] This experiment tested reduction reaction buffers at different pH values. Specifically, in HEPES buffer (40 mM) at the pH described below, 5 mg / mL mAb1 was reduced with TCEP in the presence of ZnCl2, with a Zn(II) / mAb molar ratio of 4 and a TCEP / mAb molar ratio of 3.25, and the reaction was carried out at 12°C for 20 hours. The reduction reaction solution containing the reducing antibody was used directly in the next coupling step without removing the TCEP.

[0287] A DMA solution (mc-vc-PAB-MMAE, 10 mg / mL, i.e., linker-load / mAb molar ratio = 12.0) and EDTA (final concentration 2 mM) were added to the reduction reaction solution and mixed to carry out the coupling reaction. The coupling reaction was carried out at 12℃ for 1 hour to obtain the DAR6 ADC product.

[0288] Acetylcysteine ​​(NAC) (10 mM stock solution prepared with ddH2O, NAC / mAb molar ratio = 16) was added to the first coupling reaction solution containing the ADC product, and the reaction was carried out at 12°C for 0.5 hours to remove excess linker-loading.

[0289] Next, the product was transferred to the storage buffer as described in Experiment 1. The identification and results of the crude product are shown in Tables 4-6 and 4-6. Figure 27 .

[0290] Table 4-6: pH difference test of reduction reaction buffer

[0291]

[0292] Experiment 7

[0293] This experiment tested the effects of different additives in the reduction reaction buffer. Specifically, in HEPES buffer (40 mM, pH 7), 5 mg / mL mAb1 was reduced with TCEP in the presence of ZnCl2, with a Zn(II) / mAb molar ratio of 4 and a TCEP / mAb molar ratio of 3.25, and the reaction was carried out at 12°C for 20 hours. The reduction reaction solution containing the reducing antibody was used directly in the next coupling step without the need to remove the TCEP.

[0294] A DMA solution containing the linker and load (mc-vc-PAB-MMAE, 10 mg / mL, i.e., linker-load / mAb molar ratio = 12.0) and EDTA (final concentration 2 mM) were added to the reduction reaction solution and mixed to carry out the coupling reaction. The coupling reaction was carried out at 12℃ for 1 hour to obtain the DAR6 ADC product.

[0295] Acetylcysteine ​​(NAC) (10 mM stock solution prepared with ddH2O, NAC / mAb molar ratio = 16) was added to the first coupling reaction solution containing the ADC product, and the reaction was carried out at 12°C for 0.5 hours to remove excess linker-loading.

[0296] Next, the product was transferred to the storage buffer as described in Experiment 1. The identification and results of the crude product are shown in Tables 4-7 and 4-7. Figure 28 .

[0297] Table 4-7: Tests on Additives in Reduction Reaction Buffer

[0298]

[0299] Example 5: Production of a 6+2 Dual-Load ADC

[0300] This embodiment describes... Figure 1 Exemplary processes of schemes 1-2.

[0301] Process 1

[0302] In PB buffer (40 mM, pH 6.90), 5 mg / mL antibody was reduced with TCEP (C4706-2G, Sigma-Aldrich) in the presence of ZnCl2 (14422-500G, Sigma-Aldrich), with a Zn(II) / mAb molar ratio of 4 and a TCEP / mAb molar ratio of 8, and reacted at 4°C for 20 hours. The reduction reaction solution containing the reduced antibody was used directly for the next coupling step without removing the TCEP.

[0303] A DMA solution (10 mg / mL, i.e., linker-load / mAb molar ratio = 12.0) and EDTA (5 mM final concentration) were added to the reduction reaction solution and mixed to carry out the first coupling reaction. The coupling reaction was carried out at 4°C for 2 hours to obtain the DAR6ADC product, which is an intermediate.

[0304] Acetylcysteine ​​(NAC) (acetylcysteine, A9165-100G, Sigma-Aldrich) (10mM stock solution prepared with ddH2O, NAC / mAb molar ratio = 16) was added to the first coupling reaction solution containing intermediate ADC, and the reaction was carried out at 4°C for 0.5 hours to remove excess linker-loading.

[0305] Next, the DAR6 ADC intermediate was replaced with PB buffer (40 mM, pH 6.9) using a centrifugal desalting column (40 kDa, 0.5 mL, REF: 87766, batch number SJ251704, manufacturer: Thermofisher).

[0306] Add concentrated EDTA to a final concentration of 5 mM. In the presence of EDTA, reduce the intermediate DAR6 ADC with TCEP (TCEP / mAb molar ratio = 6) and react at 37°C for 16 hours. The second reduction reaction solution containing the further reducing antibody can be used directly for the next coupling step without removing TCEP.

[0307] Add the DMA solution of linker-load 2 (total DMA 10% v / v, linker-load / mAb molar ratio = 10) to the second reduction reaction solution, and then couple the reaction at 4°C for 2 hours.

[0308] The coupling product was transferred to storage buffer (20 mM histidine-acetic acid buffer, pH 5.5) using a centrifugal desalting column (40 kDa, 0.5 mL, REF: 87766, batch number SJ251704, manufacturer: Thermofisher). The identification and results of the crude product are shown in Table 5-1 and... Figures 29 to 32 .

[0309] Table 5-1: Purity of 6+2 double-loaded molecules in crude product

[0310]

[0311] The crude product was purified by preparative hydrophobic interaction chromatography as described in Example 1.

[0312] Process 2

[0313] In HEPES buffer (40 mM, pH 7), 5 mg / mL antibody was reduced with TCEP in the presence of ZnCl2, with a Zn(II) / mAb molar ratio of 4 and a TCEP / mAb molar ratio of 3.25, and reacted at 12°C for 20 hours. The reduction reaction solution containing the reduced antibody was used directly for the next coupling step without removing the TCEP.

[0314] A DMA solution (10 mg / mL, i.e., linker-load / mAb molar ratio = 12.0) and EDTA (2 mM final concentration) of linker-load 1 were added to the reduction reaction solution and mixed to carry out the first coupling reaction. The coupling reaction was carried out at 12℃ for 1 hour to obtain the DAR6ADC product, which is an intermediate.

[0315] Acetylcysteine ​​(NAC) (acetylcysteine, A9165-100G, Sigma-Aldrich) (10mM stock solution prepared with ddH2O, NAC / mAb molar ratio = 16) was added to the first coupling reaction containing intermediate ADC, and the reaction was carried out at 12 °C for 0.5 h to remove excess linker-loading.

[0316] Next, the intermediate DAR6 ADC was replaced with HEPES buffer (40 mM, pH 7) using a centrifugal desalting column (as before).

[0317] Add concentrated EDTA to a final concentration of 2 mM. In the presence of EDTA, reduce the intermediate DAR6 ADC with DPAA (DPAA / mAb molar ratio = 6) and react at 22°C for 16 hours. The second reduction reaction solution containing the further reducing antibody can be used directly for the next coupling step without removing the DPAA.

[0318] Add the DMA solution of linker-load 2 (total DMA 10% v / v, linker-load / mAb molar ratio = 10) to the second reduction reaction solution, and then couple the reaction at 22°C for 1 hour.

[0319] The coupling product was transferred to storage buffer (20 mM histidine-acetic acid buffer, pH 5.5) using a centrifugal desalting column (40 kDa, 0.5 mL, REF: 87766, batch number #SJ251704, manufacturer: Thermofisher). The identification and results of the crude product are shown in Table 5-2 and... Figure 33 The crude product was purified and characterized by preparative hydrophobic interaction chromatography as described in Example 1. The results are shown in Table 5-2 and... Figure 33 .

[0320] Table 5-2: Purity of 6+2 dual-loaded molecules

[0321]

[0322] Example 6: Production of DAR3 ADC

[0323] This embodiment describes... Figure 2 An exemplary process of scheme (A).

[0324] In HEPES buffer (40 mM, pH 7.0), 5 mg / mL mAb1 (trastuzumab, as before) was reduced with TCEP (C4706-2G, Sigma-Aldrich) in the presence of ZnCl2 (14422-500G, Sigma-Aldrich), with a Zn(II) / mAb molar ratio of 4 and a TCEP / mAb molar ratio of 3.25, at 12°C for 20 hours. The reduction reaction solution containing the reducing antibody was used directly for the next coupling step without removing the TCEP.

[0325] A DMA solution containing the linker-loading agent and EDTA (final concentration 2 mM) were added to the reduction reaction solution to initiate a coupling reaction. The concentrations (Conc.) of the linker-loading agent solution and the LP-antibody molar ratio are described below. The temperature and time of the coupling reaction are as described below, and the reaction yielded the DAR3 ADC product. The linker-loading molecule is:

[0326] , and

[0327] Acetylcysteine ​​(NAC) (acetylcysteine, A9165-100G, Sigma-Aldrich) (10mM stock solution prepared with ddH2O, NAC / mAb molar ratio = 8) was added to the first coupling reaction containing the ADC product, and the reaction was carried out at 12°C for 0.5 hours to remove excess linker-loading.

[0328] Next, the product was transferred to storage buffer (20 mM histidine-acetic acid buffer, pH 5.5) using a centrifugal desalting column (40 kDa, 0.5 mL, REF: 87766, batch number SJ251704, manufacturer: Thermofisher). The results are shown in Table 6-1 and... Figure 34 .

[0329] Table 6-1: Production and Characterization of DAR3 ADC Products

[0330]

[0331] Example 7: Production of DAR4 ADC containing bispecific antibodies or Fc fusion proteins

[0332] This embodiment describes... Figure 2 Exemplary processes for schemes (B) and (C).

[0333] In PB buffer (40 mM, pH 6.90) or HEPES buffer (40 mM, pH 7), reduce 5 mg / mL antibody (as indicated by the ADC name) with TCEP (C4706-2G, Sigma-Aldrich) in the presence of ZnCl2 (14422-500G, Sigma-Aldrich) at a Zn(II) / mAb molar ratio of 4. The TCEP / mAb molar ratio, reaction temperature, and time are described below. The reduction reaction solution containing the reduced antibody can be used directly for the next coupling step without removing the TCEP.

[0334] A DMA solution containing the linker-loading agent (10 mg / mL, i.e., a linker-loading agent / mAb molar ratio of LP / mAb = 12.0) and EDTA (final concentration shown below) were added to the reduction reaction solution and mixed to carry out the coupling reaction. The coupling reaction was carried out at 4°C for 2 hours to obtain the DAR6 ADC product.

[0335] Acetylcysteine ​​(NAC) (acetylcysteine, A9165-100G, Sigma-Aldrich) (10mM stock solution prepared with ddH2O) was added to the first coupling reaction solution containing the ADC product. The NAC / mAb molar ratio, reaction temperature and time are described below. Excess linker-loading was removed.

[0336] Next, the product was desalted using a centrifugal desalting column (40 kDa, 0.5 mL, REF: 87766, batch number SJ251704, manufacturer: Thermofisher) to transfer it to storage buffer (20 mM histidine-acetic acid buffer, pH 5.5). The identification and results of the crude product are shown in Table 7. Figure 35 .

[0337] Table 7-1: Production and Characterization of DAR4 ADC Products

[0338]

[0339] Sequence List:

[0340]

[0341] ***********

[0342] References:

[0343] [1].Levengood, MR et al., Orthogonal Cysteine ​​Protection EnablesHomogeneous Multi-Drug Antibody–Drug Conjugates. Angew. Chem. Int. Ed. 56,733–737 (2017)

[0344] [2].Yamazaki, CM et al., Antibody-drug conjugates with dual payloads forcombating breast tumor heterogeneity and drug resistance. Nat. Commun. 12,3528 (2021)

[0345] [3].Nilchan, N. et al., Dual-mechanistic antibody-drug conjugate via site-specific selenocysteine / cysteine ​​conjugation. Antib. Ther. 2, 71–78 (2019)

[0346] [4].Boschanski, M. et al., Site-Specific Conjugation Strategy for Dual Antibody–Drug Conjugates Using Aerobic Formylglycine-Generating Enzymes. Bioconjug. Chem. 32, 1167–1174 (2021)

Claims

1. A method for producing antibody-drug conjugates, comprising: The first reduction reaction step, wherein the antibody is reduced in the presence of transition metal ions; and One or more coupling reaction steps, wherein the reduced antibody is coupled with a linker-loader to generate an antibody-drug conjugate; The method further includes an optional second reduction reaction and a subsequent coupling reaction, wherein the second reduction reaction further reduces the antibody portion of the antibody-drug conjugate; and The method further includes a demasking process to remove transition metals bound to the antibody, the demasking process being synchronized with at least one of the coupling reaction steps or with the second reduction reaction step.

2. The method as described in claim 1, wherein, The demasking process includes treatment with a chelating agent.

3. The method as described in claim 1, wherein, The method includes two or three coupling reaction steps, and the connecting sub-loads of at least two coupling reaction steps are different.

4. The method of claim 1, wherein the first reduction reaction step is followed by: In the first coupling reaction step, the antibody is coupled to a first linker-loader to generate a first antibody-drug conjugate. The second reduction reaction step of the combined simultaneous demasking process, wherein the antibody portion of the first antibody-drug conjugate is further reduced; and In the second coupling reaction step, the antibody portion of the first antibody-drug conjugate that has been further reduced is coupled to a second linker-loader to generate a second antibody-drug conjugate.

5. The method of claim 1, wherein the first reduction reaction step is followed by: In the first coupling reaction step, the antibody is coupled to a first linker-loader to generate a first antibody-drug conjugate; and The second coupling reaction step of the combined synchronous demasking process, wherein the antibody portion of the first antibody-drug conjugate is coupled with a second linker-loader to generate a second antibody-drug conjugate.

6. The method of claim 1, wherein the first reduction reaction step is followed by: The first coupling reaction step of the combined synchronous demasking process, wherein the antibody is coupled with a first linker-loader to generate a first antibody-drug conjugate.

7. The method of claim 1, wherein the first reduction reaction step is followed by: The first coupling reaction step of the combined synchronous demasking process, wherein the antibody is coupled with the first linker-loader to generate the first antibody-drug conjugate; In the second reduction reaction step, the antibody portion of the first antibody-drug conjugate is further reduced; and The second coupling reaction step involves coupling the antibody portion of the first antibody-drug conjugate with a second linker-loader to generate a second antibody-drug conjugate.

8. The method of claim 1, wherein the first reduction reaction step is followed by: In the first coupling reaction step, the antibody is coupled to a first linker-loader to generate a first antibody-drug conjugate. The second coupling reaction step of the combined synchronous demasking process, wherein the antibody portion in the first antibody-drug conjugate is coupled with a second linker-loader to generate a second antibody-drug conjugate; The second reduction reaction step, wherein the antibody portion of the second antibody-drug conjugate is further reduced; and The third coupling reaction step involves coupling the antibody portion of the second antibody-drug conjugate with a third linker-loader to generate the third antibody-drug conjugate.

9. An antibody-drug conjugate product comprising a target antibody-drug conjugate with a purity of at least 55 mol%, wherein, (i) The target antibody-drug conjugate contains two or four first linker-load molecules linked to the Fab region and at least two second linker-load molecules linked to the hinge region, wherein the first linker-load molecules are different from or the same as the second linker-load molecules; (ii) The target antibody-drug conjugate contains three or six first linker-loader molecules, and optionally further contains one or two second linker-loader molecules, wherein the first linker-loader and the second linker-loader are different from or the same as each other; or (iii) The target antibody-drug conjugate contains two or four first linker-loaders linked to the antibody Fab region and at least one (e.g., 1, 2, 3, 4) second linker-loaders linked to the antibody hinge region, and optionally may also contain one or two third linker-loaders linked to the antibody hinge region; wherein the first, second and optional third linker-loaders are the same or different.

10. The antibody product of claim 9, wherein... (i) The target antibody-drug conjugate contains four first linker-loader molecules linked to the Fab region and four second linker-loader molecules linked to the hinge region, wherein, The first connector-load is different from the second connector-load; (ii) The target antibody-drug conjugate contains four first linker-loaders linked to the Fab region and two second linker-loaders linked to the hinge region, wherein the first linker-loaders and the second linker-loaders are different; (iii) The target antibody-drug conjugate contains four first linker-loader molecules linked to the Fab region and two first linker-loader molecules linked to the hinge region, and wherein the purity is at least 65 mol% (iv) The target antibody-drug conjugate contains four first linker-load molecules linked to the Fab region, two first linker-load molecules linked to the hinge region, and two second linker-load molecules linked to the hinge region, wherein the first linker-load molecules and the second linker-load molecules are different. (v) The target antibody-drug conjugate contains four first linker-loaders linked to the Fab region, two second linker-loaders linked to the hinge region, and two third linker-loaders linked to the hinge region, wherein the first, second, and third linker-loaders are all different. (vi) The target antibody-drug conjugate contains two linker-load molecule portions loaded in the Fab region and one linker-load molecule portion loaded in the hinge region, wherein the linker-load portion is formed by a thiol-bridged linker-load molecule; or (vii) The target antibody-drug conjugate comprises two linker-loador molecule portions loaded in the antibody Fab region and two linker-loador molecule portions loaded in the antibody hinge region, wherein the antibody is a VHH-Fc protein; or (viii) The target antibody-drug conjugate contains two linker-loader molecular portions loaded in the Fab region of one of the antibody LC-HC pairs and two linker-loader molecular portions loaded in the hinge region, wherein the antibody is a bispecific antibody with a WuXiBody structure, wherein the LC+CH1 segment of one of the light and heavy chain pairs (LC-HC) is replaced with the TCR constant region, and the other LC-HC pair retains the Fab region.

11. The antibody-drug conjugate product of claim 10, wherein, The target antibody-drug conjugate (i) has a purity of at least 68 mol%; the target antibody-drug conjugate (ii) has a purity of at least 61 mol%; the target antibody-drug conjugate (iv) has a purity of at least 60 mol%; the target antibody-drug conjugate (v) has a purity of at least 56 mol%; the target antibody-drug conjugate (vi) has a purity of at least 60 mol%; the target antibody-drug conjugate (vii) has a purity of at least 60 mol%; or, the target antibody-drug conjugate (viii) has a purity of at least 60 mol%.

12. The antibody-drug conjugate product as described in claim 9, characterized in that... It is prepared by the method described in claim 1.

13. The antibody-drug conjugate product prepared by the method of claim 1, wherein, (i) The product contains a target antibody-drug conjugate, the target antibody-drug conjugate containing two or four first linker-load molecules linked to the Fab region and at least two second linker-load molecules linked to the hinge region, wherein the first linker-load molecules are different from or the same as the second linker-load molecules; (ii) The target antibody-drug conjugate contains three or six first linker-loader molecules, and optionally further contains one or two second linker-loader molecules, wherein the first linker-loader and the second linker-loader are different from or the same as each other; or (iii) The target antibody-drug conjugate contains two or four first linker-loaders linked to the antibody Fab region and at least one (e.g., 1, 2, 3, 4) second linker-loaders linked to the antibody hinge region, and optionally may also contain one or two third linker-loaders linked to the antibody hinge region; wherein the first, second and optional third linker-loaders are the same or different.

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