Method for continuously preparing antibody-drug conjugate

Abstract

Provided is a method for continuously preparing an antibody-drug conjugate, comprising: 1) providing an unpurified or partially purified antibody or an antigen-binding fragment thereof; 2) conjugating the unpurified or partially purified antibody or the antigen-binding fragment thereof with a payload via a linker to prepare the antibody-drug conjugate; and 3) purifying the antibody-drug conjugate. The method simplifies process operations, significantly shortens the production cycle, and reduces reagent consumption, with high efficiency and cost-efficiency.

Description

A method for continuous preparation of antibody conjugates

[0001] This application is based on and claims priority to PCT application No. PCT / CN2025 / 072395 filed on January 15, 2025 and PCT application No. PCT / CN2025 / 122247 filed on September 18, 2025, the disclosures of the aforementioned applications are incorporated herein by reference in their entirety. Technical Field

[0002] This invention belongs to the field of biotechnology, specifically relating to a method for the continuous preparation of antibody conjugates. Background Technology

[0003] Antibody-drug conjugates (ADCs) are a class of innovative drugs that consist of antibodies or antibody fragments targeting specific antigens and payloads linked together by a linker.

[0004] The manufacturing process of ADC products typically involves multiple stages, including naked antibody production, small molecule production, ADC drug substance production, and formulation production. Therefore, the manufacturing process of ADC products is complex, involving multiple components and process steps. During product development, process design and development should be based on principles such as "quality by design" and "risk assessment." Special attention must be paid to treating all components (naked antibody, payload / linker / payload-linker, ADC drug substance, and ADC formulation) as a whole, comprehensively considering and controlling risks such as impurities, viruses, and microbial safety in the overall manufacturing process.

[0005] The development of manufacturing processes for naked antibodies should refer to the *Chinese Pharmacopoeia*, the *Technical Guidelines for Quality Control of Human Monoclonal Antibodies*, and relevant internationally accepted technical requirements such as those from ICH and WHO. The manufacturing processes for naked antibodies are similar to those for conventional antibody drugs; however, the manufacturing processes for some genetically engineered naked antibodies used for site-specific conjugation may differ due to the characteristics of their modified groups. Therefore, in addition to addressing the risks associated with conventional antibody production, the development of naked antibodies requires careful consideration of their impact on the ADC drug production process and the final ADC product quality throughout the entire process of ADC molecule design and manufacturing.

[0006] The production process of ADC stock solution typically includes steps such as antibody modification (if applicable), coupling reaction, and ADC purification. The antibody modification process varies depending on the coupling technology used. The main purpose of antibody modification is to introduce reactive chemical groups onto the antibody. This can be achieved by using a reducing agent to open antibody disulfide bonds and generate active thiol groups, or by introducing cysteine ​​residues or non-natural amino acids containing reactive groups at specific sites on the antibody through genetic engineering. Alternatively, it can involve modifying the sugar chain with bioenzymes to introduce reactive groups for coupling reactions.

[0007] The traditional ADC production process, as shown in Figure 1, includes an antibody intermediate process stage and an ADC process stage. First, purified antibodies are obtained through fermentation, clarification, capture, virus inactivation, column chromatography, virus filtration, ultrafiltration, and elution. Then, the purified antibody is coupled with a linker-payload to obtain the ADC. The ADC is then purified again, such as through chromatography, ultrafiltration, and elution, to obtain the ADC stock solution. The antibody intermediate requires formulation development for storage, and release testing is necessary before ADC conjugation to ensure quality, which increases the ADC CMC development cycle. Furthermore, the antibody intermediate needs to be stored at -70°C, occupying storage space and increasing storage costs. In addition, after ADC conjugation, to remove unconjugated linkers-payloads and residual organic solvents, a large-volume liquid exchange is required in the ultrafiltration / elution (UF / DF) step, typically 2-4 times the volume of the liquid exchange in the antibody process, sometimes even requiring two liquid exchanges and an additional step involving the addition of organic solvents, making the ADC process complex and significantly increasing the process time. Summary of the Invention

[0008] To address the shortcomings of existing technologies, the inventors optimized the process flow, integrating antibody processing and ADC conjugation processes to establish an integrated and continuous antibody-drug conjugate (ADC) production process. The ADC process provided by this invention simplifies the process, shortens the production cycle, and reduces reagent consumption, resulting in efficiency and cost savings. Using the production process of this invention to prepare ADCs helps reduce production costs and improve the competitiveness of products after they are launched on the market.

[0009] This invention provides a method for preparing antibody conjugates, comprising:

[0010] 1) Provide unpurified or partially purified antibodies or their antigen-binding fragments;

[0011] 2) Prepare antibody conjugates by linking unpurified or partially purified antibodies or their antigen-binding fragments to a payload via a linker;

[0012] 3) Purify antibody conjugates.

[0013] In some implementations, the reaction system in step 2) does not contain Sortase ligase.

[0014] In some implementations, the enzyme is not present in the reaction system of step 2).

[0015] In some implementations, the method includes:

[0016] Provide a cell culture clarifier containing an antibody or its antigen-binding fragment, and capture the antibody or its antigen-binding fragment in the cell culture clarifier;

[0017] The obtained product is subjected to conjugation, virus inactivation and deep filtration, first chromatography, optional second chromatography, optional third chromatography, and filtration to remove potential viruses. The conjugation, virus inactivation and deep filtration, first chromatography, optional second chromatography, optional third chromatography, and filtration to remove potential viruses can be performed in any order. For example, the sequence is virus inactivation and deep filtration, conjugation, first chromatography, second chromatography, and filtration to remove potential viruses. Another example is virus inactivation and deep filtration, first chromatography, conjugation, second chromatography, and filtration to remove potential viruses.

[0018] The resulting product is concentrated and / or replaced with a buffer solution, for example by ultrafiltration / washing.

[0019] In some implementations, the method includes:

[0020] Provide cell culture clarification medium containing antibodies or their antigen-binding fragments, capture the antibodies or their antigen-binding fragments in the cell culture clarification medium, and perform virus inactivation and deep filtration;

[0021] The obtained product is subjected to coupling, first chromatography, optional second chromatography, optional third chromatography, and filtration to remove potential viruses. The coupling, first chromatography, optional second chromatography, optional third chromatography, and filtration to remove potential viruses can be performed in any order, for example, coupling, first chromatography, second chromatography, and filtration to remove potential viruses in sequence, or first chromatography, coupling, second chromatography, and filtration to remove potential viruses in sequence.

[0022] The resulting product is concentrated and / or replaced with a buffer solution, for example by ultrafiltration / washing.

[0023] In some implementations, the method includes:

[0024] Provide a cell culture clarifier containing an antibody or its antigen-binding fragment, and capture the antibody or its antigen-binding fragment in the cell culture clarifier;

[0025] The obtained product is subjected to conjugation, virus inactivation and deep filtration, first chromatography, optional second chromatography, and optional third chromatography. The conjugation, virus inactivation and deep filtration, first chromatography, optional second chromatography, and optional third chromatography can be performed in any order, for example, virus inactivation and deep filtration, conjugation, first chromatography, and second chromatography, or virus inactivation and deep filtration, first chromatography, conjugation, and second chromatography.

[0026] The resulting product is concentrated and / or replaced with a buffer solution, and filtered to remove potential viruses, for example by ultrafiltration / washing.

[0027] In some embodiments, the process flow diagram of the method is shown in Figures 8, 9, and 10. Here, "clarification" refers to the cell culture clarification solution of the antibody, "chromatography 1" refers to the first chromatography, and "chromatography 2 (optional)" refers to an optional second chromatography.

[0028] In some embodiments, the capture is performed by one or more of affinity chromatography, membrane chromatography, ion exchange chromatography, and hydrophobic chromatography. In some embodiments, the capture is performed by affinity chromatography.

[0029] In some embodiments, the chromatography is selected from size exclusion chromatography, ion exchange chromatography (e.g., anion chromatography, cation chromatography), hydrophobic interaction chromatography, mixed-mode chromatography, and ceramic hydroxyapatite chromatography.

[0030] In some implementations, step 1) includes:

[0031] 1.1) Provide a sample containing an antibody or its antigen-binding fragment;

[0032] 1.2) Capture antibodies or their antigen-binding fragments in the sample.

[0033] In some implementations, the sample in step 1.1) is a cell culture medium.

[0034] In some embodiments, the cell culture medium is a clarified cell culture medium.

[0035] In some embodiments, the clarified cell culture solution is obtained by centrifuging and / or deep filtering a cell culture containing the provided antibody or its antigen-binding fragment.

[0036] In some implementations, the capture described in step 1.2) is performed by one or more of affinity chromatography, membrane chromatography, ion exchange chromatography, and hydrophobic chromatography.

[0037] In some implementations, the capture described in step 1.2) is performed by affinity chromatography.

[0038] In some implementations, the affinity chromatography is performed in a binding-elution mode.

[0039] In some embodiments, the affinity chromatography is selected from protein A affinity chromatography, protein L affinity chromatography, protein G affinity chromatography, and any combination thereof.

[0040] In some embodiments, the equilibration solution and / or elution solution for the affinity chromatography can be selected as needed.

[0041] In some embodiments, the equilibration solution for protein A affinity chromatography is selected from Tris-HAc buffer, Tris-HCl buffer, and PB buffer. In some embodiments, the pH of the equilibration solution is 7.0–7.6, for example, 7.4.

[0042] In some embodiments, the elution solution for protein A affinity chromatography is selected from NaAc-HAc buffer and sodium citrate-citrate buffer. In some embodiments, the pH of the elution solution is 3.6 to 4.2, for example, 3.9.

[0043] In some implementations, step 1.1) is followed by step 1.2) and then includes: inactivating the antibody or its antigen-binding fragment with the virus.

[0044] In some implementations, step 1.2) is followed by: 1.3) inactivating the captured antibody or its antigen-binding fragment with the virus.

[0045] In some embodiments, the virus is inactivated by adjusting the pH value or adding a surfactant. In some embodiments, adjusting the pH value includes:

[0046] Adjust the pH to 3-4 and incubate for 0.5-3 hours, for example, adjust the pH to 3.6 and incubate for 1 hour;

[0047] Neutralize the pH to 5-7, for example 5-6, or 5.5.

[0048] In some embodiments, the surfactant is 1% Triton X-100 or a mixture containing 1% polysorbate 80 (PS80) and 0.3% tributyl phosphate (TnBP).

[0049] In some implementations, the virus is inactivated and then centrifuged and / or deep filtered.

[0050] In some implementations, the captured antibody or its antigen-binding fragment is incubated in a buffer system with a pH of 3-4, and after incubation, the pH is neutralized to 5-7 and deep filtration is performed.

[0051] In some implementations, step 1.3) is followed by ultrafiltration and / or washing.

[0052] In some implementations, step 1) includes:

[0053] 1.1) Provide a sample containing an antibody or its antigen-binding fragment;

[0054] 1.2) Capture antibodies or their antigen-binding fragments in the sample;

[0055] 1.3) Inactivate the virus by capturing the antibody or its antigen-binding fragment;

[0056] 1.4) Centrifuge and / or perform deep filtration on the product from step 1.3);

[0057] The sample, capture, virus inactivation, and deep filtration can be defined as in any embodiment of this application.

[0058] In some embodiments, centrifugation and / or deep filtration are followed by: removal of latent viruses. In some embodiments, latent viruses are removed by anion exchange chromatography. In some embodiments, the anion exchange chromatography is selected from anion exchange column chromatography, anion mixed-mode chromatography, anion membrane chromatography, and anion mixed-mode membrane chromatography. In some embodiments, the anion exchange column chromatography includes: subjecting centrifuged and / or deep-filtered antibody or its antigen-binding fragment to anion exchange column chromatography to obtain a flow-through sample. In some embodiments, the equilibration solution for the anion exchange column chromatography is selected from Tris-HAc buffer, NaAc-HAc buffer, sodium citrate-citrate buffer, sodium MES buffer, Bis-Tris buffer, and any combination thereof. In some embodiments, the pH of the NaAc-HAc buffer is 5.0 to 6.0, for example, 5.5.

[0059] In some implementations, step 1) includes:

[0060] 1.1) Provide a sample containing an antibody or its antigen-binding fragment;

[0061] 1.2) Capture antibodies or their antigen-binding fragments in the sample;

[0062] 1.3) Inactivate the virus by capturing the antibody or its antigen-binding fragment;

[0063] 1.4) Centrifuge and / or perform deep filtration on the product from step 1.3);

[0064] 1.5) Remove potential viruses from the product of step 1.4);

[0065] The sample, capture, virus inactivation, deep filtration, and removal of potential viruses may be defined as in any embodiment of this application.

[0066] Those skilled in the art will understand that when step 1) includes a partial purification operation of the antibody or its antigen-binding fragment, the "partially purified antibody or its antigen-binding fragment" mentioned in step 2) is the product of step 1).

[0067] In some implementations, step 2) includes: conjugating an unpurified or partially purified antibody or its antigen-binding fragment to a linker-payload.

[0068] In some implementations, step 2) includes:

[0069] 2.1) To conjugate unpurified or partially purified antibodies or their antigen-binding fragments to linkers.

[0070] 2.2) React the product of step 2.1) with the payload.

[0071] In some implementations, the coupling is selected from fixed-point coupling and non-fixed-point coupling.

[0072] In some embodiments, the coupling is based on one or more of cysteine, lysine, or a catalytic enzyme. The catalytic enzyme can be selected based on the coupling substrate. In some embodiments, the catalytic enzyme is selected from transglutaminase, sorting enzymes (e.g., Sortase A (derived from Staphylococcus aureus), which achieves C-terminal or N-terminal directional ligation based on LPSTG motif recognition), glycosyl modifying enzymes (e.g., glycosidases (such as EndoS, for Fc glycan excision), glycosyltransferases (such as β-1,4-galactosyltransferase, for introducing functionalized glycosyl groups for subsequent coupling)), farnesyl transferases (Ftase-modified antibodies that introduce farnesyl groups (containing olefins or alkynes) to couple the linker-toxin to the farnesyl site via click chemistry (e.g., Tetrazine-TCO reaction), and tubulin tyrosine ligases. Ligase (TTL) involves modifying the C-terminus of an engineered antibody (containing a TTL recognition sequence) with a tyrosine residue, enabling site-specific coupling of a linker to a toxin via tyrosine oxidation (generating ortho-quinones) or click chemistry (such as the tetrazine-TCO reaction), formylglycine-generating enzyme (FGE, which catalyzes the conversion of cysteine ​​to formylglycine (fGly), supporting click chemistry coupling), engineered tyrosine kinase (which generates active quinone groups by oxidizing specific tyrosine residues, achieving site-specific toxin coupling), and microbial transpeptidase (which, after modification, can recognize specific peptide tags, supporting controlled coupling). The transglutaminase is selected from microbial MTGase and mammalian tissue TG2, achieving site-specific coupling via glutamine residues.

[0073] In some embodiments, the coupling is based on one or more of cysteine, lysine, or a catalytic enzyme (such as MTGase).

[0074] In some embodiments, the coupling is based on cysteine ​​and / or lysine.

[0075] In some embodiments, the conjugation includes using a reducing agent to reduce the antibody or its antigen-binding fragment.

[0076] In some embodiments, the reducing agent is selected from phosphine reducing agents (e.g., tris(2-carboxyethyl)phosphine hydrochloride (TCEP), diphenylphosphinoacetic acid, 2-[2-(diphenylphosphino)ethyl]pyridine, 3-(diphenylphosphino)benzenesulfonic acid, 4-(diphenylphosphino)benzoic acid, 2-(diphenylphosphino)ethylamine, 3-(diphenylphosphino)propylamine, 3-(diphenylphosphino)propionic acid, 2-(diisopropylphosphino)ethylamine, 2-(diphenylphosphino)benzoic acid, (2-hydroxyphenyl)diphenylphosphine, 1,3,5-triaza-7-phosphatricyclo[3.3.1.13.7]decane, n-butyldi(1-adamantyl)phosphine); and sulfur reducing agents (e.g., dithiothreitol (DTT), dithioerythritol, ethanethiol, sodium sulfide, glutathione, cysteine) are all acceptable reducing agents.

[0077] In some embodiments, the reducing agent is selected from tris(2-carboxyethyl)phosphine (TCEP) and dithiothreitol (DTT).

[0078] In some implementations, step 2) includes:

[0079] To couple unpurified or partially purified antibodies or their antigen-binding fragments to a linker-payload; or,

[0080] 2.1) To conjugate unpurified or partially purified antibodies or their antigen-binding fragments to linkers.

[0081] 2.2) React the product of step 2.1) with the payload;

[0082] in,

[0083] The coupling is based on cysteine ​​and / or lysine;

[0084] The conjugation includes using a reducing agent to reduce the antibody or its antigen-binding fragment;

[0085] The reducing agent may be as defined in any embodiment of this application.

[0086] In some implementations, the enzyme is not present in the reaction system of step 2).

[0087] In some implementations, step 2) includes:

[0088] To couple unpurified or partially purified antibodies or their antigen-binding fragments to a linker-payload; or,

[0089] 2.1) To conjugate unpurified or partially purified antibodies or their antigen-binding fragments to linkers.

[0090] 2.2) React the product of step 2.1) with the payload;

[0091] in,

[0092] The coupling is based on one or more catalytic enzymes selected from one or more of transglutaminase (MTGase), sorting enzyme, glycosylmodifying enzyme (e.g., glycosidase, glycosyltransferase), farnesyltransferase, tubulin tyrosine ligase, formylglycine synthase, engineered tyrosine kinase, and microbial transpeptidase.

[0093] In some implementations, step 3) includes:

[0094] 3.1) The prepared antibody conjugate was subjected to column chromatography;

[0095] 3.2) Remove potential viruses from the product of the previous step;

[0096] 3.3) Concentrated and / or replacement buffer;

[0097] The order of steps 3.2) and 3.3) can be interchanged.

[0098] In some implementations, step 3) includes:

[0099] 3.1) The prepared antibody conjugate was subjected to column chromatography;

[0100] 3.2) Remove potential viruses from the product of step 3.1);

[0101] 3.3) Concentration.

[0102] In some implementations, the column chromatography in step 3.1) is selected from ion exchange chromatography, hydrophobic interaction chromatography, and any combination thereof.

[0103] In some embodiments, the ion exchange chromatography is performed in a binding-elution mode or a flow-through mode.

[0104] In some implementations, step 3.1) includes:

[0105] 3.1.1) The prepared antibody conjugate was subjected to cation exchange column chromatography to obtain the eluted sample;

[0106] 3.1.2) The eluted sample was subjected to anion exchange column chromatography to obtain a flow-through sample.

[0107] In some implementations, step 3.1) includes:

[0108] 3.1.1) The prepared antibody conjugate was subjected to anion exchange column chromatography to obtain a flow-through sample;

[0109] 3.1.2) The obtained flow-through sample is subjected to cation exchange column chromatography to obtain the eluted sample.

[0110] In some embodiments, step 3.1) includes: subjecting the prepared antibody conjugate to size exclusion chromatography, hydrophobic interaction chromatography, mixed-mode chromatography or ceramic hydroxyapatite chromatography to obtain the elution product.

[0111] In some embodiments, step 3.1) includes: subjecting the prepared antibody conjugate to hydrophobic interaction chromatography, mixed-mode chromatography, or ceramic hydroxyapatite chromatography to obtain the elution product.

[0112] In some embodiments, the buffer solution for the ion exchange column chromatography can be selected as needed.

[0113] In some embodiments, the elution solution for the cation exchange column chromatography is selected from Tris-HAc buffer, NaAc-HAc buffer, sodium citrate-citrate buffer, MES sodium salt buffer, Bis-Tris buffer, and any combination thereof.

[0114] In some embodiments, the equilibration solution for the anion exchange column chromatography is selected from Tris-HAc buffer, NaAc-HAc buffer, sodium citrate-citrate buffer, MES sodium salt buffer, Bis-Tris buffer, and any combination thereof.

[0115] In some embodiments, the elution solution for the hydrophobic interaction chromatography can be selected as needed.

[0116] In some embodiments, the elution solution for the hydrophobic interaction chromatography is selected from Tris-HAc buffer, Tris-HCl buffer, and phosphate buffer (PB buffer), and any combination thereof.

[0117] In some embodiments, the pH of the Tris-HAc buffer is 6.5 to 7.5, for example 7.0.

[0118] In some embodiments, the pH of the NaAc-HAc buffer solution is 5.0 to 6.0, for example, 5.5.

[0119] In some embodiments, the pH of the phosphate buffer is 6.0 to 7.0, for example 6.5.

[0120] In some implementations, step 3.2) includes membrane filtration of the product from the previous step.

[0121] In some implementations, step 3.2) includes nanofiltration of the product from the previous step.

[0122] In some implementations, step 3.2) includes membrane filtration of the product of step 3.1).

[0123] In some implementations, step 3.2) includes nanofiltration of the product of step 3.1).

[0124] In some embodiments, potential viruses in the product are removed by membrane filtration. In some embodiments, potential viruses in the product are removed by nanofiltration.

[0125] In some embodiments, the nanofilter is selected from Planova 20N, Planova BioEx, and Viresolve Pro.

[0126] In some embodiments, the nanofilter is a Planova 20N with an operating pressure of 0.7-1.0 bar.

[0127] In some implementations, the concentration and / or replacement buffer described in step 3.3) is performed by ultrafiltration.

[0128] In some implementations, the concentration described in step 3.3) is performed by ultrafiltration.

[0129] In some implementations, step 3.3) further includes: washing the filter.

[0130] In some implementations, step 3.3) is followed by: filter washing.

[0131] In some implementations, step 3) includes:

[0132] 3.1) The prepared antibody conjugate was subjected to column chromatography.

[0133] Step 3.1) includes:

[0134] 3.1.1) The prepared antibody conjugate was subjected to cation exchange column chromatography to obtain the eluted sample;

[0135] 3.1.2) The eluted sample was subjected to anion exchange column chromatography to obtain a flow-through sample;

[0136] or,

[0137] 3.1.1) The prepared antibody conjugate was subjected to anion exchange column chromatography to obtain a flow-through sample;

[0138] 3.1.2) The obtained flow-through sample was subjected to cation exchange column chromatography to obtain the eluted sample;

[0139] 3.2) Remove potential viruses from the product of the previous step;

[0140] 3.3) Concentrated and / or replacement buffer;

[0141] The order of steps 3.2) and 3.3) can be interchanged;

[0142] The cation exchange column chromatography, anion exchange column chromatography, concentration and / or displacement buffer, and removal of potential viruses from the product may be as defined in any embodiment of this application.

[0143] In some implementations, step 3) includes:

[0144] 3.1) The prepared antibody conjugate was subjected to column chromatography.

[0145] Step 3.1) includes:

[0146] 3.1.1) The prepared antibody conjugate was subjected to cation exchange column chromatography to obtain the eluted sample;

[0147] 3.1.2) The eluted sample was subjected to anion exchange column chromatography to obtain a flow-through sample;

[0148] or,

[0149] 3.1.1) The prepared antibody conjugate was subjected to anion exchange column chromatography to obtain a flow-through sample;

[0150] 3.1.2) The obtained flow-through sample was subjected to cation exchange column chromatography to obtain the eluted sample;

[0151] 3.2) Remove potential viruses from the product of step 3.1);

[0152] 3.3) Concentration;

[0153] Wherein, the cation exchange column chromatography, anion exchange column chromatography, concentration, and step 3.2) can be as defined in any embodiment of this application.

[0154] In some implementations, step 3) includes:

[0155] 3.1) The prepared antibody conjugate was subjected to column chromatography.

[0156] Step 3.1) includes: performing hydrophobic interaction chromatography on the prepared antibody conjugate to obtain the elution product;

[0157] 3.2) Remove potential viruses from the product of the previous step;

[0158] 3.3) Concentrated and / or replacement buffer;

[0159] The order of steps 3.2) and 3.3) can be interchanged;

[0160] The hydrophobic interaction chromatography, concentration and / or displacement buffer, and removal of potential viruses from the product can be as defined in any embodiment of this application.

[0161] In some implementations, step 3) includes:

[0162] 3.1) The prepared antibody conjugate was subjected to column chromatography.

[0163] Step 3.1) includes: performing hydrophobic interaction chromatography on the prepared antibody conjugate to obtain the elution product;

[0164] 3.2) Remove potential viruses from the product of step 3.1);

[0165] 3.3) Concentration;

[0166] The hydrophobic interaction chromatography, concentration, and step 3.2) can be defined as in any embodiment of this application.

[0167] In some implementations, the method includes:

[0168] 1) Providing unpurified or partially purified antibodies or antigen-binding fragments thereof, the steps comprising:

[0169] 1.1) Provide a sample containing an antibody or its antigen-binding fragment;

[0170] 1.2) Capture antibodies or their antigen-binding fragments in the sample;

[0171] 1.3) Inactivate the virus by capturing the antibody or its antigen-binding fragment;

[0172] 1.4) Centrifuge and / or perform deep filtration on the product from step 1.3);

[0173] 2) To prepare an antibody-drug conjugate by conjugating an unpurified or partially purified antibody or its antigen-binding fragment to a payload via a linker, the step comprising:

[0174] To couple unpurified or partially purified antibodies or their antigen-binding fragments to the linker-payload; or

[0175] 2.1) To conjugate unpurified or partially purified antibodies or their antigen-binding fragments to linkers.

[0176] 2.2) React the product of step 2.1) with the payload;

[0177] 3) Purifying the antibody-conjugate, the steps of which include:

[0178] 3.1) The prepared antibody conjugate was subjected to column chromatography.

[0179] Step 3.1) includes:

[0180] 3.1.1) The prepared antibody conjugate was subjected to cation exchange column chromatography to obtain the eluted sample;

[0181] 3.1.2) The eluted sample was subjected to anion exchange column chromatography to obtain a flow-through sample;

[0182] or,

[0183] 3.1.1) The prepared antibody conjugate was subjected to anion exchange column chromatography to obtain a flow-through sample;

[0184] 3.1.2) The obtained flow-through sample was subjected to cation exchange column chromatography to obtain the eluted sample;

[0185] 3.2) Remove potential viruses from the product of the previous step;

[0186] 3.3) Concentrated and / or replacement buffer;

[0187] The order of steps 3.2) and 3.3) can be interchanged;

[0188] The sample, capture, virus inactivation, deep filtration, removal of potential viruses, hydrophobic interaction chromatography, concentration and / or replacement buffer, and removal of potential viruses from the product may be as defined in any embodiment of this application.

[0189] In some implementations, the process flow diagram of the method is shown in Figure 3.

[0190] In some implementations, the method includes:

[0191] 1) Providing unpurified or partially purified antibodies or antigen-binding fragments thereof, the steps comprising:

[0192] 1.1) Provide a sample containing an antibody or its antigen-binding fragment;

[0193] 1.2) Capture antibodies or their antigen-binding fragments in the sample;

[0194] 1.3) Inactivate the virus by capturing the antibody or its antigen-binding fragment;

[0195] 1.4) Centrifuge and / or perform deep filtration on the product from step 1.3);

[0196] 1.5) Remove potential viruses from the product of step 1.4), for example, by removing potential viruses by anion exchange chromatography;

[0197] 2) To prepare an antibody-drug conjugate by conjugating an unpurified or partially purified antibody or its antigen-binding fragment to a payload via a linker, the step comprising:

[0198] To couple unpurified or partially purified antibodies or their antigen-binding fragments to the linker-payload; or

[0199] 2.1) To conjugate unpurified or partially purified antibodies or their antigen-binding fragments to linkers.

[0200] 2.2) React the product of step 2.1) with the payload;

[0201] 3) Purifying the antibody-conjugate, the steps of which include:

[0202] 3.1) Perform column chromatography on the prepared antibody conjugate, for example, perform hydrophobic interaction chromatography on the prepared antibody conjugate to obtain the elution product;

[0203] 3.2) Remove potential viruses from the product of the previous step;

[0204] 3.3) Concentrated and / or replacement buffer;

[0205] The order of steps 3.2) and 3.3) can be interchanged;

[0206] The sample, capture, virus inactivation, deep filtration, removal of potential viruses, hydrophobic interaction chromatography, concentration and / or replacement buffer, and removal of potential viruses from the product may be as defined in any embodiment of this application.

[0207] In some implementations, the process flow diagram of the method is shown in Figure 6.

[0208] In some implementations, column chromatography is also included between step 1) and step 2).

[0209] In some embodiments, the column chromatography is selected from ion exchange chromatography, hydrophobic interaction chromatography, and any combination thereof.

[0210] In some embodiments, the ion exchange chromatography is performed in a binding-elution mode or a flow-through mode.

[0211] In some embodiments, the ion exchange chromatography includes:

[0212] The antibody or its antigen-binding fragment is subjected to cation exchange column chromatography to obtain the eluted sample;

[0213] The eluted sample was subjected to anion exchange column chromatography to obtain a flow-through sample.

[0214] In some implementations, column chromatography is followed by the removal of potential viruses.

[0215] In some implementations, potential viruses are removed by membrane filtration.

[0216] In some embodiments, the membrane filtration is nanofiltration.

[0217] In some implementations, step 3) includes: concentrating and / or replacing the buffer solution.

[0218] In some embodiments, the concentration and / or replacement buffer is performed by ultrafiltration.

[0219] In some implementations, rinsing is performed simultaneously with concentration and / or displacement buffer, or rinsing is performed after concentration and / or displacement buffer.

[0220] In some implementations, step 3) includes: concentration.

[0221] In some implementations, the concentration is performed by ultrafiltration.

[0222] In some implementations, filtration is performed during concentration, or filtration is performed after concentration.

[0223] In some implementations, the process flow diagram of the method is shown in Figure 4.

[0224] In some implementations, column chromatography is also included between step 1) and step 2).

[0225] In some embodiments, the column chromatography is selected from ion exchange chromatography, hydrophobic interaction chromatography, and any combination thereof.

[0226] In some embodiments, the ion exchange chromatography is performed in a binding-elution mode or a flow-through mode.

[0227] In some embodiments, the ion exchange chromatography includes:

[0228] The antibody or its antigen-binding fragment is subjected to cation exchange column chromatography to obtain the eluted sample;

[0229] The eluted sample was subjected to anion exchange column chromatography to obtain a flow-through sample.

[0230] In some implementations, step 3) includes:

[0231] Remove potential viruses from the product of the previous step.

[0232] Concentrated and / or displacement buffers.

[0233] In some implementations, step 3) includes:

[0234] Remove potential viruses from the product of the previous step.

[0235] concentrate.

[0236] In some implementations, potential viruses are removed by membrane filtration.

[0237] In some embodiments, the membrane filtration is nanofiltration.

[0238] In some embodiments, the concentration and / or replacement buffer is performed by ultrafiltration.

[0239] In some implementations, rinsing is performed simultaneously with concentration and / or displacement buffer, or rinsing is performed after concentration and / or displacement buffer.

[0240] In some implementations, the concentration is performed by ultrafiltration.

[0241] In some implementations, washing is performed during concentration, or washing is performed after concentration and / or replacement of the buffer.

[0242] In some implementations, the process flow diagram of the method is shown in Figure 5.

[0243] There are no particular limitations on the antibodies that can be used in this invention.

[0244] In some embodiments, the antibody is selected from monospecific antibodies (recognizing one antigen or epitope), bispecific antibodies (recognizing two different antigens or epitopes simultaneously), and multispecific antibodies (recognizing more than two different antigens or epitopes simultaneously), such as trastuzumab, pertuzumab, sacituzumab, abciximab, adalimumab, alefacept, alemtuzumab, basiliximab, belimumab, bezlotoxumab, canakinumab, certolizumab (pegylated), and cetuximab. The following are listed: ximab, daclizumab, denosumab, efalizumab, golimumab, inflectra, ipilimumab, ixekizumab, natalizumab, nivolumab, olaratumab, omalizumab, palivizumab, panitumumab, pembrolizumab, rituximab, tocilizumab, secukinumab, and ustekinumab.In some embodiments, the antibody is selected from antibodies against any of the following antigens: BMPR1B, E16, STEAP1, MUC16, MPF, Napi2b, Sema5b, PSCA. hlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, HER2, NCA, MDP, IL20Ra, Brevican, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD22, CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH1, FcRH5, TENB2, PMEL17, TMEFF1, GDNF-Ra1, Ly6E, TMEM46, Ly6G6D, LGR5, RET, Ly6K, GPR19, GPR54, ASPHD1, tyrosinase, TMEM118, GPR172A, CD33, and CLL-1.

[0245] Specific examples of antibodies also include human antibodies, humanized antibodies, or chimeric antibodies. A “human antibody” is an antibody whose amino acid sequence corresponds to that of an antibody produced by a human or human cell, or an antibody derived from a non-human source using a human antibody library or other human antibody-encoding sequences. This definition of a human antibody explicitly excludes humanized antibodies containing non-human antigen-binding residues. A “humanized antibody” is a chimeric antibody containing amino acid residues from a non-human HVR and amino acid residues from a human FR. In some embodiments, a humanized antibody will contain substantially all or at least one (typically two) variable domains, wherein all or substantially all HVRs (e.g., CDRs) correspond to the HVRs of a non-human antibody, and all or substantially all FRs correspond to the FRs of a human antibody. A humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. A “humanized form” of an antibody, such as a non-human antibody, refers to an antibody that has undergone humanization. A “chimeric” antibody is an antibody in which a portion of the heavy chain and / or light chain originates from a particular source or species, while the remainder of the heavy chain and / or light chain originates from a different source or species.

[0246] In some embodiments, the antibody is a monoclonal antibody, such as a human antibody or a humanized antibody. Useful monoclonal antibodies are homogeneous groups of antibodies against specific antigens (e.g., cancer cell antigens, viral antigens, microbial antigens covalently linked to a second molecule). Monoclonal antibodies (mAbs) against a target antigen can be prepared using any technique known in the art, such as generating antibody molecules by culturing serial cell lines. These techniques include, but are not limited to, hybridoma techniques originally described by Kohler and Milstein (1975, Nature 256, 495-497), human-B cell hybridoma techniques (Kozbor et al., 1983, Immunology Today 4:72), and EBV-hybridoma techniques (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies can be any class of immunoglobulins, including IgG, IgM, IgE, IgA, and IgD, and any of their subclasses. Hybridomas that produce the monoclonal antibodies used in this invention can be cultured in vitro or in vivo. Useful monoclonal antibodies include, but are not limited to, human monoclonal antibodies or human-mouse (or other species) chimeric monoclonal antibodies. Human monoclonal antibodies can be prepared by a variety of techniques known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. USA 80, 7308-7312; Kozbor et al., 1983, Immunology Today 4, 72-79; and Olsson et al., 1982, Meth. Enzymol. 92, 3-16).

[0247] Furthermore, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, which contain both human and non-human portions and can be prepared using standard recombinant DNA techniques, are useful ligands. Chimeric antibodies are molecules whose different portions originate from different animal species, such as molecules having a variable region derived from a mouse monoclonal antibody and a constant region derived from human immunoglobulins. (See, for example, U.S. Patent No. 4,816,567 to Cabilly et al.; and U.S. Patent No. 4,816,397 to Boss et al., the entire contents of which are incorporated herein by reference.) Humanized antibodies are antibody molecules derived from non-human species that have one or more complementarity-determining regions (CDRs) derived from non-human species and a framework region derived from human immunoglobulin molecules. (See, for example, Queen, U.S. Patent No. 5,585,089, which is incorporated herein by reference in its entirety.) Such chimeric and humanized monoclonal antibodies can be produced using recombinant DNA techniques known in the art, for example using International Publication No. WO 87 / 02671; European Patent Publication No. 184,187; European Patent Publication No. 171,496; European Patent Publication No. 173,494; International Publication No. WO 86 / 01533; U.S. Patent No. 4,816,567; European Patent Publication No. 125,023; Berter et al., 1988, Science 240:1041-1043; Liu et al., 1987, Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al., 1987, *Journal of Immunology*, 139:3521-3526; Sun et al., 1987, *Proceedings of the National Academy of Sciences of the United States of America*, 84:214-218; Nishimura et al., 1987, *Cancer Research*, 47:999-1005; Wood et al., 1985, *Nature*, 314:446-449; and Shaw et al., 1988, *Journal of the National Cancer Institute*. Inst.) 80:1553-1559; Morrison, 1985, Science 229:1202-1207; Oi et al., 1986, Biotechnology 4:214; US Patent No. 5,225,539; Jones et al., 1986, Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et al., 1988, J. Immunol. 141:4053-4060; each of these is incorporated into this paper in its entirety by citation.

[0248] Examples of isotypes of the antibodies disclosed herein include IgG (IgG1, IgG2, IgG3, or IgG4).

[0249] In some embodiments, the antibody is a polyclonal antibody. A useful polyclonal antibody is a heterogeneous group of antibody molecules derived from the serum of an immunized animal. Polyclonal antibodies against a target antigen can be produced using various methods well known in the art. For example, to produce polyclonal antibodies, various host animals, including but not limited to rabbits, mice, rats, and guinea pigs, can be immunized by injecting a target antigen or a derivative thereof.

[0250] In some implementations, the antibody is an antibody fragment, such as Fv, Fab, Fab', scFv, a biantibody, or an F(ab')2 fragment.

[0251] There are no particular limitations on the payloads and linkers that can be used in the antibody conjugates of this invention, provided that the payload has the desired effect (e.g., diagnostic, therapeutic, labeling, etc.) and has at least one substituent or partial structure that allows linkage to the linker structure, and the linker contains at least two reactive groups, one of which can covalently bind the payload and the other capable of covalently coupling the antibody. Depending on the desired payload and the selected linker, those skilled in the art can choose appropriate methods to couple them together. For example, some conventional coupling methods, such as amine coupling, can be used to form the desired linker-payload complex, which still contains reactive groups for covalently coupling to the antibody.

[0252] In some embodiments, the linker contains a group capable of reacting with an antibody or its antigen-binding fragment, as well as a group capable of being linked to a payload. In some embodiments, the linker in the linker-payload configuration contains a group capable of reacting with an antibody or its antigen-binding fragment, for example, the linker contains a maleimide group, a bromine atom, or a methanesulfonyl group.

[0253] In some embodiments, the payload is selected from diagnostic agents, therapeutic agents, and labeling reagents. For example, the payload may be a cytotoxic agent (e.g., a chemotherapeutic agent, immunotherapeutic agent, antiviral agent, or antibacterial agent), a toxin, a radionuclide, a fluorescent agent, an oligonucleotide, a polypeptide, a molecular degrader, an immune agonist, or a radiopharmaceutical chelator. In some embodiments, the cytotoxic agent includes, but is not limited to, tubulin inhibitors, DNA alkylating agents, DNA chimeras, enzyme inhibitors, and antimetabolites. In some embodiments, the cytotoxic agent is auristatin, monomethylauristatin E (MMAE), monomethylauristatin D (MMAD), monomethylauristatin F (MMAF), maytansine or its derivatives (e.g., maytansine-like substances, DM1, DM3, DM4), paclitaxel, calcitrazine, pyroxine, doxorubicin, camptothecin-type cytotoxic agents and their derivatives (e.g., eczetidine, topotecan, irinotecan, belotecidine), PBD (pyrrolobenzodiazepine)-type cytotoxic agents and their derivatives. In some embodiments, the payload is PEG. In some embodiments, the fluorescent agent includes, but is not limited to, amine-derived fluorescent probes, such as 5-dimethylaminonaphthalene-1-(N-(2-aminoethyl))sulfonamide-dansyl ethylenediamine. 488 cadaverine (catalog number O-10465, Molecular Probes), dansyl cadaverine, N-(2-aminoethyl)-4-amino-3,6-disulfon-1,8-naphthalenediamine, dipotassium salt (fluorescent yellow ethylenediamine), or rhodamine B ethylenediamine (catalog number L-2424, Molecular Probes), or thiol-derived fluorescent probes, such as FLL-cystine (catalog number B-20340, molecular probe), etc.

[0254] In some embodiments, the payload is selected from diagnostic agents, therapeutic agents, and labeling reagents, such as cytotoxins, oligonucleotides, peptides, molecular degraders, immune agonists, and radiopharmaceutical chelators. In some embodiments, the cytotoxin is a tubulin inhibitor, DNA alkylating agent, DNA chimera, enzyme inhibitor, or antimetabolite. In some embodiments, the cytotoxin is auristatin, monomethylauristatin E (MMAE), maytansine or its derivatives (e.g., maytansine-like substances, DM1, DM3, DM4), paclitaxel, calcitrazine, pyroxine, doxorubicin, camptothecin-type cytotoxins and their derivatives (e.g., eczema, topotecan, irinotecan, belotecone), PBD (pyrrolobenzodiazepine)-type cytotoxins and their derivatives. In some embodiments, the payload is PEG.

[0255] In some embodiments, the antibody conjugate is an antibody-drug conjugate.

[0256] Terminology Definition

[0257] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the virological, biochemical, and immunological laboratory procedures used herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0258] Throughout this disclosure, unless the context otherwise requires, the words “for example,” “such as,” or variations thereof will not be considered restrictive terms but will be interpreted as meaning “including but not limited to” or “not limited to.”

[0259] Throughout this disclosure, unless the context otherwise requires, the words “comprising,” “including,” and “containing” will be understood to mean including the stated steps or elements or a group of steps or elements, but not excluding any other steps or elements or a group of steps or elements. When the words “comprising,” “including,” “containing,” or variations thereof are used herein, the meanings of the phrases “consisting of,” and “substantially consisting of,” are also provided. The phrase “consisting of,” means including and limited to what follows the phrase “consisting of.” The phrase “substantially consisting of,” means including any element listed after this phrase, and is limited to other elements that contribute to or do not impede the activity or function of the listed element.

[0260] Throughout this disclosure, unless the context otherwise requires, the words “an” and “a kind” as well as “the” and similar designations shall be interpreted to cover both the singular and the plural in the context of describing the invention (especially in the context of the following claims).

[0261] In this document, the term "antibody-drug conjugate" or ADC refers to a conjugate formed by covalently linking a payload (or drug) directly or indirectly via one or more suitable linkers to an antibody or its antigen-binding fragment. ADCs are typically in the form of "antibody-linker-payload (or drug)," where the payload includes, but is not limited to, diagnostic agents, therapeutic agents, and labeling agents. Antibody-drug conjugates combine the desirable properties of both antibodies and payloads (e.g., cytotoxic drugs), enhancing the effect of the payload by targeting it to cells expressing the antigen.

[0262] In this paper, the term "linker" refers to a molecule containing at least two reactive groups, one of which can be covalently linked to a payload (or drug) and the other can be covalently linked to an antibody.

[0263] In this document, the term "antibody" includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody, bispecific antibody, multivalent or bivalent antibody that binds to a specific antigen. A naturally occurring intact antibody consists of two heavy chains and two light chains. Each heavy chain comprises a variable region ("HCVR") and first, second, and third constant regions (CH1, CH2, and CH3), while each light chain comprises a variable region ("LCVR") and a constant region (CL). Mammalian heavy chains are classified as α, δ, ε, γ, and μ, and mammalian light chains are classified as λ or κ. Antibodies are typically "Y"-shaped, where the stem of the Y consists of the second and third constant regions of two heavy chains linked together via disulfide bonds. Each arm of the Y comprises the variable region and first constant region of a single heavy chain, which binds to the variable region and constant region of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in both chains typically contain three highly variable loops called complementarity-determining regions (CDRs) (the CDRs for light (L) chains include LCDR1, LCDR2, and LCDR3, and the CDRs for heavy (H) chains include HCDR1, HCDR2, and HCDR3). The CDR boundary of an antibody can be defined or identified by rules such as those of Kabat, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927(1997); Chothia, C. et al., J Mol Biol. Dec 5; 186(3): 651-63(1985); Chothia, C. and Lesk, AM, J. Mol. Biol., 196, 901(1987); Chothia, C. et al., Nature. Dec 21-28; 342(6252): 877-83(1989); Kabat EA et al., National Institutes of Health, Bethesda, Md.(1991)). Three CDRs are located between flank segments called framework regions (FRs), which are more conserved than the CDRs and form a scaffold to support the highly variable ring. Each HCVR and LCVR comprises four FRs, and the CDRs and FRs are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant regions of the heavy and light chains do not participate in antigen binding but exhibit various effector functions. Antibodies are designated into various classes based on the amino acid sequence of their heavy chain constant regions. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively.Several major antibody classes are divided into subclasses such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain) or IgA2 (α2 heavy chain).

[0264] In this document, the term "antibody fragment" includes a portion of a full-length antibody, typically its antigen-binding segment or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; biantibodies; linear antibodies; anti-idiotypic antibodies; and any fragment that binds to an epitope (which immune-specifically binds to cancer cell antigens, viral antigens, or microbial antigens); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0265] In this document, the term "antigen-binding fragment" refers to an antibody fragment formed from a portion of an antibody containing one or more CDRs, or any other antibody fragment that binds an antigen but does not contain the complete structure of the native antibody. Examples of antigen-binding moieties include, but are not limited to, variable domains, variable regions, biantibodies, Fab, Fab', F(ab')2, Fv fragments, scFv, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized biantibodies, multispecific antibodies, camel-derived single-domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. Antigen-binding moieties are capable of binding the same antigen as that bound to the parent antibody. In some embodiments, the antigen-binding moieties may contain one or more CDRs from a specific human antibody that are transposed to a framework region from one or more different human antibodies.

[0266] The term "Fab" in antibody refers to the following portion of the antibody, which consists of a single light chain (both the variable and constant regions) associated with a variable region and a first constant region of a single heavy chain via disulfide bonds. In some embodiments, both the first and second antigen-binding portions of the antibody to be conjugated are in Fab form. Further, the constant regions (i.e., CH1 and CL) of the two chains of the Fab are replaced with engineered or modified T-cell receptor (TCR) constant regions.

[0267] The "Fc" in antibody refers to the following portion of the antibody, which contains the second (CH2) and third constant regions (CH3) of the first heavy chain, which bind to the second and third constant regions of the second heavy chain via disulfide bonds and optionally hinge regions. The Fc portion of an antibody is responsible for various effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), but does not play a role in antigen binding.

[0268] The "hinge region" of an antibody comprises the portion of the heavy chain molecule that connects the CH1 and CH2 domains. This hinge region consists of approximately 25 amino acid residues and is flexible, thereby allowing the two N-terminal antigen-binding regions to move independently.

[0269] In this paper, the term "depth filtration" is a method of separating particles from a fluid. When the substance to be purified passes through a filter medium, the particles are deposited inside the medium, thus separating them from the fluid.

[0270] In this article, the term "ultrafiltration (UF)" refers to a membrane separation process driven by tangential flow and utilizing the principle of molecular sieves. Under certain pressure conditions, ultrafiltration allows solvents and small molecular weight substances to permeate through a microporous membrane, while larger molecules are retained. The retained portion is the concentrate, thus achieving separation, purification, and concentration. The filtration precision of ultrafiltration is 0.001-0.1 micrometers.

[0271] In this article, the term "diafiltration (DF)," also known as liquid replacement or buffer replacement, refers to the addition of a buffer solution to replace the liquid surrounding a substance with a liquid that is more conducive to achieving storage stability.

[0272] In this paper, the term "nanofiltration (NF)" refers to the process based on the principle of molecular sieves. It utilizes the size difference between viruses and proteins, allowing proteins smaller than the average pore size to pass through the filter membrane, while viruses larger than the average pore size are retained within the membrane, thus achieving virus removal. Filter membranes with different average pore sizes can be selected based on the size of the target protein. Nanofiltration simultaneously satisfies the characteristics of high virus removal efficiency, high permeability of the target protein (recovery volume, filtration time), and no change in the target protein's properties. Furthermore, regardless of whether the virus is enveloped or non-enveloped, and regardless of whether the viral genome is RNA or DNA, virus removal is primarily achieved through pore size, making it the most reliable virus removal and inactivation technology currently available.

[0273] In this article, the term "affinity chromatography," also known as affinity chromatography, refers to a chromatographic method that uses the binding properties of a stationary phase to separate molecules. Affinity chromatography packing material specifically binds to the substances to be separated, and this binding is reversible; the substances can be separated by changing the mobile phase conditions. Affinity chromatography can be used to purify or concentrate a molecule from a mixture, or to remove or reduce the concentration of a molecule in a mixture. Protein A, a cell wall surface protein derived from Staphylococcus aureus, specifically binds to antibody molecules in a sample, allowing other contaminating proteins to flow through.

[0274] In this document, the term "ion exchange chromatography (IEC)" refers to the separation of substances based on their charge using a chromatographic matrix composed of charged resins or cellulose. The term "anion exchange chromatography (AEC)" refers to the use of negatively charged resins or cellulose as a matrix to bind negatively charged substances (e.g., proteins), followed by elution by increasing the salt concentration in the eluent. In this application, "anion exchange chromatography" is also referred to as "anion exchange column chromatography" or "anion column chromatography." The term "cation exchange chromatography (CEC)" refers to the use of positively charged resins or cellulose as a matrix to bind positively charged substances (e.g., proteins), followed by elution by increasing the salt concentration or pH of the eluent. In this application, "cation exchange chromatography" is also referred to as "cation exchange column chromatography" or "cation column chromatography."

[0275] In this document, the term "Hydrophobic Interaction Chromatography (HIC)" refers to a method of separation based on differences in the hydrophobicity of the surfaces of the substances being separated. Different substances being separated (e.g., proteins) have different hydrophobic properties, resulting in varying strengths of hydrophobic interactions with the hydrophobic chromatographic medium. During elution, substances with different hydrophobic interactions are eluted sequentially and purified. In this application, "Hydrophobic Interaction Chromatography" is also referred to as "Hydrophobic Interaction Column Chromatography."

[0276] In this article, the term "size exclusion chromatography (SEC)," also known as gel filtration chromatography or molecular sieve chromatography, refers to the separation of substances based on their molecular size, utilizing the molecular sieving effect of a gel with a network structure. The chromatography column packing material is an inert, porous network structure, often composed of cross-linked polysaccharides (such as dextran or agarose). Small molecules can enter the interior, traveling a longer distance and typically eluting later, while large molecules are excluded, traveling a shorter distance and typically eluting earlier. When a mixed solution passes through a molecular sieve chromatography column, it can be separated according to its molecular weight.

[0277] In this paper, the term "mixed-mode chromatography" (MMC), also known as multimodal chromatography, refers to the simultaneous presence of two or more different types of functional ligands on the membrane matrix. These ligands bind to the substances to be separated through various interactions (such as ion exchange, hydrophobic interactions, and hydrogen bonding), thereby achieving efficient separation. "Mixed-mode membrane chromatography" (MMM) combines mixed-mode chromatography with membrane separation technology, achieving efficient separation by simultaneously carrying two or more different types of functional ligands on the membrane matrix.

[0278] In this paper, the term "ceramic hydroxyapatite chromatography (CHT)" refers to porous ceramicized hydroxyapatite (Ca). 10 (PO4)6(OH)2) is a chromatography technique using a stationary phase, on which Ca2+ is also present. 2+ (C position) and PO4 3 The two active sites (P-site) can interact with the substances to be separated through multiple processes such as metal chelation and ion exchange, making it a natural mixed-mode chromatography.

[0279] In this document, the term "site-directed coupling" refers to the introduction of reactive groups that can be used for coupling onto specific amino acid residues of an antibody through genetic engineering or chemical means, thereby achieving the site-directed connection between the antibody and a drug. Site-directed coupling techniques include the introduction of reactive cysteine ​​residues, disulfide bond rebridging, non-natural amino acid techniques, enzyme catalysis, glycosylation techniques, and proximity-induced antibody coupling techniques. Those skilled in the art can rationally select the necessary reaction reagents, such as reducing agents (including but not limited to TCEP, DTT), enzymes (including but not limited to MTGase, Sortase, EndoS), or other catalysts, according to the chosen coupling strategy to achieve efficient and specific coupling reactions.

[0280] In this document, the term "non-site coupling" refers to coupling without modifying or altering the antibody, utilizing the amino group of lysine or the thiol group of cysteine ​​obtained by breaking the interchain disulfide bond. The amino group of lysine is linked to activated carboxylic acid ester linkers or other groups via amide bonds, and the thiol group of cysteine ​​reacts with groups such as maleimide. Those skilled in the art can select appropriate reducing agents (including but not limited to TCEP, DTT) or coupling reagents (including but not limited to NHS esters, maleimide) according to the requirements of the coupling reaction to optimize reaction conditions and control coupling efficiency and uniformity.

[0281] In this paper, the term "partially purified antibody or its antigen-binding fragment" refers to an antibody or its antigen-binding fragment that has only undergone partial processing such as capture, virus inactivation, column chromatography, virus filtration, ultrafiltration, and washing. Beneficial effects

[0282] The production process provided by this invention simplifies the workflow, shortens the production cycle, and reduces reagent consumption, resulting in improved efficiency and cost-effectiveness. Furthermore, the yield and purity of the antibody-drug conjugate stock solution obtained through this integrated process are comparable to those of traditional fractional processes, and the impurity content in the process is below the detection limit. Using the ADC production process of this invention to prepare antibody-drug conjugates helps reduce production costs and improves the competitiveness of the product after it enters the market.

[0283] The beneficial effects of the process of this invention are specifically reflected in the following aspects:

[0284] 1) The production process provided by this invention saves purification steps compared to traditional processes: The integrated process provided by this invention can simultaneously remove residual impurities in the antibody raw material and impurities in the coupling step during the chromatography step. For example, linkers, payloads, linker-payload combinations, and organic solvents in the coupling step usually do not bind to the chromatography column and flow through the column during sample loading. Further elution can effectively remove them. In addition, in traditional processes, the preparation of purified antibodies requires one UF / DF process, and the preparation of antibody conjugates requires another UF / DF process, resulting in large reagent consumption and long process time. However, the integrated process provided by this invention only requires one UF / DF process for the prepared antibody conjugates, reducing reagent consumption and shortening the production cycle.

[0285] 2) The production process provided by this invention saves the development steps of antibody preparations compared with traditional processes: In traditional processes, purified antibodies need to be formulated into preparations for storage. The integrated process provided by this invention can directly couple unpurified or partially purified antibodies or their antigen-binding fragments to linkers-payloads, eliminating the need to develop purified antibody preparations, saving preparation development steps, shortening the production cycle, and saving storage space.

[0286] 3) The production process provided by this invention saves detection time compared with the traditional process: In the traditional process, after the purified antibody is prepared, the antibody needs to be released for testing, and after conjugation, the antibody conjugate needs to be released for testing. The integrated process provided by this invention only needs to be released for testing once after the antibody conjugate is obtained, which reduces the number of tests and shortens the production cycle. Attached Figure Description

[0287] Figure 1 is a schematic diagram of the traditional ADC process flow;

[0288] Figure 2 is a schematic diagram of the process flow of some comparative examples of the present invention;

[0289] Figure 3 is a schematic diagram of the process flow of some embodiments of the present invention;

[0290] Figure 4 is a schematic diagram of the process flow of some embodiments of the present invention;

[0291] Figure 5 is a schematic diagram of the process flow of some embodiments of the present invention;

[0292] Figure 6 is a schematic diagram of the process flow of some embodiments of the present invention;

[0293] Figure 7 is a schematic diagram of the process flow of some comparative examples of the present invention;

[0294] Figure 8 is a schematic diagram of the process flow of some embodiments of the present invention;

[0295] Figure 9 is a schematic diagram of the process flow of some embodiments of the present invention;

[0296] Figure 10 is a schematic diagram of the process flow of some embodiments of the present invention. Detailed Implementation

[0297] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Unless otherwise stated, reagents, materials, or instruments used that do not specify the manufacturer are all conventional products that can be purchased commercially. The features and performance of the present invention will be further described in detail below with reference to the embodiments.

[0298] Experimental materials

[0299] Clarified cell culture medium expressing antibodies

[0300] The plasmid construct encoding the anti-human HER2 antibody trastuzumab was transfected into CHO-K1 cells and cultured in HighPro 100 (ThermoFisher, A51503IQ) medium. Cell populations with high expression were selected from the transfected CHO cells and cultured for 15 days. The cell culture was centrifuged to obtain a clear cell culture medium expressing trastuzumab.

[0301] Detection methods

[0302] 1. Purity testing

[0303] An Agilent 1260 HPLC system was used with a TSK G3000SW (TOSOH, 0008541) column, PBS as the mobile phase, an injection volume of 100 μg, a flow rate of 1 mL / min, and a detection wavelength of 280 nm.

[0304] 2. DAR value detection

[0305] An Agilent 1260 HPLC system was used with a TSKgel Butyl-NPR (TOSOH, 0014947) column. Mobile phase A consisted of 1.5 M (NH4)2SO4 at pH 7.0, and mobile phase B consisted of 25% propanol at pH 7.0. A linear gradient of 10% B to 100% B was applied for 9 min.

[0306] 3. HCP content detection

[0307] This method employs a sandwich enzyme-linked immunosorbent assay (ELISA). First, 0.5 μg of HCP antibody is added to a highly absorbent 96-well plate (Corning, 42592 or equivalent) for coating. Then, 50 μL of standard and the sample to be tested are added. Finally, TMB (BD Pharmingen, 555214 or equivalent) substrate is added, followed by stop solution (2M sulfuric acid). The absorbance is then read at 450 / 650 nm using an ELISA reader.

[0308] 4. Detection of residual DNA content

[0309] DNA was extracted using the magnetic bead method (ABIPreSEQ Nucleic Acid Extraction Kit, 4400793, 4400795, 4400675). QPCR was performed on the samples and standard DNA using an Applied Biosystems 7500 system and the resDNASEQ CHO DNA RealTime PCR Reagents (4402431). The residual DNA content in the samples was calculated based on the standard curve equation.

[0310] 5. Detection of free drug content

[0311] The instrument used was an Agilent 1260 Infinity II with an Agilent Eclipse Plus C18 column. Mobile phase A consisted of 0.1% TFA in H2O, and mobile phase B consisted of 0.1% TFA in ACN. The mobile phase was increased from 10% B to 100% B over 10 min. The injection rate was 1 ml / min, the injection volume was 50 μL, and the detection wavelengths were 360 ​​nm and 376 nm.

[0312] Example 1

[0313] Using clarified cell culture medium expressing trastuzumab as the starting material, the Trastuzumab-MC-VC-PAB-MMAE conjugate was prepared using the process flow of the present invention, as shown in Figure 3 of the specification.

[0314] The specific procedures for preparing the Trastuzumab-MC-VC-PAB-MMAE conjugate are as follows:

[0315] Step (A): Trastuzumab in the cell culture clarifying medium (1444 mL, 6.449 mg / mL) was captured using a PrismA chromatography column. The equilibration solution was Tris-HAc buffer at pH 7.4, and the elution solution was NaAc-HAc solution at pH 3.9. 8676 mg of trastuzumab was captured with a purity of 99.0% and a yield of 93%.

[0316] Step (B): The pH of the captured trastuzumab solution was adjusted to 3.6 using acetic acid solution, and then incubated at 18-26°C for 1 hour. After incubation, the pH was neutralized to 5.5 using Tris base solution. 8531 mg of the neutralized sample was then filtered through a deep filter. 8122 mg of trastuzumab was obtained after deep filtration, with a purity of 99.3% and a yield of 95%.

[0317] Step (1): Nine equivalents of TCEP (tris(2-carboxyethyl)phosphonic acid hydrochloride) were added to the deep-filtered trastuzumab intermediate (1825 mg, purity: 99.3%) for reduction. After reacting at 22°C for 3.5 h, 13 equivalents of MC-VC-PAB-MMAE (CAS: 646502-53-6) were added for coupling. The reaction was carried out at 22°C for 1.5 h to obtain the coupling product.

[0318] Step (2): 1760 mg of the coupling product was purified by cation exchange column chromatography using Tris-HAc buffer at pH 7.0 as the elution solution. 1577 mg of eluted product was obtained, with a yield of 89.6% and a purity of 97.9%.

[0319] Step (3): 1380 mg of the cationic eluent was purified by anionic column chromatography using NaAc-HAc buffer at pH 5.5 as the elution solution to obtain 1369 mg of flow-through product, with a yield of 99.2% and a purity of 98.5%.

[0320] Step (4): 1092 mg of anion flow product was subjected to nanofiltration using a Planova 20N nanofilter at an operating pressure of 0.7-1.0 bar to obtain 1067 mg of nanofiltration product with a yield of 97.7% and a purity of 98.3%.

[0321] Step (5): 1035 mg of nanofiltration product was subjected to ultrafiltration and liquid exchange (UF / DF). The liquid exchange solution was sodium acetate solution with pH 5.5. The inlet flux was 350 LMH and the transmembrane pressure difference (TMP) was about 0.9 bar. 997 mg of ADC stock solution was obtained with a yield of 96.3% and a purity of 99.0% by SEC-HPLC.

[0322] In this embodiment, the total yield of ADC stock solution prepared from the starting material, the clarified cell culture medium expressing trastuzumab, was 73%. The ADC stock solution had a DAR value of 8.0, HCP content <1ppm, residual DNA content <0.3pg / mg, and process-related impurities HCP / DNA were all below the detection limit. Free drug residue was <0.004 (%), which was below the detection limit.

[0323] Example 2

[0324] Using clarified cell culture medium expressing trastuzumab as the starting material, the Trastuzumab-MC-PEG conjugate was prepared by using the process flow of the present invention, as shown in Figure 3 of the specification.

[0325] The specific experimental protocol for preparing the Trastuzumab-MC-PEG conjugate is as follows:

[0326] Step (A): Trastuzumab in the cell culture clarifying medium (1382 mL, 6.807 mg / mL) was captured using a PrismA chromatography column. The equilibration solution was Tris-HAc buffer at pH 7.4, and the elution solution was NaAc-HAc solution at pH 3.9. 8815 mg of trastuzumab was captured, with a purity of 98.1% and a yield of 93.7%.

[0327] Step (B): The pH of the captured trastuzumab solution was adjusted to 3.6 using acetic acid solution and incubated for 1 hour. After incubation at 18-26°C, the pH was neutralized to 5.5 using Tris base solution. 8668 mg of the neutralized solution was then filtered through a deep filter. 8281 mg of trastuzumab was obtained after deep filtration, with a purity of 98.1% and a yield of 95.5%.

[0328] Step (1): Add 7 equivalents of TCEP to the deep-filtered trastuzumab intermediate (2100mg, purity: 98.1%) for reduction, react at 22°C for 3.5h, then add 18 equivalents of MC-PEG (CAS No.: 640267-62-5) for coupling, react at 22°C for 1.5h to obtain the coupling product.

[0329] Step (2): 1905 mg of the coupling product was purified by cation column chromatography using NaAc-HAc buffer at pH 5.5 as the elution solution. 1865 mg of eluted product was obtained, with a yield of 97.9% and a purity of 98.9%.

[0330] Step (3): 1742 mg of cation elution product was purified by anion exchange column chromatography using NaAc-HAc buffer at pH 5.5 as the elution solution to obtain 1737 mg of flow-through product with a yield of 99.7% and a purity of 99.0%.

[0331] Step (4): 908 mg of anion flow product was subjected to nanofiltration using a Viresolve Pro nanofilter to obtain 902 mg of nanofiltration product, with a yield of 99.3% and a purity of 99.0%.

[0332] Step (5): 871 mg of nanofiltration product was subjected to ultrafiltration and liquid exchange (UF / DF). The liquid exchange solution was sodium acetate solution with pH 5.5. The inlet flux was 350 LMH and the transmembrane pressure difference (TMP) was about 0.9 bar. 872 mg of ADC stock solution was obtained with a yield of 100% and a purity of 98.9%.

[0333] In this embodiment, the total yield of ADC stock solution prepared from the starting material, the clarified cell culture medium expressing trastuzumab, was 86%. The ADC stock solution had a DAR value of 7.9, HCP content <0.4ppm, residual DNA content <0.5pg / mg, and process-related impurities HCP / DNA were all below the detection limit. Free drug residue was <1.3 (%), which was below the detection limit.

[0334] Compare with Example 1

[0335] Using the trastuzumab intermediate obtained through deep filtration in step (B) of Example 1 as the starting material, the Trastuzumab-MC-VC-PAB-MMAE conjugate was prepared using a conventional ADC process. The process flow for the comparative example is shown in Figure 2 of the specification.

[0336] The specific procedures for preparing the Trastuzumab-MC-VC-PAB-MMAE conjugate are as follows:

[0337] Step (1): The trastuzumab intermediate (3420 mg, purity: 99.3%) after deep filtration was purified by anion exchange column chromatography. NaAc-HAc buffer at pH 5.5 was used as the equilibration solution to obtain 3431 mg of flow-through product with a yield of 100% and a purity of 99.1%.

[0338] Step (2): 3152 mg of anion-flowed product was purified by cation column chromatography. NaAc-HAc buffer at pH 5.5 was used as the equilibration and elution solution to obtain 3011 mg of elution product, with a yield of 96% and a purity of 99.8%.

[0339] Step (3): 2933 mg of cation elution product was subjected to nanofiltration using a Planova BioEx nanofilter at an operating pressure of 2-3 bar to obtain 2912 mg of nanofiltration product with a yield of 99% and a purity of 99.8%.

[0340] Step (4): The 2842 mg nanofiltration product was subjected to ultrafiltration and liquid exchange (UF / DF) to prepare 2785 mg of trastuzumab with a yield of 98.0% and a purity of 99.8%. Excipients were added to 2767 mg of trastuzumab, and after filtration, 2693 mg of trastuzumab antigen solution was obtained with a yield of 97.3% and a purity of 99.8%. The trastuzumab antigen solution was frozen at -70°C.

[0341] Step (5): Thaw the trastuzumab antigen solution, add 9 equivalents of TCEP (tris(2-carboxyethyl)phosphonic acid hydrochloride) to trastuzumab (1315mg, purity: 99.8%) for reduction, react at 22°C for 3.5h, then add 13 equivalents of MC-VC-PAB-MMAE (CAS: 646502-53-6) for conjugation, react at 22°C for 1.5h to obtain 1315mg of conjugated product.

[0342] Step (6): 1305 mg of the coupling product was subjected to ultrafiltration and liquid exchange (UF / DF). The liquid exchange solution was sodium acetate solution with pH 5.5. The inlet flux was 350 LMH and the transmembrane pressure difference (TMP) was about 0.9 bar. 1239 mg of ADC stock solution was obtained with a yield of 95% and a purity of 99.5%.

[0343] In this control example, the total yield of ADC stock solution prepared from the starting material, the clarified cell culture medium expressing trastuzumab, was 76%. The ADC stock solution had a DAR value of 8.0, HCP content <1 ppm, residual DNA content <0.3 pg / mg, and process-related impurities HCP / DNA were all below the detection limit. Free drug residues were <0.004 (%), below the detection limit.

[0344] Compare with Example 2

[0345] Using the trastuzumab intermediate obtained through deep filtration in step (B) of Example 2 as the starting material, the Trastuzumab-MC-PEG conjugate was prepared using a conventional ADC process. The process flow for the control example is shown in Figure 2 of the specification.

[0346] The specific steps for preparing the Trastuzumab-MC-PEG conjugate are as follows:

[0347] Step (1): The trastuzumab intermediate (1966 mg, purity: 98.1%) after deep filtration was purified by anion exchange column chromatography. NaAc-HAc buffer at pH 5.5 was used as the equilibration and elution solution to obtain 1945 mg of flow-through product with a yield of 98.9% and a purity of 97.9%.

[0348] Step (2): 1771 mg of anion-flowed product was purified by cation column chromatography using NaAc-HAc buffer at pH 5.5 as the elution solution. 1606 mg of eluted product was obtained, with a yield of 90.7% and a purity of 99.9%.

[0349] Step (3): 1566 mg of cation elution product was nanofiltered using a Viresolve Pro nanofilter to obtain 1555 mg of nanofiltration product, with a yield of 99.3% and a purity of 99.9%.

[0350] Step (4): 1534 mg of nanofiltration product was subjected to ultrafiltration and liquid exchange (UF / DF) to prepare 1517 mg of trastuzumab intermediate with a yield of 98.9% and a purity of 99.9%. Excipients were added to 1384 mg of trastuzumab, and after filtration, 1424 mg of trastuzumab antigen solution was obtained with a yield of 102.9% and a purity of 99.9%. The trastuzumab antigen solution was frozen at -70°C.

[0351] Step (5): Thaw the trastuzumab antigen solution, add 9 equivalents of TCEP (tris(2-carboxyethyl)phosphonium hydrochloride) to 1302mg trastuzumab (1302mg, purity: 99.9%) for reduction, react at 22℃ for 3.5h, then add 13 equivalents of MC-PEG (CAS No.: 640267-62-5) for coupling, react at 22℃ for 1.5h to obtain the coupling product.

[0352] Step (6): 1293 mg of the coupling product was subjected to ultrafiltration and liquid exchange (UF / DF). The liquid exchange solution was sodium acetate solution with pH 5.5. The inlet flux was 350 LMH and the transmembrane pressure difference (TMP) was about 0.9 bar. 1242 mg of ADC stock solution was obtained with a yield of 96.1% and a purity of 99.9%.

[0353] In this control example, the total yield of ADC stock solution prepared from the starting material, the clarified cell culture medium expressing trastuzumab, was 82%. The DAR value of the ADC stock solution was 7.9, the HCP content was <0.4 ppm, the residual DNA content was <0.5 pg / mg, and the process-related impurities HCP / DNA were all below the detection limit. The free drug residue was <1.3 (% mg / mg), which was below the detection limit.

[0354] Example 3

[0355] Using clarified cell culture medium expressing trastuzumab as the starting material, the Trastuzumab-MMAE conjugate was prepared by using the process flow of the present invention, as shown in Figure 6 of the specification.

[0356] The specific experimental protocol for preparing Trastuzumab-MMAE conjugates is as follows:

[0357] Step (A): Trastuzumab in the cell culture clarifying medium (2160 mL, 5.086 mg / mL) was captured using a Prism A chromatography column. The equilibration solution was Tris-HAc buffer at pH 7.4, and the elution solution was NaAc-HAc solution at pH 3.9. 10097 mg of trastuzumab was captured, with a yield of 91.9%.

[0358] Step (B): The pH of the captured trastuzumab solution was adjusted to 3.6 using acetic acid solution, and then incubated at 18-26°C for 1 hour. After incubation, the pH was neutralized to 5.5 using Tris base solution. 10004 mg of the neutralized solution was then filtered through a deep filter. 9666 mg of trastuzumab was obtained after deep filtration, with a yield of 96.6% and a purity of 99.0%.

[0359] Step (C): The trastuzumab intermediate, after deep filtration, was purified by anion exchange column chromatography using 3918 mg of NaAc-HAc buffer at pH 5.5 as the equilibration solution to obtain 3874 mg of flow-through product, with a yield of 98.9% and a purity of 99.0%.

[0360] Step (1): Add 1940 units of transglutaminase (MTGase) and 5 equivalents of NH2-PEG3-VC-PAB-MMAE to the trastuzumab intermediate (1940 mg, purity: 99.0%) of the anion-flowed product at a ratio of 1 U / mg antibody to obtain the conjugated product.

[0361] Step (2): 1852 mg of the coupling product was purified by hydrophobic interaction column chromatography using phosphate buffer at pH 6.5 as the elution solution to obtain 1106 mg of eluted product, with a yield of 59.7% and a product purity of 99.7%.

[0362] Step (3): 1096 mg of hydrophobic interaction chromatography elution product was subjected to nanofiltration using a Viresolve Pro nanofilter to obtain 1082 mg of nanofiltration product, with a yield of 98.7% and a purity of 99.6%.

[0363] Step (4): 1067 mg of nanofiltration product was subjected to ultrafiltration and liquid exchange (UF / DF). The liquid exchange solution was a NaAc-HAc solution with pH 5.5. The inlet flux was 300 LMH and the transmembrane pressure difference (TMP) was about 1.0 bar. 981 mg of ultrafiltration and liquid exchange product was obtained, with a yield of 91.9% and a purity of 99.7%. 673 mg of ultrafiltration and liquid exchange product was subjected to an excipient addition step to prepare 675 mg of ADC stock solution, with a yield of 100.2% and a purity of 99.8%.

[0364] In this embodiment, the total yield of ADC stock solution prepared from the starting material, the clarified cell culture medium expressing trastuzumab, was 49%. The ADC stock solution had a DAR value of 1.8, an HCP content of 2.5 ppm, a residual DNA content of <0.5 pg / mg, a process-related impurity protein A content of <3 ppm, a residual MTGase content of 44 ppm, and a free drug residue of <0.01% (mg / mg), which was below the detection limit.

[0365] Compare with Example 3

[0366] Using the trastuzumab intermediate obtained from anion exchange column chromatography in step (C) of Example 3 as the starting material, the Trastuzumab-MMAE conjugate was prepared using a conventional ADC process. The process flow for the comparative example is shown in Figure 7 of the specification.

[0367] The specific steps for preparing the Trastuzumab-MMAE conjugate are as follows:

[0368] Step (1): The trastuzumab intermediate (2083 mg, purity: 99.0%) after anion exchange column chromatography was purified by cation exchange column chromatography using NaAc-HAc buffer at pH 5.5 as the elution solution. 2041 mg of cation exchange column chromatography product was obtained, with a yield of 98.0% and a purity of 99.4%.

[0369] Step (2): 2015 mg of cation exchange column chromatography product was subjected to nanofiltration using a Viresolve Pro nanofilter to obtain 2015 mg of nanofiltration product with a yield of 100.0% and a purity of 99.4%.

[0370] Step (3): 2001 mg of nanofiltration product was subjected to ultrafiltration and liquid exchange (UF / DF) to prepare 1879 mg of trastuzumab intermediate, with a yield of 93.9% and a purity of 99.9%. The 1879 mg ultrafiltration and liquid exchange product was subjected to excipient addition step to prepare 1844 mg of trastuzumab intermediate stock solution, with a yield of 98.1% and a purity of 99.6%. The trastuzumab stock solution was frozen at -70℃.

[0371] Step (4): Thaw the trastuzumab antigen solution, add 1810 units of transglutaminase (MTGase, the same batch of enzyme as the MTGase used in Example 3) and 5 equivalents of NH2-PEG3-VC-PAB-MMAE to 1810 mg of trastuzumab intermediate (purity: 99.6%) at a rate of 1 U / mg antibody, and react at 30°C for 48 h to obtain the conjugated product.

[0372] Step (5): 1871 mg of the coupling product was purified by hydrophobic interaction column chromatography using phosphate buffer at pH 6.5 as the elution solution to obtain 1165 mg of eluted product, with a yield of 62.3% and a product purity of 99.8%.

[0373] Step (6): 1153 mg of hydrophobic interaction column chromatography product was subjected to ultrafiltration and liquid exchange (UF / DF). The liquid exchange solution was a NaAc-HAc solution with pH 5.5. The inlet flux was 300 LMH and the transmembrane pressure difference (TMP) was about 1.0 bar. 1097 mg of ultrafiltration and liquid exchange product was obtained with a yield of 95.2% and a purity of 99.8%. 799 mg of ADC stock solution was prepared by adding excipients to 806 mg of ultrafiltration and liquid exchange product with a yield of 99.2% and a purity of 99.9%.

[0374] In this control example, the total yield of ADC stock solution prepared from the starting material, the clarified cell culture medium expressing trastuzumab, was 48%. The ADC stock solution had a DAR value of 1.9, an HCP content of 2.5 ppm, a residual DNA content of <0.5 pg / mg, a process-related impurity protein A content of <3 ppm, a residual MTGase content of 46 ppm, and a free drug residue of <0.01% (mg / mg), which was below the detection limit.

[0375] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them; although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications can still be made to the specific implementation of the present invention or equivalent substitutions can be made to some technical features without departing from the spirit of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the technical solutions claimed in the present invention.

Claims

1. A method for preparing antibody-conjugates, comprising: 1) Provide unpurified or partially purified antibodies or their antigen-binding fragments; 2) Prepare antibody conjugates by linking unpurified or partially purified antibodies or their antigen-binding fragments to a payload via a linker; 3) Purify antibody conjugates.

2. The method of claim 1, comprising: Provide a cell culture clarifier containing an antibody or its antigen-binding fragment, and capture the antibody or its antigen-binding fragment in the cell culture clarifier; The obtained product is subjected to coupling, virus inactivation and deep filtration, first chromatography, optional second chromatography, optional third chromatography, and filtration to remove potential viruses. The coupling, virus inactivation and deep filtration, first chromatography, optional second chromatography, optional third chromatography, and filtration to remove potential viruses can be performed in any order. The resulting product is concentrated and / or replaced with a buffer solution.

3. The method of claim 1, comprising: Provide a cell culture clarifier containing an antibody or its antigen-binding fragment, and capture the antibody or its antigen-binding fragment in the cell culture clarifier; The resulting product is subjected to coupling, virus inactivation and deep filtration, first chromatography, optional second chromatography, and optional third chromatography, wherein the coupling, virus inactivation and deep filtration, first chromatography, optional second chromatography, and optional third chromatography can be performed in any order. The resulting product is concentrated and / or the buffer is replaced, and the product is filtered to remove potential viruses.

4. The method according to any one of claims 1-3, wherein, Step 1) includes: 1.1) Provide a sample containing an antibody or its antigen-binding fragment; 1.2) Capture antibodies or their antigen-binding fragments in the sample.

5. The method of claim 4, wherein, The sample mentioned in step 1.1) is a cell culture medium; Preferably, the cell culture medium is a clarified cell culture medium; Preferably, the clarified cell culture solution is obtained by centrifuging and / or deep filtering a cell culture containing the provided antibody or its antigen-binding fragment.

6. The method of claim 4 or 5, wherein, The capture described in step 1.2) is performed by one or more of affinity chromatography, membrane chromatography, ion exchange chromatography, and hydrophobic chromatography. Preferably, the affinity chromatography is performed in a binding-elution mode; Preferably, the affinity chromatography is selected from protein A affinity chromatography, protein L affinity chromatography, protein G affinity chromatography, and any combination thereof.

7. The method according to any one of claims 2-6, wherein, Step 1.1) followed by step 1.2) includes: inactivating the virus by inactivating the antibody or its antigen-binding fragment; or, Step 1.2) is followed by: 1.3) Virus inactivation of the captured antibody or its antigen-binding fragment; Preferably, the virus is inactivated by adjusting the pH value or adding a surfactant; Preferably, the virus is inactivated and then centrifuged and / or deep filtered; Preferably, the captured antibody or its antigen-binding fragment is incubated in a buffer system with a pH of 3-4, and after incubation, the pH is neutralized to 5-7 and deep filtration is performed. Optionally, step 1.3) may be followed by: ultrafiltration and / or filtration washing; Optionally, centrifugation and / or deep filtration may be followed by removal of potential viruses, preferably by anion exchange chromatography, preferably selected from anion exchange column chromatography, anion mixed-mode chromatography, anion membrane chromatography and anion mixed-mode membrane chromatography.

8. The method according to any one of claims 1-7, wherein, Step 1) includes: 1.1) Provide a sample containing an antibody or its antigen-binding fragment; 1.2) Capture antibodies or their antigen-binding fragments in the sample; 1.3) Inactivate the virus by capturing the antibody or its antigen-binding fragment; 1.4) Centrifuge and / or deep filter the product from step 1.3).

9. The method according to any one of claims 1-7, wherein, Step 1) includes: 1.1) Provide a sample containing an antibody or its antigen-binding fragment; 1.2) Capture antibodies or their antigen-binding fragments in the sample; 1.3) Inactivate the virus by capturing the antibody or its antigen-binding fragment; 1.4) Centrifuge and / or perform deep filtration on the product from step 1.3); 1.5) Remove potential viruses from the product of step 1.4).

10. The method according to any one of claims 1-9, wherein, Step 2) includes: To couple unpurified or partially purified antibodies or their antigen-binding fragments to a linker-payload; or, Step 2) includes: 2.1) To conjugate unpurified or partially purified antibodies or their antigen-binding fragments to linkers. 2.2) Connect the product of step 2.1) to the payload.

11. The method of claim 10, preferably, wherein the coupling is selected from fixed-point coupling and non-fixed-point coupling. Preferably, the coupling is based on one or more of cysteine, lysine, or catalytic enzymes (such as transglutaminase (MTGase), sorting enzyme, glycosylmodifying enzyme (e.g., glycosidase, glycosyltransferase), farnesyltransferase, tubulin tyrosine ligase, formylglycine synthase, engineered tyrosine kinase, microbial transpeptidase). Preferably, the conjugation includes reducing the antibody or its antigen-binding fragment using a reducing agent, preferably selected from tris(2-carboxyethyl)phosphine (TCEP) and dithiothreitol (DTT).

12. The method according to any one of claims 1-11, wherein, Step 3) includes: 3.1) The prepared antibody conjugate was subjected to column chromatography; 3.2) Remove potential viruses from the product of the previous step; 3.3) Concentrated and / or replacement buffer; The order of steps 3.2) and 3.3) can be interchanged.

13. The method of claim 12, wherein, Step 3.1) The column chromatography is selected from ion exchange chromatography, hydrophobic interaction chromatography, and any combination thereof; Preferably, the ion exchange chromatography is performed in a binding-elution mode or a flow-through mode; Preferably, step 3.1) include: 3.1.1) The prepared antibody conjugate was subjected to cation exchange column chromatography to obtain the eluted sample; 3.1.2) The eluted sample was subjected to anion exchange column chromatography to obtain a flow-through sample; Alternatively, step 3.1) include: 3.1.1) The prepared antibody conjugate was subjected to anion exchange column chromatography to obtain a flow-through sample; 3.1.2) The obtained flow-through sample was subjected to cation exchange column chromatography to obtain the eluted sample; Alternatively, step 3.1) includes: subjecting the prepared antibody conjugate to size exclusion chromatography, hydrophobic interaction chromatography, mixed-mode chromatography, or ceramic hydroxyapatite chromatography to obtain the elution product.

14. The method of claim 12 or 13, wherein, Step 3.2) includes membrane filtration of the product from the previous step; Preferably, the product from the previous step is subjected to nanofiltration.

15. The method according to any one of claims 12-14, wherein, Step 3.3) describes the concentration and / or replacement of the buffer solution, performed via ultrafiltration. Preferably, step 3.3) further includes: washing and filtering, or Step 3.3) is followed by: washing and filtering.

16. The method according to any one of claims 1-15, wherein, Step 3) includes: 3.1) The prepared antibody conjugate was subjected to column chromatography. Step 3.1) includes: 3.1.1) The prepared antibody conjugate was subjected to cation exchange column chromatography to obtain the eluted sample; 3.1.2) The eluted sample was subjected to anion exchange column chromatography to obtain a flow-through sample; or, 3.1.1) The prepared antibody conjugate was subjected to anion exchange column chromatography to obtain a flow-through sample; 3.1.2) The obtained flow-through sample was subjected to cation exchange column chromatography to obtain the eluted sample; 3.2) Remove potential viruses from the product of the previous step; 3.3) Concentrated and / or replacement buffer; The order of steps 3.2) and 3.3) can be interchanged.

17. The method according to any one of claims 1-15, wherein, Step 3) includes: 3.1) The prepared antibody conjugate was subjected to hydrophobic interaction chromatography to obtain the elution product; 3.2) Remove potential viruses from the product of the previous step; 3.3) Concentrated and / or replacement buffer; The order of steps 3.2) and 3.3) can be interchanged.

18. The method according to any one of claims 1-11, wherein, Column chromatography is also included between steps 1) and 2). Preferably, the column chromatography is selected from ion exchange chromatography, hydrophobic interaction chromatography, and any combination thereof; Preferably, the ion exchange chromatography is performed in a binding-elution mode or a flow-through mode; Preferably, the ion exchange chromatography includes: The antibody or its antigen-binding fragment is subjected to cation exchange column chromatography to obtain the eluted sample; The eluted sample was subjected to anion exchange column chromatography to obtain a flow-through sample; Preferably, the process after column chromatography also includes the removal of potential viruses. Preferably, potential viruses are removed by membrane filtration. Preferably, the membrane filtration is nanofiltration.

19. The method of claim 18, wherein, Step 3) includes: Concentrated and / or displaced buffer solution, preferably, the concentrated and / or displaced buffer solution is obtained by ultrafiltration; Preferably, washing is performed simultaneously with concentration and / or displacement buffer, or washing is performed after concentration and / or displacement buffer.

20. The method according to any one of claims 1-11, wherein, Column chromatography is also included between steps 1) and 2). Preferably, the column chromatography is selected from ion exchange chromatography, hydrophobic interaction chromatography, and any combination thereof; Preferably, the ion exchange chromatography is performed in a binding-elution mode or a flow-through mode; Preferably, the ion exchange chromatography includes: The antibody or its antigen-binding fragment is subjected to cation exchange column chromatography to obtain the eluted sample; The eluted sample was subjected to anion exchange column chromatography to obtain a flow-through sample.

21. The method of claim 20, wherein, Step 3) includes: Remove potential viruses from the product of the previous step. Concentrated and / or displacement buffers; Preferably, potential viruses are removed by membrane filtration; Preferably, the membrane filtration is nanofiltration; Preferably, the concentration and / or replacement buffer is obtained by ultrafiltration; Preferably, washing is performed simultaneously with concentration and / or displacement buffer, or washing is performed after concentration and / or displacement buffer.

22. The method according to any one of claims 1-21, wherein, The method includes: 1) Providing unpurified or partially purified antibodies or antigen-binding fragments thereof, the steps comprising: 1.1) Provide a sample containing an antibody or its antigen-binding fragment; 1.2) Capture antibodies or their antigen-binding fragments in the sample; 1.3) Inactivate the virus by capturing the antibody or its antigen-binding fragment; 1.4) Centrifuge and / or perform deep filtration on the product from step 1.3); 2) To prepare an antibody-drug conjugate by conjugating an unpurified or partially purified antibody or its antigen-binding fragment to a payload via a linker, the step comprising: To couple unpurified or partially purified antibodies or their antigen-binding fragments to the linker-payload; or 2.1) To conjugate unpurified or partially purified antibodies or their antigen-binding fragments to linkers. 2.2) React the product of step 2.1) with the payload; 3) Purifying the antibody-conjugate, the steps of which include: 3.1) The prepared antibody conjugate was subjected to column chromatography. Step 3.1) includes: 3.1.1) The prepared antibody conjugate was subjected to cation exchange column chromatography to obtain the eluted sample; 3.1.2) The eluted sample was subjected to anion exchange column chromatography to obtain a flow-through sample; or, 3.1.1) The prepared antibody conjugate was subjected to anion exchange column chromatography to obtain a flow-through sample; 3.1.2) The obtained flow-through sample was subjected to cation exchange column chromatography to obtain the eluted sample; 3.2) Remove potential viruses from the product of the previous step; 3.3) Concentrated and / or replacement buffer; The order of steps 3.2) and 3.3) can be interchanged.

23. The method according to any one of claims 1-21, wherein the method comprises: 1) Providing unpurified or partially purified antibodies or antigen-binding fragments thereof, the steps comprising: 1.1) Provide a sample containing an antibody or its antigen-binding fragment; 1.2) Capture antibodies or their antigen-binding fragments in the sample; 1.3) Inactivate the virus by capturing the antibody or its antigen-binding fragment; 1.4) Centrifuge and / or perform deep filtration on the product from step 1.3); 1.5) Remove potential viruses from the product of step 1.4), for example, by removing potential viruses by anion exchange chromatography; 2) To prepare an antibody-drug conjugate by conjugating an unpurified or partially purified antibody or its antigen-binding fragment to a payload via a linker, the step comprising: To couple unpurified or partially purified antibodies or their antigen-binding fragments to the linker-payload; or 2.1) To conjugate unpurified or partially purified antibodies or their antigen-binding fragments to linkers. 2.2) React the product of step 2.1) with the payload; 3) Purifying the antibody-conjugate, the steps of which include: 3.1) The prepared antibody conjugate was subjected to column chromatography; 3.2) Remove potential viruses from the product of the previous step; 3.3) Concentrated and / or replacement buffer; The order of steps 3.2) and 3.3) can be interchanged.

24. The method according to any one of claims 1-23, wherein, The antibodies are selected from monospecific antibodies, bispecific antibodies, and multispecific antibodies, such as trastuzumab, pertuzumab, sacituzumab, abciximab, adalimumab, alefacept, alemtuzumab, basiliximab, belimumab, bezlotoxumab, canakinumab, certolizumab (pegylated), cetuximab, daclizumab, and dinosumab. The following are listed: enosumab, efalizumab, golimumab, inflectra, ipilimumab, ixekizumab, natalizumab, nivolumab, olaratumab, omalizumab, palivizumab, panitumumab, pembrolizumab, rituximab, tocilizumab, secukinumab, and ustekinumab.

25. The method according to any one of claims 1-24, wherein, The linker contains a group capable of reacting with an antibody or its antigen-binding fragment and a group capable of being linked to a payload, or the linker in the linker-payload configuration contains a group capable of reacting with an antibody or its antigen-binding fragment, for example, the linker contains a maleimide group, a bromine atom, or a methanesulfonyl group.

26. The method according to any one of claims 1-25, wherein, The payload is selected from diagnostic agents, therapeutic agents, and labeling reagents; Preferably, the payload is selected from cytotoxins, oligonucleotides, peptides, molecular degraders, immune agonists, radiopharmaceutical chelators, and PEG. For example, the cytotoxin is a tubulin inhibitor, DNA alkylating agent, DNA chimera, enzyme inhibitor, or antimetabolite. For example, the cytotoxin is auristatin, monomethyl auristatin E (MMAE), maytansine or its derivatives (e.g., maytansine-like substances, DM1, DM3, DM4), paclitaxel, calcitrazine, pyroxine, doxorubicin, camptothecin-type cytotoxins and their derivatives (e.g., eczetidine, topotecan, irinotecan, belotecidine), PBD (pyrrolobenzodiazepine)-type cytotoxins and their derivatives.

27. The method according to any one of claims 1-26, wherein, The antibody-drug conjugate is an antibody-drug conjugate.

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