Method for improving product quality of antibody drug conjugate

Abstract

The invention relates to a method for improving the product quality of an antibody drug conjugate, which comprises the following steps: coupling a drug to be coupled with an antibody to obtain the antibody drug conjugate, and purifying the antibody drug conjugate: adding a regulating solution into the antibody drug conjugate, collecting a product through column chromatography flow-through, and further performing ultrafiltration and / or percolation concentration on the product to obtain the antibody drug conjugate with improved product quality. The obtained antibody drug conjugate is high in purity, uniform in DAR, free of solvent residues and stable in quality, meanwhile, the steps of ultrafiltration, percolation and the like are not adopted in antibody pretreatment, the antibody purification method is simplified, and the production cost is saved.

Description

[0001] This application is a divisional application of Chinese patent application No. 202280054509.7, filed on August 22, 2022, entitled "A method for improving the quality of antibody-drug conjugate products". Technical Field

[0002] This application relates to the field of medicinal chemistry, specifically to a method for improving the quality of antibody-drug conjugate products. Background Technology

[0003] With the development of the field of antibody-drug conjugates, the development of purification and conjugation production process conditions for bioactive drugs containing target antibodies has become particularly important. The development and effective control of antibody separation and purification processes as well as antibody-drug conjugate ...

[0004] The design and parameter control of antibody separation and purification processes directly affect the product quality and yield of antibody-drug conjugates. Existing purification processes for the antibody portion of antibody-drug conjugates often require ultrafiltration and / or percolation steps to bring the antibody to conjugation requirements. However, this step increases the number of purification steps and production costs, affecting the yield of the antibody and ultimately the conjugate.

[0005] On the other hand, the drug conjugation process has a significant impact on the quality of antibody-drug conjugates (ADCs). Currently, commonly used conjugation methods for ADCs include lysine conjugation, light-heavy chain disulfide bond reversion, and site-directed conjugation. Due to the diversity of conjugation sites and methods, ADCs obtained through lysine conjugation, light-heavy chain disulfide bond reversion, or site-directed conjugation processes will all produce certain non-target products and may even experience protein aggregation, affecting the pharmaceutical safety and efficacy of the ADCs. The number of toxins linked to the antibody (Drug-antibody ratio, DAR) determines the homogeneity of the drug. Ensuring product quality while controlling DAR homogeneity as much as possible is a challenge in the manufacturing process of ADCs. In existing technologies, conjugation and purification processes for ADCs with DAR values ​​between 3 and 4 are relatively common. Therefore, developing purification and conjugation processes for ADCs with high DAR values ​​(e.g., 6-8) is of great significance for improving product quality, reducing costs, and mitigating safety risks. Summary of the Invention

[0006] The inventors of this application conducted extensive experiments and repeated refinements on the upstream antibody purification process and antibody-drug conjugate process, improving the reaction conditions and process parameters of each step in the production process. The antibody product obtained by this method meets the conjugation requirements, improves the purity and quality of the final antibody-drug conjugate, significantly reduces the content of related substances and potential drug risks, and ensures stable and reliable product quality, making it suitable for continuous industrial-scale production.

[0007] This invention provides a method for improving the quality of antibody-drug conjugate products, comprising:

[0008] (1) Provide antibody-drug conjugates;

[0009] (2) Purification of the antibody-drug conjugate: Add conditioning solution to the antibody-drug conjugate, collect the product by column chromatography flow-through, and concentrate the product by further ultrafiltration and / or percolation to obtain an antibody-drug conjugate with improved product quality;

[0010] The structure of the drug to be coupled is shown in formula (I):

[0011] D-[L1-(L2) m1 -(L3) m2 -(L4) m3 -E]-G

[0012] Formula (I)

[0013] in,

[0014] L1 is or Each of R1 and R2 is independently hydrogen, halogen, carboxylic acid group, sulfonic acid group, cyano group, or C. 1-6 Alkyl, Halogenated C 1-6 alkyl and cyano substituted C 1-6 Alkyl groups (e.g., -CH2CN), C 1-6 Alkoxy, C 2-10 alkenyl or C 2-10 Alkyne group; Z1 is an amino acid or a peptide composed of 2-10 amino acids; x1 and x2 are each independently 0, 1, 2, 3, 4, 5 or 6; and L1 is connected to D at position 1 and L2 at position 2.

[0015] L2 is Or it does not exist; where y1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and L2 at position 1 is connected to L1, and L2 at position 2 is connected to L3;

[0016] L3 is selected from 5-12 membered heteroaryl rings or is absent;

[0017] L4 is , or Z2 is selected from C 1-6 Alkylene, C 2-10 imidene group, C 2-10 Ethyne group, C 3-8 Cycloalkylene and 5-6-membered heteroaryl groups; R3 is selected from H and C. 1-6 Alkyl group; Z3 is absent or selected from C 1-6 Alkylene; or, R3 and Z3 together with the nitrogen atom to which they are attached form a 4-8 membered heterocyclic group; α is 0, 1, 2, 3, 4, 5 or 6, and L4 is attached to E at the 2 position and to L3 at the 1 position;

[0018] E is In this context, each R4 is independently hydrogen, β is 0, 1 or 2, and the 2 position of E is connected to G, and the 1 position of E is connected to L4.

[0019] m1, m2 and m3 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0020] D represents a bioactive molecular fragment;

[0021] G is a leaving group for nucleophilic substitution; preferably, G is selected from halogen, sulfonyl, sulfonate, or nitro.

[0022] In some embodiments, the present invention provides a method for improving the quality of antibody-drug conjugate products, characterized in that 5-20 mM of reducing agent is added to the pretreated antibody, mixed and subjected to a reduction reaction, then the drug to be conjugated is added, stirred and allowed to stand, and then an acidic solution is added.

[0023] Add conditioning solution to the product of the above steps, collect the product by column chromatography flow-through, and concentrate the product by further ultrafiltration and / or percolation to obtain antibody-drug conjugate;

[0024] The structure of the drug to be coupled is shown in formula (I) above.

[0025] In some embodiments, the antibody-drug conjugate in step (1) is obtained by adding a reducing agent solution with a concentration of 5-20 mM to the pretreated antibody, then adding the drug to be conjugated to the reaction system, stirring and letting it stand, and then adding an acidic solution to obtain the antibody-drug conjugate.

[0026] In some embodiments, the method further includes a sterilization filtration step.

[0027] In some embodiments, the antibody undergoes a sterilization filtration step prior to the reduction reaction.

[0028] In some embodiments, the reducing agent is tris(2-carboxyethyl)phosphine.

[0029] In some embodiments, the reduction reaction includes treating the antibody with the reducing agent for 10 min–2 h, for example, 30 min–1 h.

[0030] In some embodiments, the reduction reaction is the treatment of the antibody with the reducing agent at room temperature; and / or, the reduction reaction is carried out at 4-37°C, for example, 18-26°C. In some embodiments, the column chromatography method is hydrophobic chromatography, preferably, the hydrophobic chromatography includes equilibration and elution steps.

[0031] In some embodiments, the hydrophobic chromatography equilibration step is performed at pH 6-7 using a 20 mmol / L phosphate buffer-ammonium sulfate solution as the equilibration solution, with the conductivity controlled at 50-80 mS / cm.

[0032] In some embodiments, the hydrophobic chromatography equilibration step is performed at pH 6-7 using a 20 mmol / L phosphate buffer-ammonium sulfate solution as the equilibration solution, with the conductivity controlled at 62-70 mS / cm.

[0033] In some embodiments, the elution step of the hydrophobic chromatography is performed at pH 6-7 using the same solution as the equilibration buffer as the eluent, with the conductivity controlled at 22-30 mS / cm.

[0034] In some implementations, the chromatography column uses Butyl Sepharose High Performance packing.

[0035] In some embodiments, the ultrafiltration and / or percolation concentration uses a Pellicon 3 Cassette Biomax membrane.

[0036] In some embodiments, the percolation solution used is a 10 mmol / L histidine-histidine hydrochloride solution with a pH of 5.7-6.3.

[0037] In some embodiments, after adding the drug to be coupled to the reaction system, the mixture is stirred for 5-15 minutes and then allowed to stand for reaction. Preferably, the mixture is allowed to stand for 1-12 hours, for example, 1-6 hours, 1-4 hours, 1-3 hours, 1.5-3 hours, or 1.5-2.5 hours.

[0038] In some embodiments, the stirring time is 5-15 minutes, the settling temperature is 4-37°C, for example 18-26°C, and the settling time is 1.5-3 hours.

[0039] In some embodiments, the drug to be coupled is a small molecule compound with a linker arm, preferably selected from DNA topoisomerase inhibitors, tubulin inhibitors, or derivatives thereof.

[0040] In some embodiments, the DNA topoisomerase inhibitor is a topoisomerase I inhibitor, preferably camptothecin, SN-38, irinotecan, topotecan, belotetane, rubotecan, or derivatives thereof.

[0041] In some embodiments, the DNA topoisomerase inhibitor is a topoisomerase II inhibitor, preferably, the topoisomerase II inhibitor is actinomycin D, doxorubicin, doxorubicin, docalimicin, daunorubicin, mitoxantrone, podophyllotoxin, etoposide, or derivatives thereof.

[0042] In some embodiments, the microtubule inhibitor is a vinca alkaloid, vincristine, vinblastine, paclitaxel, docetaxel, cabazitaxel, or derivatives thereof.

[0043] In some embodiments, the small molecule compound with the linker arm is selected from N-((S)-1-(((S)-1-((4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionamido)methyl)phenyl)amino)-1-oxo-5-ureido-2-yl)amino)-3-methyl-1-oxybutane-2-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)-5-hexyneamide;

[0044] (S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4',6,7]indolazino[1,2-b]quinoline-4-yl(4-((S)-42-(2-(methylsulfonyl)pyrimidin-5-yl)-4,8,37-trioxo-2-(3-ureopropyl)-6,12,15,18,21,24,27,30,33-nonoxy-3,9,36-azatetracosane-41-amido)benzyl)carbonate;

[0045] 4-((S)-2-(4-aminobutyl)-42-(2-(methanesulfonyl)pyrimidin-5-yl)-4,8,37-trioxo-6,12,15,18,21,24,27,30,33-nonoxa-3,9,36-triazatetrazolyl-41-acetylamidoyl)benzyl-((S)-4-ethyl-11-(2-(N-isopropylmethanesulfonyl)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyranone[3',4':6,7]indolazin[1,2-b]quinoline-4-yl) carbonate;

[0046] (S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolazino[1,2-b]quinoline-4-yl-4-((2S,5S)-5-isopropyl-2-methyl-38-(4-((6-(2-(methanesulfonyl)pyrimidin-5-yl)yl)hex-5-ynylamido)methyl)-1H-1,2,3-triazol-1-yl)-4,7,11-trioxo-9,15,18,21,24,27,30,33,36-nonoxy-3,6,12-triazatriaconitamido)benzyl carbonate;

[0047] 4–((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxa-3,9-diazatriapentadecanoamide)benzyl((S)-4-ethyl-11-(2-(N-isopropylacetamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indo[1,2-b]quinoline-4-yl) carbonate;

[0048] 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-amido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxa-3,9-diazapentadecanoamide)benzyl((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indo[1,2-b]quinoline-4-yl)carbonate

[0049] 4-((S)-2-(4-aminobutyric acid)-35-(4-((2-(2-(methanesulfonyl)pyrimidin-5-yl)-oxazol-4-formamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxy-3,9-diazapentadecanoamide)benzyl-((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indo[1,2-b]quinoline-4-yl) carbonate;

[0050] N-((1-((6S,9S)-1-amino-6-((4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S))) -2-((S)-2-(dimethylamino)-3-methylbutyramide)-N,3-dimethylbutyramide)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropionamide)-3-phenylpropionamide)methyl)phenyl)carbamoyl)-9-isopropyl-1,8,11,15-tetraoxy-13,19,22,25,28,31,34,37,40-nonoxa-2,7,10,16-tetraazaanthraalkyl-42-yl)-1H-1,2,3-triazol-4-yl)methyl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)-5-hexyneamide;

[0051] 4-((2S,5S)-5-isopropyl-38-(4-((6-(2-(methanesulfonyl)pyrimidin-5-yl)-5-hexylenamide)methyl)-1H-1,2,3-triazol-1-yl)-4,7,11-trioxo-2-(3-ureopropyl)-9,15,18,21,24,27,30,33,36-nonazo-3,6,12-triazatetradodecyl)benzyl-((S)-1-(((S)-1-((( 3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidone-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate;

[0052] (S)-2-((2R,3R)-3-((2S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl(((4-((S)-2-((S)-3-methyl-2-(32-(4-(((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-carbamoyl)methyl)-1H-1, 2,3-Triazol-1-yl)-5-oxo-3,9,12,15,18,21,24,27,30-nonoxy-6-azatriacontamido)butyrylamino)-5-ureidopentanoylamino)benzyl)oxy)carbonyl)amino)butyrylamino)butyrylamino)-3-methoxy-5-methylheptyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropionyl)-L-phenylalanine;

[0053] One of the 4-((S)-2-(4-aminobutyl)-35-(4-((4-(2-(methanesulfonyl)pyrimidin-5-yl)benzamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxy-3,9-diazapentapentadecanoamide)benzyl((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7] indene[1,2-b]quinoline-4-yl) carbonates.

[0054] In some embodiments, the drug to be coupled is a small molecule compound with a linker arm, and the small molecule compound with the linker arm is selected from compounds with the following structures:

[0055] ;

[0056] ;

[0057] ;

[0058] ;

[0059] ;

[0060] ;

[0061] ;

[0062] ;

[0063] ;

[0064] ;

[0065] .

[0066] In some embodiments, the antibody is Sacituzumab; the drug to be conjugated is a small molecule compound with a linker arm, the structure of which is shown below:

[0067] .

[0068] In some embodiments, the antibody-drug conjugate has the following structure:

[0069]

[0070] Wherein, γ is 1-10; preferably, γ is 5-8; preferably, the connecting arm is connected to the thiol group of Sacituzumab.

[0071] In some embodiments, the concentration of the drug to be coupled is 10-70 mM, preferably 50 mM.

[0072] In some embodiments, the pH of the tris(2-carboxyethyl)phosphine solution is 7.0-8.0, for example 7.4-8.0;

[0073] In some embodiments, the acidic solution is selected from citric acid or acetic acid.

[0074] In some embodiments, the acidic solution adjusts the pH of the reaction system to acidic; preferably, the acidic solution adjusts the pH of the reaction system to 6.0-7.0; more preferably, the acidic solution adjusts the pH of the reaction system to 4.5-7.0.

[0075] In some embodiments, antibody pretreatment includes adding a disodium edetate solution to the antibody and adjusting the pH with a Tris solution. Preferably, the concentration of the disodium edetate solution is 2-10 mM and the concentration of the Tris solution is 1-3 M. More preferably, the concentration of the disodium edetate solution is 2-10 mM and the concentration of the Tris solution is 2 M.

[0076] In some embodiments, antibody pretreatment includes adding a disodium edetate solution to the antibody and adjusting the pH with a phosphate. Preferably, the phosphate is a sodium phosphate; more preferably, the sodium phosphate is disodium hydrogen phosphate. Preferably, the concentration of the disodium edetate solution is 2-10 mM, and the concentration of the disodium hydrogen phosphate solution is 0.2-2 M, preferably 1 M.

[0077] In some embodiments, the antibody is an anti-Her2 antibody, an anti-EGFR antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-Trop-2 antibody.

[0078] In some embodiments, the anti-Her2 antibody is Trastuzumab or Pertuzumab, the anti-EGFR antibody is Cetuximab or Nimotuzumab, the anti-PD-1 monoclonal antibody is Nivolumab or Pembrolizumab, the anti-PD-L1 monoclonal antibody is Atezolizumab or Durvalumab, and the anti-Trop-2 antibody is Sacituzumab or Datopotamab.

[0079] In some embodiments, the concentration of the antibody in the reduction reaction is 10-25 g / L.

[0080] In some embodiments, the conditioning solution is selected from ammonium sulfate. In some embodiments, the conditioning solution is added to the antibody-drug conjugate prior to the hydrophobic chromatography step.

[0081] In some embodiments, the conditioning solution adjusts the antibody-drug conjugate to a high conductivity; preferably, the conditioning solution adjusts the conductivity of the antibody-drug conjugate to 62-70 mS / cm.

[0082] In some implementations, the antibody is further purified before pretreatment.

[0083] In some embodiments, the purification includes steps of chromatography and filtration, wherein the chromatography is selected from affinity chromatography, anion exchange chromatography, and / or cation exchange chromatography.

[0084] In some embodiments, the purification process sequentially includes the steps of affinity chromatography, incubation, deep filtration, anion exchange chromatography, and cation exchange chromatography.

[0085] In some implementations, the purification process does not include steps such as ultrafiltration or percolation.

[0086] In some embodiments, the packing material for the affinity chromatography is selected from Cytiva Mabselect Sure LX, Cytiva MabSelect SuRe, Cytiva MabSelect PrismA, Merck Eshmuno A, and Tosoh AFrProteinA-HC 650F.

[0087] In some embodiments, the affinity chromatography further includes equilibration, washing, and elution steps. Preferably, the equilibration step uses NaCl-phosphate buffer. Preferably, the washing can be performed once, twice, or multiple times in a pH range of 5.4-6.2. Preferably, the washing solution is selected from phosphate buffer-NaCl and sodium acetate-acetic acid solution. Preferably, the elution uses sodium acetate-acetic acid buffer.

[0088] In some embodiments, the elution step of the affinity chromatography needs to be controlled at pH 3.5-3.7.

[0089] In some embodiments, the incubation includes incubation at room temperature and pH 3.6-3.8.

[0090] In some implementations, the depth filter is a Millipore X0HC.

[0091] In some embodiments, the anion exchange chromatography packing material is selected from Cytiva Q Sepharose FastFlow, Thermo POROS 50HQ, Thermo POROS XQ, and Bio-Rad Nuvia HP-Q.

[0092] In some embodiments, the pH of the anion exchange chromatography operating system is 5.5-6.5, preferably 5.8-6.2.

[0093] In some embodiments, the cation exchange chromatography packing material is selected from Tosoh Gigacap S 650M, MerckEshmuno CPX, Thermo POROS 50HS, Thermo POROS XS, and Bio-Rad Nuvia HP-S.

[0094] In some embodiments, the pH of the cation exchange chromatography operating system is 5.5-6.5, preferably 5.9-6.1.

[0095] In some embodiments, the purification also includes steps of virus removal filtration and sterilization filtration.

[0096] The method of the present invention has at least the following beneficial effects:

[0097] (1) By optimizing the process steps and reaction parameters, the present invention uses hydrophobic chromatography and optimizes the parameters after the coupling is completed, so that the antibody-drug conjugate obtained by the present invention has high purity, uniform DAR value, and almost no residual solvent in the product, thus achieving controllability and stability of product quality.

[0098] (2) The present invention purifies and pretreats the conjugated antibodies used in the antibody-conjugate reaction. Conventional antibody pretreatment steps often require ultrafiltration and / or percolation, but the present invention omits these operations and reduces process time and cost by controlling the process parameters of chromatography, filtration and other steps, thereby improving the overall yield of antibody products. Detailed Implementation

[0099] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention or its application or use. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the reagents used in the following embodiments are all commercially available products, and the solutions can be prepared using conventional techniques in the art.

[0100] Example 1 Antibody Pretreatment

[0101] 1. Pretreatment of the first batch of antibodies

[0102] The affinity chromatography column (Mabselect Sure LX packing material) was equilibrated with 20 mM phosphate buffer (pH 7.2) and 150 mM NaCl solution. A solution containing Sacituzumab antibody was loaded at a concentration of 42.7 g / L. After washing, at least three column volumes were performed with 20 mM PB and 1 M NaCl solution (pH 6.0), followed by at least three column volumes of a second wash with 20 mM sodium acetate-acetic acid buffer (pH 5.5). Finally, elution was performed with 20 mM sodium acetate-acetic acid solution (pH 3.57).

[0103] The collected eluent was incubated at room temperature with citric acid to adjust the pH to 3.7 for 2 hours to inactivate the sample, followed by neutralization with 2M Tris. The sample was then clarified by deep filtration at a loading of 1366 g / m³. 2The membrane material used was Millipore X0HC. The processed sample was first subjected to anion exchange chromatography with a loading capacity of 108.5 g / L, using Cytiva QSepharose Fast Flow as the packing material. The loading conductivity was 2.68 mS / cm, and the pH was 6.09. The flow-through peak was collected after loading. The flow-through sample was then subjected to cation exchange chromatography using a Gigacap S 650M packing column with a loading capacity of 49.2 g / L. The elution pH was 6.02, and the eluent was 20 mM PB + 105 mM sodium chloride solution. The product was collected for further use. After pre-filtration, the collected product was subjected to virus removal and sterilization filtration using Millipore Viresolve Pro to obtain the final pretreated antibody.

[0104] 2. Second batch of antibody pretreatment

[0105] The affinity chromatography column (Mabselect Sure LX packing material) was equilibrated with 20 mM phosphate buffer (pH 7.2) and 150 mM NaCl solution. A solution containing Sacituzumab antibody was loaded at a concentration of 41.9 g / L. After washing, the column was washed with at least three column volumes of 20 mM PB and 1 M NaCl solution (pH 6.0), followed by a second wash of at least three column volumes of 20 mM sodium acetate-acetic acid buffer (pH 5.5). Finally, the column was eluted with 20 mM sodium acetate-acetic acid solution (pH 3.62).

[0106] The collected eluent was incubated at room temperature with citric acid to adjust the pH to 3.69 for 2 hours to inactivate the sample. The resulting sample was then neutralized with 2M Tris solution. The sample was clarified by deep filtration with a loading of 1394.9 g / m³. 2 The membrane material used was Millipore X0HC. The processed sample was first subjected to anion exchange chromatography with a loading capacity of 107.7 g / L using GE Q Sepharose Fast Flow column as the packing material. The loading conductivity was 2.71 mS / cm, and the pH was 6.07. The flow-through peak was collected after loading. The flow-through sample was then subjected to cation exchange chromatography using a Gigacap S 650M column as the packing material, with a loading capacity of 48.6 g / L. The elution pH was 6.03, and the eluent was 20 mM PB + 105 mM sodium chloride solution. The product was collected for further use. After pre-filtration, the collected product was subjected to virus removal and sterilization filtration using Millipore Viresolve Pro to obtain the final pretreated antibody.

[0107] 3. Pretreatment of the third batch of antibodies

[0108] The affinity chromatography column (Mabselect Sure LX packing material) was equilibrated with 20 mM phosphate buffer (pH 7.2) and 150 mM NaCl solution. A solution containing Sacituzumab antibody was loaded at a concentration of 43.2 g / L. After washing, at least three column volumes were performed with 20 mM PB and 1 M NaCl solution (pH 6.0), followed by at least three column volumes of a second wash with 20 mM sodium acetate-acetic acid buffer (pH 5.5). Finally, elution was performed with 20 mM sodium acetate-acetic acid solution (pH 3.58).

[0109] The collected eluent was incubated at room temperature with citric acid to adjust the pH to 3.66 for 2 hours to inactivate the sample. The resulting sample was then neutralized with 2M Tris solution. The sample was clarified by deep filtration at a loading of 1447.8 g / m³. 2 The membrane material used was Millipore X0HC. The processed sample was first subjected to anion exchange chromatography with a loading capacity of 116 g / L using a GE Q Sepharose Fast Flow column. The loading conductivity was 2.69 mS / cm, and the pH was 6.06. The flow-through peak was collected after loading. The flow-through sample was then subjected to cation exchange chromatography using a Gigacap S 650M column with a loading capacity of 52.8 g / L. The elution pH was 6.0, and the eluent was 20 mM PB + 105 mM sodium chloride solution. The product was collected for further use. After pre-filtration, the collected product was subjected to virus removal and sterilization filtration using Millipore Viresolve Pro to obtain the final pretreated antibody.

[0110] The samples collected from each step of the above three batches were subjected to IEC purity testing using ion exchange chromatography and SEC purity testing using SEC-HPLC. The results are shown in the table below.

[0111] Table 1 Purity test results

[0112] As shown in Table 1, the three batches of antibodies produced using the process of this invention all met the purity requirements and can be used for the preparation of subsequent antibody-drug conjugates.

[0113] DNA and HCP residues were detected in samples collected from each step of the three batches mentioned above. Residual DNA was detected by quantitative real-time PCR, and residual HCP was detected by ELISA. The results are shown in the table below.

[0114] Table 2 Results of Residual Substance Detection

[0115] As shown in Table 2, the three batches of antibodies produced using the process of this invention all met the requirements for DNA and HCP residues and can be used for the preparation of subsequent antibody-drug conjugates.

[0116] Example 2 Preparation of antibody-drug conjugates

[0117] Sacituzumab antibody was diluted with 0.25 mL of a solution containing 20 mM PB, 150 mM NaCl, and 5 mM disodium edetate (pH 7.6). The pH was adjusted to 7.4, and 10 mM TCEP was added as a reducing agent. The drug to be conjugated was then conjugated with the Sacituzumab antibody. After mixing, the mixture was allowed to stand at room temperature for 2 hours. Cysteine ​​was added to terminate the reaction. Finally, the buffer solution was replaced using a G-25 gel column to obtain the conjugated product.

[0118] In this embodiment, antibody-drug conjugates were obtained by conjugating the drug to be conjugated with Sacituzumab using the following structures:

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129] Example 3: Improving the Product Quality of Antibody-Drug Conjugates

[0130] A 5 mM sodium edetate solution was added to the pretreated antibody to be conjugated (prepared according to the method in Example 1) under slow stirring. The pH was adjusted to alkaline using 2 M Tris. 10 mM TCEP solution was added to the antibody solution and stirred until homogeneous. After reacting at 23.3°C for 35 min, 50 mM of the drug solution to be conjugated was added to the reaction system, stirred until homogeneous, and allowed to stand at room temperature for 150 min. The pH was then adjusted to acidic (pH 6.0-7.0) using 1 M citric acid to obtain the target product. The structural formula of the drug to be conjugated is [insert structural formula here].

[0131]

[0132] Ammonium sulfate was added to the target product for conditioning, followed by flow-through hydrophobic column chromatography to collect the product. The hydrophobic column packing material was Butyl Sepharose High Performance, and the equilibration buffer was a 20 mmol / L phosphate buffer-ammonium sulfate solution at pH 6.59 with a conductivity of 66.51 mS / cm. Elution was performed using the same elution buffer, with a conductivity of 26.93 mS / cm. The hydrophobic chromatographic product was further concentrated by ultrafiltration to obtain the antibody-drug conjugate.

[0133] Intermediates from each of the above preparation steps were collected, and their purity was determined by SEC-HPLC, DAR was determined by RP-HPLC, and free toxins were determined by RP-HPLC. Bacterial endotoxins were also tested according to the provisions of General Chapter 1143 of the 2015 edition of the Chinese Pharmacopoeia for the gel method. The results are shown in the table below.

[0134] Table 3. Results of purity, DAR, free toxins, and bacterial endotoxins of the coupling intermediate.

[0135] As shown in Table 3, the antibody-drug conjugates produced by the conjugation process of this invention have high purity, uniform DAR values, and effectively controlled free toxin content and endotoxin, which significantly improves the quality of the prepared antibody-drug conjugate products.

[0136] Example 4: Improving the Product Quality of Antibody-Drug Conjugates

[0137] A 5 mM sodium edetate solution was added to the pretreated antibody to be conjugated (the antibody was prepared according to the method in Example 1) under slow stirring. The pH was adjusted to alkaline using 2 M Tris. 10 mM TCEP solution was added to the antibody solution and stirred until homogeneous. After reacting at 23.5 °C for 32 min, 50 mM of the drug solution to be conjugated was added to the reaction system, stirred until homogeneous, and allowed to stand at room temperature for 2.5 h. The pH was then adjusted to acidic (pH 6.0-7.0) using 1 M citric acid to obtain the target product. The structural formula of the drug to be conjugated is [insert structural formula here].

[0138]

[0139] Ammonium sulfate was added to the target product for conditioning, followed by flow-through hydrophobic column chromatography to collect the product. The hydrophobic column packing material was Butyl Sepharose High Performance (GE). The equilibration buffer was a 20 mmol / L phosphate buffer-ammonium sulfate solution at pH 6.59 with a conductivity of 66.69 mS / cm. The eluent was the same as the equilibration buffer, with a conductivity of 27.16 mS / cm. The hydrophobic chromatographic product was further concentrated by ultrafiltration to obtain the antibody-drug conjugate.

[0140] The purity, DAR, free toxins, and bacterial endotoxins of the intermediates prepared in each step were tested (using the same testing method as in Example 3), and the results are shown in the table below.

[0141] Table 4. Results of purity, DAR, free toxins, and bacterial endotoxins of the coupling intermediate.

[0142] As shown in Table 4, the antibody-drug conjugates produced using the conjugation process of this invention have high purity, uniform DAR values, and effectively controlled free toxin content and endotoxins, significantly improving the quality of the prepared antibody-drug conjugate products.

[0143] Example 5: Improving the Product Quality of Antibody-Drug Conjugates

[0144] Add 5 mM disodium edetate solution to the pretreated antibody to be conjugated (the antibody was prepared according to the method in Example 1) under slow stirring, and adjust the pH to alkaline using 2 M Tris. Add 10 mM TCEP solution to the antibody solution, stir to mix, and react at 22.3 °C for 35 min. Then add 50 mM of the drug solution to be conjugated to the reaction system, stir to mix, and let stand at room temperature for 153 min. Adjust the pH to acidic (pH 6.0-7.0) with 1 M citric acid to obtain the target product. The structural formula of the drug to be conjugated is [insert structural formula here].

[0145] ,

[0146] Ammonium sulfate was added to the target product for conditioning, followed by flow-through hydrophobic column chromatography to collect the product. The hydrophobic column packing material was Butyl Sepharose High Performance (GE). The equilibration buffer was a 20 mmol / L phosphate buffer-ammonium sulfate solution at pH 6.51 with a conductivity of 66.66 mS / cm. The eluent was the same as the equilibration buffer, with a conductivity of 26.98 mS / cm. The hydrophobic chromatographic product was further concentrated by ultrafiltration to obtain the antibody-drug conjugate.

[0147] The purity, DAR, free toxins, and bacterial endotoxins of the intermediates prepared in each step were tested (using the same testing method as in Example 3), and the results are shown in the table below.

[0148] Table 5. Results of purity, DAR, free toxins, and bacterial endotoxins of the coupling intermediate.

[0149] As shown in Table 5, the antibody-drug conjugates produced by the conjugation process of this invention have high purity, uniform DAR values, and effectively controlled free toxin content and endotoxin, which significantly improves the quality of the prepared antibody-drug conjugate products.

[0150] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

Claims

1. A method for improving the quality of antibody-drug conjugate products, comprising: (1) The drug to be conjugated is conjugated with the antibody to obtain the antibody-drug conjugate; (2) Purification of the antibody-drug conjugate: Add conditioning solution to the antibody-drug conjugate, collect the product by column chromatography flow-through, and concentrate the product by further ultrafiltration and / or percolation to obtain an antibody-drug conjugate with improved product quality; The structure of the drug to be coupled is shown in formula (I): D - [L1- (L2) m1 -(L3) m2 -(L4) m3 -E]-G Formula (I) in, L1 is or Each of R1 and R2 is independently hydrogen, halogen, carboxylic acid group, sulfonic acid group, cyano group, or C. 1-6 Alkyl, Halogenated C 1-6 alkyl and cyano substituted C 1-6 Alkyl groups (e.g., -CH2CN), C 1-6 Alkoxy, C 2-10 alkenyl or C 2-10 Alkyne group; Z1 is an amino acid or a peptide composed of 2-10 amino acids; x1 and x2 are each independently 0, 1, 2, 3, 4, 5 or 6; and L1 is connected to D at position 1 and L2 at position 2. L2 is Or it does not exist; where y1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and L2 at position 1 is connected to L1, and L2 at position 2 is connected to L3; L3 is selected from 5-12 membered heteroaryl rings or is absent; L4 is , or Z2 is selected from C 1-6 Alkylene, C 2-10 imidene group, C 2-10 Ethyne group, C 3-8 Cycloalkylene and 5-6-membered heteroaryl groups; R3 is selected from H and C. 1-6 Alkyl group; Z3 is absent or selected from C 1-6 Alkylene; or, R3 and Z3 together with the nitrogen atom to which they are attached form a 4-8 membered heterocyclic group; α is 0, 1, 2, 3, 4, 5 or 6, and L4 is attached to E at the 2 position and to L3 at the 1 position; E is In this context, each R4 is independently hydrogen, β is 0, 1 or 2, and the 2 position of E is connected to G, and the 1 position of E is connected to L4. m1, m2 and m3 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; D represents a bioactive molecular fragment; G is the leaving group in a nucleophilic substitution reaction.

2. A method for improving the quality of antibody-drug conjugate products, characterized in that, Add a reducing agent with a concentration of 5-20 mM to the pretreated antibody, mix well and carry out the reduction reaction, then add the drug to be conjugated, stir and let stand, then add an acidic solution; add an adjustment solution to the product of the above steps, collect the product by column chromatography flow-through, and the product is further concentrated by ultrafiltration and / or percolation to obtain an antibody-drug conjugate with improved product quality. The structure of the drug to be coupled is shown in formula (I) of claim 1.

3. The method as described in claim 1 or 2, characterized in that, The drug to be coupled is selected from the following compounds: ； ； ； ； ； ； ； ； ； ； 。 4. The method according to any one of claims 1-3, wherein the column chromatography is hydrophobic chromatography.

5. The method of claim 4, wherein the hydrophobic chromatography includes the steps of equilibration and elution.

6. The method according to any one of claims 1-5, wherein G is selected from halogen, sulfonyl, sulfonate, or nitro.

7. The method according to any one of claims 1-6, wherein the adjusting solution is an ammonium sulfate solution.

8. The method according to any one of claims 1-7, wherein the antibody-drug conjugate in step (1) is obtained by the following method: adding a reducing agent solution with a concentration of 5-20 mM to the pretreated antibody, then adding the drug to be conjugated to the reaction system, stirring and letting stand, and then adding an acidic solution to obtain the antibody-drug conjugate.

9. The method of claim 8, characterized in that... One or more of the following: 1) The reducing agent is TCEP (tris(2-carboxyethyl)phosphine); 2) Treat the antibody with the reducing agent for 10 min to 2 h; 3) Treat the antibody with the reducing agent at room temperature; and / or carry out the reaction at 4-37°C; 4) After adding the drug to be coupled to the reaction system, stir for 5-15 minutes, and then let it stand to react; 5) The acidic solution is a citric acid solution or an acetic acid solution, and the pH of the reaction system is adjusted to acidic using the acidic solution; preferably, the acidic solution adjusts the pH of the reaction system to 4.5-7.0; 6) In the reduction reaction, the concentration of the antibody is 10-25 g / L; 7) The concentration of the drug to be coupled is 10-70 mM.

10. The method of claim 8 or 9, wherein the antibody pretreatment step comprises adding a sodium edetate solution to the antibody and adjusting the pH using a Tris solution.

11. The method of claim 8 or 9, wherein the antibody pretreatment step comprises adding a disodium edetate solution to the antibody and adjusting the pH using a Tris solution, wherein the concentration of the disodium edetate solution is 2-10 mM and the concentration of the Tris solution is 2 M.

12. The method of claim 8 or 9, wherein the pretreatment step comprises adding a disodium edetate solution to the antibody and adjusting the pH using phosphate.

13. The method of any one of claims 1-12, wherein the antibody is further purified prior to pretreatment, the purification comprising steps of chromatography and filtration, the chromatography being selected from affinity chromatography, anion exchange chromatography and / or cation exchange chromatography; Preferably, the purification process includes affinity chromatography, incubation, deep filtration, anion exchange chromatography, and cation exchange chromatography in sequence, but does not include ultrafiltration or diafiltration steps.

14. The method according to any one of claims 1-13, wherein the antibody is an anti-Her2 antibody, an anti-EGFR antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-Trop-2 antibody; preferably, the anti-Her2 antibody is Trastuzumab or Pertuzumab, the anti-EGFR antibody is Cetuximab or Nimotuzumab, the anti-PD-1 monoclonal antibody is Nivolumab or Pembrolizumab, the anti-PD-L1 monoclonal antibody is Atezolizumab or Durvalumab, and the anti-Trop-2 antibody is Sacituzumab or Datopotamab.

15. The method according to any one of claims 1-14, wherein the drug to be coupled is a small molecule compound with a linker arm, the small molecule compound being selected from DNA topoisomerase inhibitors, tubulin inhibitors, or derivatives thereof; preferably, the topoisomerase inhibitor is a topoisomerase I inhibitor or a topoisomerase II inhibitor; preferably, the topoisomerase I inhibitor is camptothecin, SN-38, irinotecan, topotecan, belotetan, rubotecan, or derivatives thereof; preferably, the topoisomerase II inhibitor is actinomycin D, doxorubicin, doxorubicin, docarmicin, daunorubicin, mitoxantrone, podophyllotoxin, etoposide, or derivatives thereof; preferably, the tubulin inhibitor is vinca alkaloids, vincristine, vinblastine, paclitaxel, docetaxel, cabazitaxel, or derivatives thereof.

16. The method according to any one of claims 1-15, wherein the antibody is Sacituzumab; and the structure of the drug to be conjugated is shown below: ； Preferably, the antibody-drug conjugate has the following structure: in, γ is 1-10; preferably, γ is 5-8; preferably, the connecting arm is connected to the thiol group of Sacituzumab.

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