WO2022068898A1 - 一种双取代桥连抗体偶联药物的制备方法 - Google Patents

一种双取代桥连抗体偶联药物的制备方法 Download PDF

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WO2022068898A1
WO2022068898A1 PCT/CN2021/121825 CN2021121825W WO2022068898A1 WO 2022068898 A1 WO2022068898 A1 WO 2022068898A1 CN 2021121825 W CN2021121825 W CN 2021121825W WO 2022068898 A1 WO2022068898 A1 WO 2022068898A1
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antibody
buffer
hydrolysis
preparation
phosphate
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French (fr)
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周伟
朱会凯
王珍珍
徐辉
谭小钉
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Jiangsu Mabwell Health Pharmaceutical R&d Co Ltd
Mabwell Shanghai Bioscience Co Ltd
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Jiangsu Mabwell Health Pharmaceutical R&d Co Ltd
Mabwell Shanghai Bioscience Co Ltd
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Priority to EP21874553.7A priority Critical patent/EP4223741A4/en
Priority to US18/247,056 priority patent/US20230372526A1/en
Publication of WO2022068898A1 publication Critical patent/WO2022068898A1/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6855—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from breast cancer cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids

Definitions

  • the invention belongs to the field of antibody drug preparation technology, in particular, the invention relates to a method for preparing antibody conjugated drugs by using a double-substituted maleimide linker.
  • Antibody-drug conjugates are a new type of tumor therapeutic drug composed of antibodies or antibody-like ligands, small molecule drugs and linkers that couple the two.
  • the high selectivity, stability and good pharmacokinetic characteristics of antibodies or antibody-like ligands are currently the focus of attention in the field of tumor therapy.
  • Conjugation technology and preparation process are extremely critical for the development of ADC drugs.
  • the third-generation ADC coupling technology is widely used, which is mainly divided into three categories: the site-specific coupling technology of unnatural amino acids, the enzyme-catalyzed site-specific coupling technology, and the chemical modification site-specific coupling technology.
  • the first two types of technologies usually require special modification of the antibody. Therefore, relatively speaking, the chemical modification site-directed coupling technology is a kind of antibody-drug conjugate preparation technology that is more general to ordinary antibody molecules.
  • patent application CN201380025774.3 discloses a preparation scheme for preparing ADC drugs using bridge linkers
  • patent application CN201310025021.4 discloses a preparation scheme for ADC drugs using tridentate linkers.
  • the ADC drug prepared by sub-preparation (see WO2018/095422A1), the obtained ADC drug is based on the disulfide chain bridge of the bis-substituted maleimide, has better stability, does not easily undergo thioether exchange in vivo, and has good drug in vivo performance. In vitro activity.
  • ADC drugs prepared using the process route disclosed in WO2018/095422A1 often have the problem of poor product uniformity, and it is speculated that relatively serious small molecule drug shedding occurs.
  • the shedding of small-molecule drugs can lead to non-targeted toxicity, which brings greater safety hazards to drug development.
  • the purpose of the present invention is to provide a new preparation method of double-substituted bridged antibody conjugated drug.
  • a preparation method of a double-substituted bridged antibody-drug conjugate comprising: hydrolyzing an antibody-conjugated product obtained by coupling an antibody and a compound shown in formula I:
  • Ar is phenyl or substituted phenyl, and the substituent in the substituted phenyl is selected from alkyl (such as C1-C6 alkyl, preferably C1-C3 alkyl, more preferably methyl), alkoxy (eg C1-C6 alkoxy, preferably C1-C3 alkoxy, more preferably methoxy), halogen (F, Cl, Br or I), ester, cyano and -C(O)NR1R2-, wherein R1 and R2 are independently selected from chemical bonds, H and alkyl (eg C1-C6 alkyl, preferably C1-C3 alkyl, more preferably methyl), or R1 and R2 form a 5-7 membered heterocycle, and the heteroatom is selected from one or more of O, N and S;
  • alkyl such as C1-C6 alkyl, preferably C1-C3 alkyl, more preferably methyl
  • alkoxy eg C1-C6 alkoxy
  • X and Y are independently hydrogen, halogen (F, Cl, Br or I), trifluoromethyl or alkoxy (eg C1-C6 alkoxy, preferably C1-C3 alkoxy, preferably methoxy);
  • n is any integer from 1 to 24;
  • L-CTD is a cytotoxic drug and a linked release structure used in antibody-drug conjugates.
  • L is for example selected from Val-Ala-PAB (VA-PAB), Val-Cit-PAB (VC-PAB), Phe-Lys-PAB and MAC glucuronide phenol linker;
  • CTD is for example cytotoxic drug, preferably microtubule Inhibitors (eg, microtubule inhibitors MMAE, DM1, DM4, Tublysin); topoisomerase inhibitors (eg, topoisomerase inhibitors SN38, Ixinotecan, and Ixinotecan derivatives) or DNA binding agents (eg, DNA binding agents PBD and derivatives thereof).
  • PAB is p-aminobenzyloxycarbonyl.
  • R1 and R2 are independently selected from H and C1-C3 alkyl; or R1 and R2 form a 6-membered heterocycle, and the heteroatom is selected from one or more of O and N, preferably morpholine.
  • Ar is phenyl, 4-methylformamido substituted phenyl or 4-formylmorpholine substituted phenyl
  • X and Y are independently hydrogen, fluorine, trifluoromethyl or methoxy; more preferably, X and Y are independently hydrogen, or X and Y are independently located on a benzene ring relative to maleimide interposition;
  • n is any integer from 1 to 10, preferably from 3 to 5;
  • the L-CTD is VC-PAB-MMAE or VC-seco-DUBA.
  • the hydrolysis is performed before or after purification of the antibody-conjugated product.
  • the hydrolysis comprises: heating the antibody-conjugated product in a hydrolysis buffer at pH 7.4-9.0 at 25-45° C. for 1-24 h.
  • the hydrolysis buffer may contain sodium dihydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, citric acid, glycine, tromethamine, arginine hydrochloride, hydrochloric acid, phosphoric acid, hydroxide One or more of sodium and potassium hydroxide.
  • the hydrolysis buffer is sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer or tromethamine buffer.
  • the concentration of the antibody conjugated product in the hydrolysis buffer is 2-30 mg/mL, preferably 5-20 mg/mL.
  • the hydrolysis comprises: heating the antibody-conjugated product in the hydrolysis buffer at 25-35° C. for 1-6 h, preferably 1-3 h.
  • the hydrolysis buffer is pH 7.5-8.5, preferably pH 7.8.
  • the hydrolysis buffer is a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer at pH 7.5-8.5.
  • the hydrolysis buffer is: 50mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, pH 7.8; 50mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer+3% arginine, pH 7.8; 50 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer, pH 7.8; or 50 mM tromethamine-hydrochloric acid buffer, pH 7.8.
  • the antibody is IgG, preferably IgG1.
  • the antibody-drug conjugate is prepared according to the method disclosed in WO2018/095422A1.
  • the preparation method provided by the invention comprises the following steps:
  • Antibody reduction add a reducing agent with a molar equivalent of ⁇ 5.5 times the antibody to the buffer containing the antibody at a concentration of 5-30 mg/mL, and react at 30-40 ° C for 1.5-2 h, wherein the reducing agent is selected from TCEP , one or more of DTT, 2-MEA and DTBA;
  • Antibody coupling replace the reduced antibody in step a into a buffer of pH 6.5-7.6, and dilute the antibody to a concentration of 3.5-15 mg/mL, dissolve 4.5-6.5 times the antibody in an organic co-solvent
  • a molar equivalent of the compound represented by formula I is added to the antibody diluent, so that the organic co-solvent accounts for 5%-30% of the volume of the reaction system, and the reaction is stirred at 15-35 ° C for ⁇ 0.5h, wherein the The organic co-solvent is one or more selected from DMA, DMSO, DMF and ACN;
  • Hydrophobic chromatography The antibody conjugated product is purified by hydrophobic chromatography using a hydrophobic filler, the hydrophobic filler is Butyl Sepharose HP (GE), Capto Phenyl Impres (GE), Toyopearl Butyl 650M (TOSOH) or Butyl Sepharose 4FF ( GE), the stationary phase uses sodium chloride or ammonium sulfate as a buffer for chromatography salts; and
  • the preparation method further comprises the following steps after step b or after step c:
  • Hydrolysis Replace the antibody conjugated product into a hydrolysis buffer with pH 7.4-9.0, and heat at 25-45°C for 1-24h.
  • the hydrolysis buffer may contain sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate, citric acid, glycine, tromethamine, arginine hydrochloride, hydrochloric acid, phosphoric acid, sodium hydroxide and one or more of potassium hydroxide.
  • the displacements in steps a, b and d are carried out using molecular sieves, ultrafiltration centrifuge tubes or ultrafiltration membrane packs.
  • step a of the preparation method of the present invention
  • the reducing agent is TCEP or DTT;
  • the reducing agent is 6.5-10 times the molar equivalent of the antibody
  • the reducing agent is added in the form of an aqueous solution of 5-15 mg/mL, preferably 10 mg/mL;
  • the antibody is IgG, preferably IgG1;
  • the concentration of the antibody is 10-15 mg/mL, preferably 12 mg/mL;
  • the buffer is phosphate buffer at pH 6.5-7.6.
  • the buffer is a pH 7.4, 50 mM phosphate buffer.
  • step b of the preparation method of the present invention
  • the buffer is pH 7.2-7.4; preferably, the buffer is phosphate buffer or phosphate-EDTA buffer at pH 7.2-7.4. According to a specific embodiment of the present invention, the buffer is 50mM sodium dihydrogen phosphate-disodium hydrogen phosphate+100mM NaCl+2mM EDTA buffer, pH 7.2-7.4;
  • the antibody is diluted to a concentration of 3.5-10 mg/mL during displacement;
  • the compound represented by the formula I is 5.0-6.0 times the molar equivalent of the antibody
  • the organic co-solvent is DMA, DMSO or ACN;
  • the organic co-solvent accounts for 6%-30% of the volume of the reaction system
  • the reaction is stirred at 15-35°C for 0.5-1 h.
  • step c of the preparation method of the present invention
  • the stationary phase uses a buffer with ammonium sulfate as the chromatographic salt.
  • the concentration of ammonium sulfate is preferably 0.45mol/L;
  • the stationary phase uses phosphate buffered saline with ammonium sulfate as the chromatographic salt, and the elution phase is phosphate buffered saline.
  • the phosphate is 0-100 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution, pH 7.4-8.0.
  • step d of the preparation method of the present invention
  • the concentration of the antibody conjugated product in the hydrolysis buffer is 2-30 mg/mL, preferably 5-20 mg/mL;
  • the hydrolysis buffer is sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer or tromethamine buffer;
  • the hydrolysis comprises: heating the antibody conjugated product in the hydrolysis buffer at 25-35° C. for 1-6 h, preferably 1-3 h.
  • the hydrolysis buffer is pH 7.5-8.5, preferably pH 7.8.
  • the hydrolysis buffer is a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer at pH 7.5-8.5.
  • the hydrolysis buffer is: 50mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, pH 7.8; 50mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer+3% arginine, pH 7.8; 50 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer, pH 7.8; or 50 mM tromethamine-hydrochloric acid buffer, pH 7.8.
  • the present invention provides the disubstituted bridged antibody drug conjugate obtained by the method of the present invention.
  • WO2018/095422A1 describes the following ADC preparation process:
  • ADC mixed products of two structures were prepared.
  • the mixed drug has poor stability in vitro and in vivo, and has severe input reactions during administration. This may be due to the shedding of small-molecule drugs from antibodies, which has negative effects on the efficacy and safety of drugs to varying degrees.
  • a hydrolysis step is further introduced, and this step can be performed before or after the column chromatography purification of the ADC drug.
  • the product of each of the two steps can be incubated in a pH 8.0 ⁇ 0.5 hydrolysis buffer (eg, 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer) so that one molecule of maleimide can react with one molecule of water
  • the addition reaction generates ring-opened carboxylic acid compounds, thereby obtaining ADC drugs with a uniform ring-opening structure.
  • the method of the present invention further screened the reaction parameters used in the operation steps of antibody reduction, coupling and chromatographic purification, so that the final ADC drug can achieve higher purity, and the non-reducing capillary electrophoresis purity is greater than 95%.
  • this process it has been verified by experiments that this process can be successfully scaled up, and the physicochemical properties of the coupled samples obtained after amplification are basically the same as those of the small-scale coupled samples.
  • Fig. 1 shows the characterization results of ADC product samples obtained by using the preparation method of the present invention with different antibodies in Example 7, wherein:
  • 1A to 1C are HIC-HPLC, NR-CE-SDS and SEC-HPLC characterization results of the product obtained from Pertuzumab, respectively.
  • Fig. 2 is the ADC product sample characterization results of group A and group B without hydrolysis and after hydrolysis in Example 8, wherein:
  • 2A to 2B are the HIC-HPLC characterization results of group A and group B samples, respectively;
  • 2C to 2D are the SEC-HPLC characterization results of Group A and Group B samples, respectively;
  • 2E to 2F are the NR CE-SDS characterization results of Group A and Group B samples, respectively.
  • the method of the present invention provides a more stable and homogeneous ADC product as shown below:
  • the “equivalents” mentioned in the present invention are all molar equivalent ratios compared with antibodies.
  • PB used in the present invention specifically refers to a sodium phosphate buffer with disodium hydrogen phosphate-sodium dihydrogen phosphate as the main component.
  • Disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solutions with different pH values are usually obtained by intermodulation of sodium dihydrogen phosphate and disodium hydrogen phosphate solutions of the same concentration.
  • Sample preparation The sample was diluted to 2.0 mg/ml with mobile phase B, centrifuged at 12000 rpm for 10 min, and the supernatant was taken for HPLC analysis;
  • Sample preparation the sample was diluted to 3.0 mg/ml with mobile phase B, centrifuged at 12000 rpm for 10 min, and the supernatant was taken for HPLC analysis;
  • Sample preparation The sample was diluted to 2.0 mg/ml with mobile phase B, centrifuged at 12000 rpm for 10 min, and the supernatant was taken for HPLC analysis;
  • the mentioned D3%, D4%, and D5% represent the percentages of ADCs with DAR values of 3, 4, and 5, respectively.
  • sample processing Dilute the sample to 2.0mg/ml (sample buffer: 40mM PB, 1% SDS, pH 6.5), take 50 ⁇ L+45 ⁇ L sample buffer+5 ⁇ L IAM (0.5M), mix well, water bath at 70°C for 10min, Take the supernatant for injection analysis.
  • sample buffer 40mM PB, 1% SDS, pH 6.5
  • IAM 0.5M
  • Capillary activation under 20psi pressure, rinse with 0.1mol/L sodium hydroxide, 0.1mol/L hydrochloric acid, and injection water for 10min, 5min, and 2min respectively; under 70psi pressure, rinse with SDS-MW-Gel Buffer for 10min; -15kv voltage, 20psi, 25°C column temperature, pre-separation for 10min; running program: 70psi, rinse the capillary with 0.1mol/L sodium hydroxide, 0.1mol/L hydrochloric acid, injection water for 3min, 1min, 1min respectively; then pour glue for 10min , inject 40s in -5kv condition, run the separation program (-18kv, 20psi). The column temperature was 25°C, the separation voltage was -15kv and the separation time was 40min.
  • Sample processing After the sample was diluted to about 2.0-3.0 mg/ml with mobile phase, centrifuged at 12,000 rpm for 10 min, and the supernatant was taken for injection and analysis.
  • Injection volume 2 ⁇ L
  • Source temperature 125°C
  • Liquid chromatography mass spectrometry (LC-MS) coupling is an orthogonal technique for analyzing DAR and drug distribution in ADCs.
  • Sample treatment Use 1 ⁇ L PNGase F/100 ⁇ g mAb to deglycosylate the sample and react at 37°C overnight. Before the analysis, reduction was performed with 20 mmol/L dithiothreitol (DTT) and incubated at 37°C for 30 min. Samples were diluted to 1 mg/mL with water before loading.
  • DTT dithiothreitol
  • Chromatographic column PLRP-S column (Agilent), particle size 5 ⁇ m, Inner diameter 2.1mm, length 50mm;
  • Mobile phase is an aqueous solution of 0.1% formic acid and 0.025% trifluoroacetic acid
  • mobile phase B is an acetonitrile solution of 0.1% formic acid and 0.025% trifluoroacetic acid
  • the electrospray voltage is set to 4500-5000V, the original temperature is set to 350°C, and the air curtain and atomizing gas are set to 40;
  • Pertuzumab was displaced into 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer pH 7.4 and diluted to 12.2 mg/ml.
  • An aqueous solution with a concentration of 1.0 mg/ml of dithiothreitol (DTT) or tricarboxyethyl phosphine (TCEP) with 8.5 times the molar equivalent of antibody was added, and the reaction was carried out at 35° C. for 1.5 h. Then, it was stirred with C-3 pre-dissolved in DMA in 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer at 25°C for 1 h. After coupling, the sample was changed to pH 7.4 50mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, and sampling was carried out for HIC-HPLC detection. The test results are shown in Table 1-1.
  • the pertuzumab was replaced with pH7.4 50mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, and diluted to 12mg/ml.
  • the aqueous solutions of different equivalents of TECP and DTT were added to the reaction system, and the reaction was carried out at 35°C for 1.5h. Samples were taken for D-SEC detection, and the remaining amount of unreduced antibody was analyzed. The test results are shown in Table 1-2.
  • pertuzumab into pH7.4 50mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, and dilute to 12mg/ml, add TECP aqueous solution with a concentration of 10mg/ml to the reaction system, incubate at 35°C for 2h, after reduction
  • the pertuzumab was substituted into pH 6.8-7.8 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate + 100 mM NaCl + 2 mM EDTA buffer, respectively, and diluted to 5 mg/ml.
  • pH HIC-HPLC D3%/D4%/D5%
  • NR-CE-SDS purity 6.5 7.57/78.76/13.49 93.8 6.7 5.89/83.12/10.98 94.8 7.0 5.62/81.82/12.56 94.7
  • Example 2b for the reduction process of pertuzumab.
  • the antibodies were exchanged, diluted to about 5.0 mg/ml, and grouped.
  • organic solvents N,N-dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), and acetonitrile (ACN) were used, and compound C-3 was used any of these solvents.
  • DMA dimethyl sulfoxide
  • ACN acetonitrile
  • One is dissolved to 20 mg/mL, and the feeding volume of the compound C-3 solution is calculated according to 5 times the molar equivalent of the total amount of antibodies, and the corresponding solvent is added to the reaction system of the two to achieve the solvent ratio shown in Table 2-3.
  • the proportion of DMA is greater than 4% or the proportion of DMSO and ACN is greater than 6%, the proportion of organic co-solvent is less than or equal to 30%, the coupling reaction system is clear, and the reaction result is good.
  • Example 2a For antibody reduction. After the reduction, replace with pH 7.4 50mM disodium hydrogen phosphate-sodium dihydrogen phosphate+100mM NaCl+2mM EDTA buffer, and dilute the antibody to 2.5-13mg/ml, compound C-3 into a 20mg/ml DMA solution. 5.0 equivalents of small molecule DMA solution was added to the reaction system and stirred at 25 °C for 1 h. After the coupling, the solution system was replaced with 50mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer at pH 7.4, and the components of the sample were analyzed by hydrophobic chromatography HIC-HPLC and NR-CE-SDS. The results are shown in Table 2-4. .
  • the concentration of the conjugated-reactive antibody can be selected at 3.5-10 mg/ml.
  • the antibody reduction coupling process refers to Example 2a.
  • the solution system is replaced with pH 7.4 50mM disodium hydrogen phosphate-sodium dihydrogen phosphate+100mM NaCl+2mM EDTA buffer and the antibody concentration is diluted to about 5mg/ml, Then the reaction system was preheated to 25 ⁇ 10°C, the DMA solution of compound C-1 was added to the reaction system, and the mixture was stirred at the corresponding temperature for 1 h.
  • Example 2a for the reductive coupling process of pertuzumab. After coupling, the antibody conjugated drug is replaced in 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer at pH 7.4-9.0, stirred at 25-45 °C for 3 hours, and observed. The color change of the reaction solution. The results are shown in Table 3-1.
  • Example 2a for the reductive coupling process of pertuzumab.
  • the samples after coupling with different small molecular compounds were hydrolyzed in pH7.8 50mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution at 35°C for 3-18h. After sampling at different time points, the samples were sent to Native Ms to detect the hydrolysis of the samples. ratio, the results are shown in Table 3-2.
  • Example 2a for the reductive coupling process of pertuzumab.
  • the samples after coupling with different small molecular compounds were replaced in different hydrolysis solution systems (A: 50mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, pH 7.8; B: 50mM disodium hydrogen phosphate-sodium dihydrogen phosphate Buffer + 3% arginine, pH 7.8; C: 50mM potassium dihydrogen phosphate-potassium hydrogen phosphate buffer, pH 7.8; D: 50mM tromethamine-hydrochloric acid buffer, pH 7.8) Sampling at different time points Detect Native Ms, and detect the hydrolysis ratio of the sample. The results are shown in Table 3-3.
  • Example 2a for the reductive coupling process of pertuzumab.
  • the coupled sample was replaced with a hydrolysis system of 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate, pH 7.8. After sampling at different time points, the Native Ms was sent for inspection to detect the hydrolysis ratio of the sample. The results are shown in Table 3-4.
  • the antibody reductive coupling process refers to Example 2a.
  • the sample is changed to pH 7.8 50mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, incubated at 35 ° C for more than 3 hours, and the obtained antibody conjugated product is used for Filler screening test.
  • the fillers with different ligands and different scaffolds were subjected to column chromatography under the same chromatographic conditions. The types of fillers and the screening results are shown in Table 4-1.
  • Butyl Sepharose 4FF, Butyl Sepharose HP and Capto Phenyl Impres could purify the coupled samples.
  • Butyl Sepharose 4FF is preferentially used, and the D3 flow-through protocol is used to purify the coupled sample.
  • Antibody reductive coupling process Referring to Example 2a, the sample was replaced with a solution of 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate, pH 7.8, and diluted to 12 mg/mL. After hydrolysis at 35°C for 3 hours, use a chromatographic salt solution of 50mM disodium hydrogen phosphate-sodium dihydrogen phosphate+1.0-2.0mol/L sodium chloride, or 50mM disodium hydrogen phosphate-sodium dihydrogen phosphate+0.325-0.45mol The chromatographic salt solution of /L ammonium sulfate was used as the stationary phase, and the chromatographic salt solution of 50 mM sodium dihydrogen phosphate-disodium hydrogen phosphate was used as the elution phase. The samples after the coupling and hydrolysis of pertuzumab and the compound were subjected to column layering with Butyl Sepharose 4FF. The separation results are shown in Table 4-2.
  • Pertuzumab was exchanged to pH 7.4 in 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, diluted to approximately 12 mg/ml protein concentration, and 10 mg/ml of TCEP in water (TCEP was 6.5 times the antibody equivalent) Added to the reaction system, incubated at 35°C for 2 hours, and then changed to pH 7.2 50mM disodium hydrogen phosphate-sodium dihydrogen phosphate+100mM NaCl+2mM EDTA buffer, and diluted to 8mg/mL, then added to The small molecule compound C-3 pre-dissolved in DMA in the equivalent ratio eq shown in the following table was added to the reaction system, and DMA was added to the reaction system to make it reach 10%, and the mixture was heated and stirred for 1 hour.
  • the results show that: the scale of ⁇ 100 mg, the method of the present invention can obtain the coupling results basically consistent with the small test, and the coupling process can be successfully scaled up.
  • the solution was changed to pH 7.8 by ultrafiltration, centrifugation, and 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, and the concentration of the antibody conjugated product was 12 mg/mL, and incubated at 35 °C for 3 h.
  • the samples were conductance-adjusted with 3M ammonium sulfate, using Butyl Sepharose 4FF packing and 0.45M ammonium sulfate as chromatographic salts 50mM sodium dihydrogen phosphate-disodium hydrogen phosphate stationary phase, 50mM disodium hydrogen phosphate-sodium dihydrogen phosphate As the mobile phase, the pH of both the stationary phase and the mobile phase was 7.5.
  • IgG1 antibodies against different targets such as HER2 (Pertuzumab) and CD20 (Rituximab) were coupled with small molecular compounds, and detected after preparation. The results show that this process is suitable for IgG1 antibodies of different targets. Good applicability sex. The results are shown in Table 7-1 and Figure 1.
  • IgG1 antibodies with different antibody sequences against different targets are suitable for the ADC preparation method provided by the present invention.
  • Example 8 Physicochemical properties, in vitro/in vivo stability, in vivo efficacy and safety of ADC samples with different preparation methods.
  • Example 6 According to the method of Example 6 above, 2.5 g of Pertuzumab was reduced and coupled with small molecule compound C-1 in 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate + 100 mM NaCl + 2 mM EDTA buffer at pH 7.2. The ultrafiltration membrane was used to replace it in the buffer of pH 7.8, and the final product was obtained and divided into two groups, A and B.
  • the group A samples were directly purified by Butyl Sepharose 4FF packing, and the obtained purified solution was concentrated and exchanged to pH 7.4 50mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer to obtain the group A sample stock solution with a concentration of 10 mg/mL.
  • the samples of group B were incubated at 35°C for 3 hours, they were purified by Butyl Sepharose 4FF packing using the same method as group A, and the obtained purified solution was concentrated and exchanged to pH 7.4 50mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer to obtain group B.
  • B sample stock solution the concentration is about 10mg/mL.
  • the samples of group A and group B were diluted to 1 mg/ml with physiological saline, and administered to cynomolgus monkeys at a dose of 6 mg/kg. Blood was collected at 30min, 1h, 2h, 4h, 12h, 24h, 48h, 72h and 128h. Using the calibrated MMAE (MCE: HY-15162) as the standard substance, the free MMAE in the plasma was detected. The results showed that although the maximum peak value of the samples in group A and group B were both 24h, the exposure of free MMAE in group B was significantly lower than that in group A, suggesting that the samples in group B after hydrolysis treatment were more stable in vivo. The results are shown in Table 8-2.
  • the cynomolgus monkeys over 2.5 weeks of age were divided into groups, and two groups of cynomolgus monkeys, one male and one female, were randomly selected.
  • the samples of group A and group B were diluted to 1 mg/ml with physiological saline. It was administered by intravenous drip at a dose of 6 mg/kg. Seven days after administration, the biochemical parameters in monkey blood were analyzed. The results showed that abnormal indexes and severity in group A were more serious than those in group B samples.

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Abstract

一种双取代桥连抗体偶联药物的制备方法，所述制备方法包括：使抗体和式I所示的化合物偶联得到的抗体偶联产物进行水解。还提供通过所述制备方法得到的抗体偶联产物。

Description

一种双取代桥连抗体偶联药物的制备方法
相关申请的交叉引用
本专利申请要求于2020年9月29日提交的申请号为CN202011046911X的中国发明专利申请的优先权权益，在此将其全部内容引入作为参考。
技术领域
本发明属于抗体药物制备工艺领域，具体而言，本发明涉及一种采用双取代马来酰亚胺连接子制备抗体偶联药物的方法。
背景技术
抗体偶联药物(Antibody-drug conjugates，ADC)是由抗体或抗体类配体与小分子药物以及将二者偶联起来的连接子共同组成的一种新型肿瘤治疗药物，其结合了小分子药物的抗肿瘤活性和抗体或抗体类配体的高选择性、稳定性和良好的药代动力学特征，是目前肿瘤治疗领域的关注热点。
偶联技术与制备工艺对于发展ADC药物极为关键。为了确保这类药物的临床安全性和有效性，不仅需要实现药物与抗体的稳定连接，而且需尽量降低ADC产物的异质性。目前普遍应用第三代ADC偶联技术，主要分为非天然氨基酸的定点偶联技术、酶催化定点偶联技术以及化学修饰定点偶联技术这三类。其中，前两类技术通常需要对抗体进行特殊改造，因此相对而言，化学修饰定点偶联技术是一类对普通抗体分子更通用的抗体偶联药物制备技术，其主要利用特定结构的连接子以及与其相适应的偶联工艺以实现定点偶联。例如，专利申请CN201380025774.3公开了一种采用桥状连接子制备ADC药物的制备方案；专利申请CN201310025021.4公开了一种采用三齿型连接子的ADC药物制备方案。
迈威(上海)生物技术有限公司和下属子公司江苏迈威康生物开发了一种连接子新技术，基于该技术提供了一种新型的双取代马来酰亚胺类连接子以及利用该连接子制备的ADC药物(参见WO2018/095422A1)，得到的ADC药物基于双取代马来酰亚胺的二硫链桥接，具有更好的稳定性，在体内不易发生巯醚交换，具有良好的药物体内体外活性。但是，已发现使用WO2018/095422A1中公开的工艺路线制备的ADC药物往往会有产品均一性不佳的问题，推测出现了较为严重的小分子药物脱落。在药物安全性方面， 小分子药物脱落会导致非靶向毒性，为药物开发带来较大的安全隐患。
因此，本领域亟需开发一种与双取代马来酰亚胺类连接子相适应的、简单、高效、稳定且具有应用价值的定点偶联药物制备工艺。
发明内容
实验证明，在WO2018/095422A1所公开的工艺条件下制备的ADC药物仍然无法满足临床用药的安全、有效、稳定等质量要求，无法实现产品商业化生产。为了解决这一问题，本发明的目的是提供一种双取代桥连抗体偶联药物的新的制备方法。
本发明的技术方案如下：
一种双取代桥连抗体偶联药物的制备方法，所述制备方法包括：使抗体和式I所示的化合物偶联得到的抗体偶联产物进行水解：
Figure PCTCN2021121825-appb-000001
式I中，Ar为苯基或取代苯基，所述取代苯基中的取代基选自烷基(例如C1-C6烷基、优选C1-C3烷基，更优选甲基)、烷氧基(例如C1-C6烷氧基、优选C1-C3烷氧基，更优选甲氧基)、卤素(F、Cl、Br或I)、酯基、氰基和-C(O)NR1R2-，其中R1和R2独立地选自化学键、H和烷基(例如C1-C6烷基、优选C1-C3烷基，更优选甲基)，或R1和R2形成5-7元杂环，杂原子选自O、N和S中的一个或多个；
X和Y独立地为氢、卤素(F、Cl、Br或I)、三氟甲基或烷氧基(例如C1-C6烷氧基、优选C1-C3烷氧基，优选甲氧基)；
n为1～24中的任意整数；
L-CTD为应用于抗体偶联药物中的细胞毒药物及连接释放结构。其中，L例如选自Val-Ala-PAB(VA-PAB)、Val-Cit-PAB(VC-PAB)、Phe-Lys-PAB和MAC glucuronide phenol连接子；CTD例如为细胞毒药物，优选微管抑制剂(例如，微管抑制剂类MMAE、DM1、DM4、Tublysin)；拓扑异构酶抑制剂(例如，拓扑异构酶抑制剂类SN38、依喜替康以及依喜替康衍生物) 或DNA结合剂(例如，DNA结合剂PBD及其衍生物)。PAB为对-氨基苄氧羰基。
优选地，R1和R2独立地选自H和C1-C3烷基；或者R1和R2形成6元杂环，杂原子选自O和N中的一个或多个，优选为吗啉。
更优选地，Ar为苯基、4-甲基甲酰胺基取代苯基
Figure PCTCN2021121825-appb-000002
或4-甲酰基吗啉取代苯基
Figure PCTCN2021121825-appb-000003
优选地，X和Y独立地为氢、氟、三氟甲基或甲氧基；更优选地，X和Y独立地为氢，或者X和Y独立地位于苯环上相对马来酰亚胺的间位；
优选地，n为1～10、优选3～5中的任意整数；
优选地，L-CTD为VC-PAB-MMAE或VC-seco-DUBA。
优选地，所述水解在所述抗体偶联产物的纯化之前或之后进行。
优选地，所述水解包括：将抗体偶联产物在pH 7.4-9.0的水解缓冲液中在25-45℃下加热1-24h。其中所述水解缓冲液可包含磷酸二氢钠、磷酸氢二钠、磷酸氢二钾、磷酸二氢钾、枸橼酸、甘氨酸、氨基丁三醇、盐酸精氨酸、盐酸、磷酸、氢氧化钠和氢氧化钾中的一种或多种。优选地，所述水解缓冲液为磷酸二氢钠-磷酸氢二钠缓冲液、磷酸二氢钾-磷酸氢二钾缓冲液或氨基丁三醇缓冲液。
优选地，所述抗体偶联产物在水解缓冲液中的浓度为2-30mg/mL、优选5-20mg/mL。
更优选地，所述水解包括：将抗体偶联产物在所述水解缓冲液中在25-35℃下加热1-6h、优选1-3h。其中优选地，所述水解缓冲液为pH 7.5-8.5，优选pH7.8。例如，所述水解缓冲液为pH 7.5-8.5的磷酸二氢钠-磷酸氢二钠缓冲液。
根据本发明的具体实施方案，所述水解缓冲液为：50mM磷酸二氢钠-磷酸氢二钠缓冲液，pH 7.8；50mM磷酸二氢钠-磷酸氢二钠缓冲液+3％精氨酸，pH 7.8；50mM磷酸二氢钾-磷酸氢二钾缓冲液，pH 7.8；或50mM氨基丁三醇-盐酸缓冲液，pH 7.8。
优选地，所述抗体为IgG，优选IgG1。
根据本发明的具体实施方式，所述抗体偶联药物根据WO2018/095422A1中公开的方法制备。
具体而言，本发明提供的制备方法包括以下步骤：
a.抗体还原：将≥5.5倍抗体摩尔当量的还原剂加入到含5-30mg/mL浓度抗体的缓冲液中，在30-40℃下反应1.5-2h，其中所述还原剂为选自TCEP、DTT、2-MEA和DTBA中的一种或多种；
b.抗体偶联：将步骤a中经还原的抗体置换到pH 6.5-7.6的缓冲液中，并将抗体稀释到3.5-15mg/mL浓度，将溶解于有机共溶剂中的4.5-6.5倍抗体摩尔当量的式I所示的化合物加入到所述抗体稀释液中，使得所述有机共溶剂占反应体系体积的5％-30％，在15-35℃下搅拌反应≥0.5h，其中所述有机共溶剂为选自DMA、DMSO、DMF和ACN中的一种或多种；
c.疏水层析：使用疏水填料对抗体偶联产物进行疏水层析纯化，所述疏水填料为Butyl Sepharose HP(GE)、Capto Phenyl Impres(GE)、Toyopearl Butyl 650M(TOSOH)或Butyl Sepharose 4FF(GE)，固定相使用氯化钠或硫酸铵作为层析盐的缓冲液；并且
所述制备方法在步骤b之后或步骤c之后还包括以下步骤：
d.水解：将抗体偶联产物置换到pH 7.4-9.0的水解缓冲液中，在25-45℃下加热1-24h。其中所述水解缓冲液可包含磷酸二氢钠、磷酸氢二钠、磷酸氢钾、磷酸二氢钾、枸橼酸、甘氨酸、氨基丁三醇、盐酸精氨酸、盐酸、磷酸、氢氧化钠和氢氧化钾中的一种或多种。
在本发明的方法中，步骤a、b和d中的置换采用分子筛、超滤离心管或超滤膜包进行。
在本发明的制备方法的步骤a中：
优选地，所述还原剂为TCEP或DTT；
优选地，所述还原剂为6.5-10倍抗体摩尔当量；
优选地，所述还原剂以5-15mg/mL、优选10mg/mL的水溶液形式加入；
优选地，所述抗体为IgG，优选IgG1；
优选地，所述抗体的浓度为10-15mg/mL、优选12mg/mL；
优选地，所述缓冲液为pH 6.5-7.6的磷酸盐缓冲液。根据本发明的具体实施方式，所述缓冲液为pH7.4、50mM的磷酸盐缓冲液。
在本发明的制备方法的步骤b中：
优选地，所述缓冲液为pH 7.2-7.4；优选地，所述缓冲液为pH 7.2-7.4 的磷酸盐缓冲液或磷酸盐-EDTA缓冲液。根据本发明的具体实施方式，所述缓冲液为50mM磷酸二氢钠-磷酸氢二钠+100mM NaCl+2mM EDTA缓冲液，pH 7.2-7.4；
优选地，置换时将抗体稀释到3.5-10mg/mL浓度；
优选地，所述式I所示的化合物为5.0-6.0倍抗体摩尔当量；
优选地，所述有机共溶剂为DMA、DMSO或ACN；
优选地，使得所述有机共溶剂占反应体系体积的6％-30％；
优选地，在15-35℃下搅拌反应0.5-1h。
在本发明的制备方法的步骤c中：
优选地，固定相使用以硫酸铵作为层析盐的缓冲液。根据本发明的具体实施方式，硫酸铵浓度优选为0.45mol/L；
优选地，固定相使用以硫酸铵作为层析盐的磷酸盐缓冲液，洗脱相为磷酸盐缓冲液。
更优选地，磷酸盐为0-100mM磷酸氢二钠-磷酸二氢钠缓冲溶液，pH7.4-8.0。
在本发明的制备方法的步骤d中：
优选地，所述抗体偶联产物在水解缓冲液中的浓度为2-30mg/mL、优选5-20mg/mL；
优选地，所述水解缓冲液为磷酸二氢钠-磷酸氢二钠缓冲液、磷酸二氢钾-磷酸氢二钾缓冲液或氨基丁三醇缓冲液；
优选地，所述水解包括：将抗体偶联产物在所述水解缓冲液中在25-35℃下加热1-6h、优选1-3h。其中优选地，所述水解缓冲液为pH 7.5-8.5，优选pH7.8。例如，所述水解缓冲液为pH 7.5-8.5的磷酸二氢钠-磷酸氢二钠缓冲液。
根据本发明的具体实施方案，所述水解缓冲液为：50mM磷酸二氢钠-磷酸氢二钠缓冲液，pH 7.8；50mM磷酸二氢钠-磷酸氢二钠缓冲液+3％精氨酸，pH 7.8；50mM磷酸二氢钾-磷酸氢二钾缓冲液，pH 7.8；或50mM氨基丁三醇-盐酸缓冲液，pH 7.8。
另一方面，本发明提供通过本发明的方法得到的双取代桥连抗体偶联药物。
WO2018/095422A1描述了如下ADC制备工艺：
Figure PCTCN2021121825-appb-000004
因此，制得两种结构的ADC混合产物。实验证明，该混合药物体外、体内稳定性不佳，在给药过程中具有严重的输入反应。这可能是由于小分子药物从抗体上脱落，从而对药物的药效和安全性带来不同程度的负面影响。
Figure PCTCN2021121825-appb-000005
为了改善这一问题，本发明的方法中，在抗体偶联得到ADC药物的步骤后，进一步引入了水解步骤，该步骤可在ADC药物的柱色谱纯化之前或之后进行。例如，可将两个步骤各自的产物在pH 8.0±0.5水解缓冲液(例如50mM磷酸氢二钠-磷酸二氢钠缓冲液)中孵育，使得一分子马来酰亚胺可以和一分子水发生加成反应，生成开环的羧酸类化合物，从而获得均一的开环结构的ADC药物。实验证明，经过水解处理后，ADC药物的产物结构更加均一，该均一ADC药物产物在血浆中37℃孵育过程中，小分子药物的脱落明显减少。
此外，本发明的方法就抗体还原、偶联与色谱纯化操作步骤中采用的反应参数进行了进一步筛选，使得最终ADC药物可以实现更高的纯度，非还原毛细管电泳纯度大于95％。同时，经实验验证，此工艺流程可顺利放大，且放大后得到的偶联样品理化性质与小试偶联样品基本一致。
附图说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1为实施例7中以不同抗体采用本发明的制备方法得到的ADC产物样品表征结果，其中：
1A至1C分别是以Pertuzumab得到的产物的HIC-HPLC、NR-CE-SDS和SEC-HPLC表征结果。
图2为实施例8中不经过水解与经过水解的组A和组B的ADC产物样品表征结果，其中：
2A至2B分别是组A和组B样品的HIC-HPLC表征结果；
2C至2D分别是组A和组B样品的SEC-HPLC表征结果；
2E至2F分别是组A和组B样品的NR CE-SDS表征结果。
实施发明的最佳方式
本发明的方法提供如下所示的质量更加稳定、更加均一的ADC产物：
Figure PCTCN2021121825-appb-000006
本发明中提到的“当量”均为与抗体相比的摩尔当量比。
本发明中提到的“式I所示的化合物”在本文中与“小分子”或“小分子化合物”可互换使用，结构示例如下：
A系列分子：
Figure PCTCN2021121825-appb-000007
Figure PCTCN2021121825-appb-000008
Figure PCTCN2021121825-appb-000009
B系列分子：
Figure PCTCN2021121825-appb-000010
Figure PCTCN2021121825-appb-000011
C系列分子：
Figure PCTCN2021121825-appb-000012
Figure PCTCN2021121825-appb-000013
D系列分子：
Figure PCTCN2021121825-appb-000014
本发明中使用的“PB”特指磷酸氢二钠-磷酸二氢钠为主要成分的磷酸钠缓冲液。不同pH值的磷酸氢二钠-磷酸二氢钠缓冲溶液通常使用相同浓度的磷酸二氢钠、磷酸氢二钠溶液互调得到。
通用检测方法包括：
(1)疏水作用色谱HIC-HPLC：
(a1)定点偶联药物疏水色谱HIC-HPLC测定DAR值
样品制备：样品用流动相B稀释至2.0mg/ml后，12000rpm离心10min，取上清用于HPLC分析；
色谱柱：Sepax Proteomix HIC Butyl-NP5，5μm，4.6mm×35mm；
流动相A：0.025M磷酸盐+1.2M硫酸铵(pH 7.0)；
流动相B：0.025M磷酸盐(pH 7.0)；
流动相C：100％IPA；
流速：0.8mL/min；
检测波长：280nm；
柱温：30℃；
上样量：20μL。
HIC色谱梯度
Figure PCTCN2021121825-appb-000015
DAR计算公式：
DAR＝∑(加权峰面积)/100，即DAR＝(D0峰面积比×0+D1峰面积比×1+D2峰面积比×2+D3峰面积比×3+D4峰面积比×4+D5峰面积比×5+D6峰面积比×6+D7峰面积比×7+D8峰面积比×8)/100。
注：A、B、及C系列分子采用(a1)分析方法测定DAR值。
(a2)定点偶联药物疏水色谱HIC-HPLC测定DAR值
样品制备：样品用流动相B稀释至3.0mg/ml后，12000rpm离心10min，取上清用于HPLC分析；
色谱柱：Sepax Proteomix HIC Butyl-NP5，5μm，4.6mm×35mm；
流动相A：125mM PB+2.5M(NH4) 2SO 4(pH6.7)；
流动相B：125mM PB(pH6.7)；
流动相C：IPA；
流动相D：H 2O；
流速：0.5mL/min；
检测波长：280nm；
柱温：25℃；
上样量：10μL。
HIC色谱梯度
Figure PCTCN2021121825-appb-000016
DAR值计算如(a1)方法中所述。
注：L系列分子采用(a2)分析方法测定DAR值。
(a3)随机偶联药物疏水色谱HIC-HPLC测定DAR值
样品制备：样品用流动相B稀释至2.0mg/ml后，12000rpm离心10min，取上清用于HPLC分析；
色谱柱：TOSOH Butyl NPR，2.5μm，4.6mm×100mm；
流动相A：125mM PB+2.5M(NH4) 2SO 4(pH 6.8)；
流动相B：125mM PB(pH 6.8)；
流动相C：100％IPA；
流动相D：H 2O；
流速：0.7mL/min；
检测波长：280nm；
柱温：30℃；
上样量：10μL。
HIC色谱梯度
Figure PCTCN2021121825-appb-000017
DAR值计算如(a1)方法中所述。
注：D系列分子采用(a3)分析方法测定DAR值。
所提到的D3％、D4％、D5％分别代表DAR值为3、4、5的ADC所占的百分比。
(2)D-SEC：
色谱柱：Zenix-C SEC-300；
流动相：水:乙腈＝65:35(均含0.1％TFA和0.1％FA)；
流速：0.2mL/min；
柱温：25℃；
检测波长：280nm；
洗脱梯度：等度洗脱。
(3)非还原毛细管电泳NR-CE-SDS：
样品处理：将样品稀释至2.0mg/ml(样品缓冲液：40mM PB，1％SDS，pH 6.5)，取50μL+45μL样品缓冲液+5μL IAM(0.5M)，混匀，70℃水浴10min，取上清进样分析。
分析条件：
毛细管活化：20psi压力下，0.1mol/L的氢氧化钠、0.1mol/L的盐酸、注射水分别冲洗10min、5min、2min；70psi压力下，SDS-MW-Gel Buffer冲洗10min；-15kv电压、20psi，25℃的柱温下，预分离10min；运行程 序：70psi，分别以0.1mol/L的氢氧化钠、0.1mol/L的盐酸、注射水冲洗毛细管3min、1min、1min；随后灌胶10min，在-5kv条件进样40s，运行分离程序(-18kv、20psi)。柱温25℃、分离电压-15kv分离时间40min。
(4)分子排阻色谱SEC-HPLC：
样品处理：样品用流动相稀释至约2.0-3.0mg/ml后，12000rpm离心10min，取上清进样分析。
色谱柱：TOSOH，TSKgel G3000SW XL，5μm，7.8mm×300mm；
流动相：100mM PB+200mM盐酸精氨酸，5％异丙醇(pH 6.8)；
流速：0.6mL/min；
检测波长：280nm；
柱温：30℃；
上样量：10uL；
洗脱梯度：等度洗脱。
(5)Native Ms：
色谱柱：ACQUITY UPLC Protein BEH SEC 200A(1.7μm,2.1mm×150mm)；
流动相：50mM乙酸铵；
运行时间：12min；
流速：0.065mL/min；
进样体积：2μL；
柱温：30℃；
检测波长：280nm；
毛细管：3.0kV；
锥孔电压：140V；
源偏离：80V；
源温度：125℃；
脱溶剂气温度：250℃；
脱溶剂气流量：600L/h；
(6)LC-MS：
液相色谱质谱(LC-MS)偶联是一种分析ADC中DAR和药物分布的正交技术。
样品处理：使用1μL PNGase F/100μg mAb，对样品进行去糖基化处理， 37℃下过夜反应。分析开始前，利用20mmol/L二硫苏糖醇(DTT)进行还原，37℃孵育30min。样品上样之前，用水稀释至1mg/mL。
LC-MS色谱条件：
色谱柱：PLRP-S色谱柱(Agilent)，颗粒大小5μm，
Figure PCTCN2021121825-appb-000018
内径2.1mm，长度50mm；
流动相：流动相A为0.1％甲酸和0.025％三氟乙酸的水溶液，流动相B为0.1％甲酸和0.025％三氟乙酸的乙腈溶液；
流速：0.25mL/min；
电喷雾电压设为4500～5000V，原温设为350℃，气帘和雾化气设置为40；
进样量：15～20μg。
LC-MS色谱梯度
Figure PCTCN2021121825-appb-000019
(7)溶液置换：
无特殊说明情况下，使用30k超滤离心管，6500r/min离心换液。
以下参照具体的实施例来说明本发明。本领域技术人员能够理解，这些实施例仅用于说明本发明，其不以任何方式限制本发明的范围。
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的原料、试剂材料等，如无特殊说明，均为市售购买产品。
实施例1 抗体还原条件实验筛选
a.还原剂选择
将pertuzumab置换至pH7.4 50mM磷酸氢二钠-磷酸二氢钠缓冲液中，并稀释至12.2mg/ml。加入8.5倍抗体摩尔当量的二硫苏糖醇(DTT)或三羧基乙基膦(TCEP)的浓度为1.0mg/ml的水溶液，35℃下反应1.5h。然后，与预先溶解在DMA中的C-3在50mM磷酸氢二钠-磷酸二氢钠缓冲液中25℃下搅拌1h。偶联后样品换液至pH 7.4 50mM磷酸氢二钠-磷酸二氢 钠缓冲液中，取样进行HIC-HPLC检测。检测结果见表1-1。
表1-1.不同种类还原剂还原抗体后偶联结果
还原剂 HIC-HPLC(D3％/D4％/D5％)
DTT 12.93/77.93/8.24
TCEP 7.59/81.87/10.05
结果显示：还原剂TCEP与DTT得到的最终结果无显著差异。
b.还原剂用量选择
将pertuzumab置换至pH7.4 50mM磷酸氢二钠-磷酸二氢钠缓冲液中，并稀释至12mg/ml，将不同当量TECP、DTT的水溶液加入反应体系，35℃下反应1.5h。分别取样进行D-SEC检测，分析未还原抗体剩余量。检测结果见表1-2。
表1-2.不同当量还原剂还原抗体后的未还原抗体剩余量
Figure PCTCN2021121825-appb-000020
结果显示：TCEP当量≥6.5或DTT当量≥5.5，抗体充分还原，还原剂的优选当量范围为6.5-10。
实施例2 抗体偶联条件实验筛选
a.小分子当量选择：
将pertuzumab置换至pH7.4 50mM磷酸氢二钠-磷酸二氢钠缓冲液中， 并稀释至12mg/ml，加入10mg/ml浓度的TECP水溶液，35℃下孵育2h，然后将还原后的pertuzumab置换至pH7.4 50mM磷酸氢二钠-磷酸二氢钠+100mM NaCl+2mM EDTA缓冲液中，并稀释至8mg/ml。将4.5-6当量的小分子化合物C-3溶解在有机溶剂DMA中，加入到反应体系中，其中DMA占整个反应体积10％，加热搅拌60min。反应结束后，使用超滤离心管将抗体偶联药物置换至pH 7.4 50mM磷酸氢二钠-磷酸二氢钠缓冲液中，除去偶联反应中过量的化合物C-3，同时降低溶液中的DMA溶剂含量。使用疏水色谱HIC-HPLC对样品各组分进行分析，结果见表2-1。
表2-1.不同小分子当量对偶联结果的影响
小分子当量 D3％ D4％ D5％
6.5 5.63 66.02 26.21
6.0 7.82 73.22 18.81
5.5 6.97 75.63 17.23
5.0 6.67 78.83 13.91
4.5 15.32 65.25 6.54
结果显示：当反应当量在5.5±0.5的范围内，反应体系较澄清，HIC-HPLC图谱中主峰(D4)的比例较高。
b.反应pH选择：
将pertuzumab置换至pH7.4 50mM磷酸氢二钠-磷酸二氢钠缓冲液中，并稀释至12mg/ml，将10mg/ml浓度的TECP水溶液加入反应体系，在35℃下孵育2h，将还原后的pertuzumab分别置换至pH 6.8-7.8 50mM磷酸氢二钠-磷酸二氢钠+100mM NaCl+2mM EDTA缓冲液中，并稀释至5mg/ml。将5.0当量的化合物C-3溶解在机溶剂DMA中，加入到反应体系中，其中DMA占整个反应体积10％，加热搅拌60min。反应结束后，用pH7.4 50mM磷酸氢二钠-磷酸二氢钠缓冲液进行置换。使用疏水色谱HIC-HPLC、NR-CE-SDS对样品各组分进行分析，结果见表2-2。
表2-2.不同pH对偶联结果的影响
pH HIC-HPLC(D3％/D4％/D5％) NR-CE-SDS纯度(％)
6.5 7.57/78.76/13.49 93.8
6.7 5.89/83.12/10.98 94.8
7.0 5.62/81.82/12.56 94.7
7.3 5.38/80.72/13.91 94.3
7.6 3.88/82.47/10.65 93.9
7.8 8.12/76.53/15.17 90.3
结果显示：当反应pH值在6.5-7.6的范围内，反应体系较澄清，HIC-HPLC主峰的比例较高，且NR-CE-SDS纯度良好。
c.反应共溶剂与比例选择：
pertuzumab还原过程参照实施例2b。还原后抗体换液、稀释至约5.0mg/ml，并进行分组。各组根据表2-3所示，使用有机溶剂N,N-二甲基乙酰胺(DMA)、二甲基亚砜(DMSO)、乙腈(ACN)，将化合物C-3使用这些溶剂中任一种溶解至20mg/mL，根据抗体总量5倍摩尔当量计算化合物C-3溶液的投料体积，并向二者的反应体系中补加相应溶剂达到表2-3中所示的溶剂比例。在25℃下搅拌1h，置换反应体系至pH7.4 50mM磷酸氢二钠-磷酸二氢钠缓冲液中，分别取样，使用疏水色谱HIC-HPLC、NR-CE-SDS对样品各组分进行分析，结果见表2-3。
表2-3.不同有机溶剂作为共溶剂的偶联结果
Figure PCTCN2021121825-appb-000021
Figure PCTCN2021121825-appb-000022
结果显示：DMA、DMSO和ACN皆可作为该工艺步骤共溶剂，但优选DMA、DMSO。DMA比例＞4％或DMSO、ACN比例＞6％，有机共溶剂比例≤30％，偶联反应体系澄清，反应结果良好。
d.抗体浓度选择：
抗体还原参见实施例2a，还原结束后，用pH 7.4 50mM磷酸氢二钠-磷酸二氢钠+100mM NaCl+2mM EDTA缓冲液置换，并将抗体稀释到2.5-13mg/ml，化合物C-3配制成20mg/ml DMA溶液。将5.0当量小分子的DMA溶液加入到反应体系中，25℃下搅拌1h。偶联结束后用pH 7.4的50mM磷酸氢二钠-磷酸二氢钠缓冲液置换溶液体系，使用疏水色谱HIC-HPLC、NR-CE-SDS对样品各组分进行分析，结果见表2-4。
表2-4.抗体浓度对偶联结果的影响
抗体浓度(mg/ml) HIC-HPLC(D3％/D4％/D5％) NR-CE-SDS(％)
2.5 15.21/74.68/8.99 89.4
3.5 11.58/80.12/8.19 91.7
5.0 11.32/81.23/7.36 91.1
7 10.54/83.72/5.67 90.6
10 11.31/81.47/7.56 90.5
13 14.31/75.68/9.56 88.5
从表2-4可以看出，抗体浓度在3.5-10mg/ml皆可取得良好的偶联结果。 因此，偶联反应抗体浓度可选择在3.5-10mg/ml。
e.反应温度选择：
抗体还原偶联过程参照实施例2a，抗体还原结束后，用pH 7.4 50mM磷酸氢二钠-磷酸二氢钠+100mM NaCl+2mM EDTA缓冲液置换溶液体系并将抗体浓度稀释至约5mg/ml，然后反应体系预热到25±10℃，向反应体系中加入化合物C-1的DMA溶液，在相应温度下搅拌1h。反应结束后，用pH 7.450mM磷酸氢二钠-磷酸二氢钠缓冲液置换溶液体系，使用疏水色谱HIC-HPLC、SEC-HPLC、NR-CE-SDS对样品各组分进行分析，结果见表2-5。
表2-5.不同反应温度对偶联结果的影响
反应温度(℃) HIC-HPLC(D3％/D4％/D5％) SEC主峰比例(％) NR-CE-SDS纯度(％)
15 6.77/81.93/11.30 98.0 95.3
20 7.13/81.63/11.24 97.9 95.9
25 9.37/80.80/9.41 97.7 95.0
30 8.05/80.84/10.77 97.2 94.9
35 8.16/79.94/10.76 97.3 95.1
结果显示：在25±10℃范围内，反应皆取得较好的反应结果。因此，25±10℃皆为可以选择的温度。
实施例3 抗体水解条件实验筛选
a.反应温度、pH和时间选择：
pertuzumab还原偶联过程参照实施例2a，偶联后将抗体偶联药物置换到pH 7.4-9.0的50mM磷酸氢二钠-磷酸二氢钠缓冲液中，在25-45℃条件下搅拌3h，观察反应液颜色变化情况。结果见表3-1。
表3-1.抗体偶联药物在不同温度、pH条件下水解情况
Figure PCTCN2021121825-appb-000023
Figure PCTCN2021121825-appb-000024
结果显示：温度35±10℃，pH8.0±0.5范围内，反应时间≥2hr，抗体偶联产物均可顺利水解。
b.小分子选择：
pertuzumab还原偶联过程参照实施例2a。将采用不同小分子化合物偶联后的样品在pH7.8 50mM磷酸氢二钠-磷酸二氢钠缓冲液中，35℃水解3-18h，不同时间点取样后送检Native Ms，检测样品的水解比例，结果见表3-2。
表3-2.不同小分子化合物偶联样品水解后Native Ms结果
Figure PCTCN2021121825-appb-000025
结果显示：在小分子化合物为C-1、C-3时，3小时或6小时即可几乎全部水解，A-2、A-4、B-6、B-7时的偶联样品在35℃条件下在缓冲液中也能顺利水解，但水解比例相对较低。
c.水解体系的选择：
pertuzumab还原偶联过程参照实施例2a。将采用不同小分子化合物偶联后的样品分别置换于不同的水解溶液体系(A：50mM磷酸氢二钠-磷酸二氢钠缓冲液，pH 7.8；B：50mM磷酸氢二钠-磷酸二氢钠缓冲液+3％精氨酸，pH 7.8；C：50mM磷酸二氢钾-磷酸氢钾缓冲液，pH 7.8；D：50mM氨基丁三醇-盐酸缓冲液，pH 7.8)不同时间点取样后送检Native Ms，检测样品的水解比例，结果见表3-3。
表3-3.不同水解体系水解比例
Figure PCTCN2021121825-appb-000026
结果显示：C-3、C-1以及A-3在几种缓冲液中均可迅速水解，在含有精氨酸体系中水解速度更快。而A-4、B-7化合物在pH值相同的情况下则可能需要更长的水解时间。
d.ADC药物浓度的选择
pertuzumab还原偶联过程参照实施例2a。将偶联后的样品置换至50mM磷酸氢二钠-磷酸二氢钠，pH 7.8的水解体系中，不同时间点取样后送检Native Ms，检测样品的水解比例，结果见表3-4。
表3-4.不同抗体偶联产物浓度水解速率
浓度(mg/ml) 1h水解比例(％) 2h水解比例(％) 3h水解比例(％)
2 74.53 86.63 89.51
5 87.14 97.25 98.22
10 97.43 98.62 99.07
12 98.01 99.19 99.63
15 98.05 99.23 99.58
20 93.26 99.27 99.22
30 75.93 79.82 83.12
结果显示：当ADC浓度低于5mg/mL时，或浓度大于20mg/mL时，尽管延长了水解反应时间，水解比例仍低于5-20mg/mL ADC浓度时。
实施例4 抗体纯化条件实验筛选
使用高浓度的硫酸铵对经水解后的ADC药物进行电导调节，将电导调节至固定相的电导值附近后，样品经过滤上样至不同填料的层析柱，使用固定相进行洗脱，合并收集纯化后的样品。
a.填料选择：
抗体还原偶联过程参照实施例2a，偶联后样品换液至pH 7.8 50mM磷酸氢二钠-磷酸二氢钠缓冲液中，在35℃下孵育大于3小时，得到的抗体偶联产物用于填料筛选试验。不同配基和不同支架的填料在相同层析条件下进行柱层析，筛选填料的种类及筛选结果见表4-1。
表4-1.不同种类填料筛选结果
Figure PCTCN2021121825-appb-000027
结果显示，Butyl Sepharose 4FF、Butyl Sepharose HP、Capto Phenyl Impres均可以纯化偶联后样品。综合考虑收率和样品性质，优先使用Butyl Sepharose 4FF，采用D3流穿的方案纯化偶联样品。
b.层析盐及其浓度选择：
抗体还原偶联过程参照实施例2a，将样品置换至50mM磷酸氢二钠-磷酸二氢钠，pH 7.8的溶液，并稀释至12mg/mL。35℃水解3小时后，使用50mM磷酸氢二钠-磷酸二氢钠+1.0-2.0mol/L氯化钠的层析盐溶液、或50mM磷酸氢二钠-磷酸二氢钠+0.325-0.45mol/L硫酸铵的层析盐溶液作为固定相， 使用50mM磷酸二氢钠-磷酸氢二钠的层析盐溶液作为洗脱相，对pertuzumab与化合物偶联水解后样品用Butyl Sepharose 4FF进行柱层析，分离结果见表4-2。
表4-2.不同种类盐及盐浓度对纯化结果的影响
Figure PCTCN2021121825-appb-000028
结果显示：氯化钠和硫酸铵两种盐均可用于该柱层析工艺，考虑收率和样品异质性，优选0.45mol/L硫酸铵作为固定相的层析盐溶液。
实施例5 抗体工艺放大条件实验筛选
将反应规模放大：≥100mg，考察该工艺可放大性并进一步调整偶联工艺条件。
在≥100mg规模，将Pertuzumab换液至pH7.4 50mM磷酸氢二钠-磷酸二氢钠缓冲液中，稀释至约12mg/ml蛋白浓度，将10mg/ml TCEP水溶液(TCEP为6.5倍抗体当量)加入到反应体系中，在35℃下孵育2小时，然后换液至pH7.2 50mM磷酸氢二钠-磷酸二氢钠+100mM NaCl+2mM EDTA缓冲液中，并稀释至8mg/mL，随后向反应体系中加入如下表所示当量比eq的预先溶解在DMA中的小分子化合物C-3，并向反应体系中加入DMA使之达到10％，加热搅拌1小时。偶联结束后，用pH7.8 50mM磷酸氢二钠-磷酸二氢钠缓冲液置换溶液体系，并使抗体偶联产物浓度为12mg/mL，经35℃加热3小时后，使用HIC-HPLC检测，结果见表5-1。
表5-1.≥100mg反应规模不同反应条件实验结果
Figure PCTCN2021121825-appb-000029
结果显示：≥100mg规模，采用本发明的方法皆可取得与小试基本一致的偶联结果，该偶联工艺可顺利放大。
实施例6 不同小分子化合物与抗体偶联实验
将Pertuzumab换液至pH7.4 50mM磷酸氢二钠-磷酸二氢钠缓冲液中，稀释至约12mg/ml蛋白浓度，将10mg/ml TCEP水溶液(TCEP为6.5倍抗体当量)加入到反应体系中，在35℃下孵育2小时，然后换液至pH7.2 50mM磷酸氢二钠-磷酸二氢钠+100mM NaCl+2mM EDTA缓冲液中，并稀释至8mg/mL，随后分别向反应体系中加入溶有小分子化合物A-1或C-3(0.6eq)的DMA有机共溶剂，加热搅拌1h。然后超滤离心换液至pH7.8 50mM磷酸氢二钠-磷酸二氢钠缓冲液中，并使抗体偶联产物浓度为12mg/mL，在35℃孵育3h。水解后样品经3M硫酸铵进行电导调节后，使用Butyl Sepharose 4FF填料和0.45M硫酸铵作为层析盐的50mM磷酸二氢钠-磷酸氢二钠固定相，50mM磷酸氢二钠-磷酸二氢钠作为流动相，固定相与流动相的pH值均为7.5。纯化，得到的纯化液浓缩换液至pH 7.4 50mM磷酸氢二钠-磷酸二氢钠 缓冲液中，分别检测HIC-HPLC、SEC-HPLC、NR-CE-SDS，结果见表6-1。
表6-1 不同小分子化合物与抗体偶联实验结果
Figure PCTCN2021121825-appb-000030
从表中数据可以看出，不同化合物A-1、C-3均适合此偶联工艺，结果一致性良好。
实施例7 不同小分子化合物与不同抗体大规模偶联实验
参照实施例6，将针对HER2(Pertuzumab)、CD20(Rituximab)这些不同靶点的IgG1抗体与小分子化合物偶联，制备后检测，结果显示该工艺对于不同靶点的IgG1抗体均具有良好的适用性。结果见表7-1和图1。
表7-1 不同小分子化合物与不同抗体大规模偶联实验结果
Figure PCTCN2021121825-appb-000031
可以看出，针对不同靶点不同抗体序列的IgG1类抗体均适合本发明提供的ADC制备方法。
实施例8 不同制备方法ADC样品理化性质、体外/体内稳定性、体内药效及安全性。
如上述实施例6方法，将2.5g的Pertuzumab进行还原并于pH7.2 50mM磷酸氢二钠-磷酸二氢钠+100mM NaCl+2mM EDTA缓冲液中，与小分子化合物C-1进行偶联并使用超滤膜包置换至pH 7.8的缓冲液中，得到终产物进行分组为A、B两组。
组A样品直接经Butyl Sepharose 4FF填料纯化，得到的纯化液浓缩换液至pH 7.4 50mM磷酸氢二钠-磷酸二氢钠缓冲液中，获得组A样品原液，浓 度为10mg/mL。组B样品在35℃下孵育3h后，再使用组A相同方法经Butyl Sepharose 4FF填料纯化，得到的纯化液浓缩换液至pH 7.4 50mM磷酸氢二钠-磷酸二氢钠缓冲液中，获得组B样品原液，浓度约10mg/mL。
a.理化性质考察：
分别对组A以及组B样品进行质谱、非还原电泳、疏水层析以及SEC分析。上述结果表明，尽管通过纯化手段，同样能够提高组A与组B样品在HIC、SEC上的纯度比例，但组B样品的NR CE-SDS纯度更高。结果见表8-1和图2。
表8-1 不同工艺的偶联理化结果分析
Figure PCTCN2021121825-appb-000032
b.体内血浆稳定性考察：
将组A、组B的样品使用生理盐水稀释至1mg/ml，分别对食蟹猴进行给药，给药剂量为6mg/kg。在30min、1h、2h、4h、12h、24h、48h、72h、128h进行采血。取用标定好的MMAE(MCE：HY-15162)作为标准物质，对血浆中的游离MMAE进行检测。结果显示，尽管组A与组B样品的最大峰值均为24h，但组B的游离MMAE暴露量要显著小于组A的游离MMAE，提示经过水解处理的组B样品在体内具有更加稳定。结果见表8-2。
表8-2 不同工艺的偶联游离MMAE结果分析
Figure PCTCN2021121825-appb-000033
c.体内生化指标检测
对2.5周龄以上的食蟹猴分组，随机选取2组雌雄各一只的食蟹猴。将组A、组B的样品使用生理盐水稀释至1mg/ml。使用静脉滴注方式进行给 药，给药剂量为6mg/kg。给药后七天，对猴血液中的生化指标进行分析。结果表明，组A中异常指标及严重程度均较组B样品更为严重。
表8-3 不同工艺的偶联体内药效结果分析
Figure PCTCN2021121825-appb-000034
以上对本发明具体实施方式的描述并不限制本发明，本领域技术人员可以根据本发明作出各种改变或变形，只要不脱离本发明的精神，均应属于本发明所附权利要求的范围。

Claims (11)

  1. 一种双取代桥连抗体偶联药物的制备方法，所述制备方法包括：使抗体和式I所示的化合物偶联得到的抗体偶联产物进行水解：
    Figure PCTCN2021121825-appb-100001
    式I中，Ar为苯基或取代苯基，所述取代苯基中的取代基选自烷基、烷氧基、卤素、酯基、氰基和-C(O)NR1R2-，其中R1和R2独立地选自化学键、H和烷基，或R1和R2形成5-7元杂环，杂原子选自O、N和S中的一个或多个；
    X和Y独立地为氢、卤素、三氟甲基或烷氧基；
    n为1～24中的任意整数；
    L-CTD为应用于ADC中细胞毒药物及连接释放结构。
  2. 根据权利要求1所述的制备方法，其特征在于，所述烷基为C1-C6烷基、优选C1-C3烷基，更优选甲基；
    优选地，所述烷氧基为C1-C6烷氧基、优选C1-C3烷氧基，更优选甲氧基；
    优选地，L选自Val-Ala-PAB(VA-PAB)、Val-Cit-PAB(VC-PAB)、Phe-Lys-PAB和MAC glucuronide phenol连接子；CTD例如为细胞毒药物，优选微管抑制剂(例如，微管抑制剂类MMAE、DM1、DM4、Tublysin)；拓扑异构酶抑制剂(例如，拓扑异构酶抑制剂类SN38、依喜替康以及依喜替康衍生物)或DNA结合剂(例如，DNA结合剂PBD及其衍生物)。
  3. 根据权利要求1或2所述的制备方法，其特征在于，所述式I中：
    R1和R2独立地选自H和C1-C3烷基；或者R1和R2形成6元杂环，杂原子选自O和N中的一个或多个，优选为吗啉；
    优选地，Ar为苯基、4-甲基甲酰胺基取代苯基
    Figure PCTCN2021121825-appb-100002
    或4-甲 酰基吗啉取代苯基
    Figure PCTCN2021121825-appb-100003
    优选地，X和Y独立地为氢、氟、三氟甲基或甲氧基；更优选地，X和Y独立地为氢，或者X和Y独立地位于苯环上相对马来酰亚胺的间位；
    优选地，n为1～10、优选3～5中的任意整数；
    优选地，L-CTD为VC-PAB-MMAE或VC-seco-DUBA。
  4. 根据权利要求1至3中任一项所述的制备方法，其特征在于，所述水解在所述抗体偶联产物的纯化之前或之后进行；
    优选地，所述水解包括：将抗体偶联产物在pH 7.4-9.0的水解缓冲液中在25-45℃下加热1-24h，其中所述水解缓冲液包含磷酸二氢钠、磷酸氢二钠、磷酸氢钾、磷酸二氢钾、枸橼酸、甘氨酸、氨基丁三醇、盐酸精氨酸，盐酸盐酸、磷酸、氢氧化钠和氢氧化钾中的一种或多种；
    优选地，所述水解缓冲液为磷酸二氢钠-磷酸氢二钠缓冲液、磷酸二氢钾-磷酸氢二钾缓冲液或氨基丁三醇缓冲液；
    优选地，所述抗体偶联产物在水解缓冲液中的浓度为2-30mg/mL、优选5-20mg/mL。
  5. 根据权利要求1至4中任一项所述的制备方法，其特征在于，所述水解包括：将抗体偶联产物在所述水解缓冲液中在25-35℃下加热1-6h、优选1-3h；
    优选地，所述水解缓冲液为pH 7.5-8.5，优选pH7.8；
    优选地，所述抗体为IgG，优选IgG1。
  6. 根据权利要求1至5中任一项所述的制备方法，其特征在于，所述制备方法包括以下步骤：
    a.抗体还原：将≥5.5倍抗体摩尔当量的还原剂加入到含5-30mg/mL浓度抗体的缓冲液中，在30-40℃下反应1.5-2h，其中所述还原剂为选自TCEP、DTT、2-MEA和DTBA中的一种或多种；
    b.抗体偶联：将步骤a中经还原的抗体置换到pH 6.5-7.6的磷酸盐缓冲液中，并将抗体稀释到3.5-15mg/mL浓度，将溶解于有机共溶剂中的4.5-6.5倍抗体摩尔当量的式I所示的化合物加入到所述抗体稀释液中，并使得所述有机共溶剂占反应体系体积的5％-30％，在15-35℃下搅拌反应≥0.5h，其中所述有机共溶剂为选自DMA、DMSO、DMF和ACN中的一种或多种；
    c.疏水层析：使用疏水填料对抗体偶联产物进行疏水层析纯化，其中所述疏水填料为Butyl Sepharose HP(GE)、Capto Phenyl Impres(GE)、Toyopearl Butyl 650M(TOSOH)或Butyl Sepharose 4FF(GE)，固定相使用氯化钠或硫酸铵作为层析盐的缓冲液；并且
    所述制备方法在步骤b之后或步骤c之后还包括以下步骤：
    d.水解：将抗体偶联产物置换到pH 7.4-9.0的水解缓冲液中，在25-45℃下加热2-24h，其中所述水解缓冲液包含磷酸二氢钠、磷酸氢二钠、磷酸氢钾、磷酸二氢钾、枸橼酸、甘氨酸、氨基丁三醇、盐酸精氨酸、盐酸、磷酸、氢氧化钠和氢氧化钾中的一种或多种。
  7. 根据权利要求1至6中任一项所述的制备方法，其特征在于，在所述步骤a中：
    优选地，所述还原剂为TCEP或DTT；
    优选地，所述还原剂为6.5-10倍抗体摩尔当量；
    优选地，所述还原剂以5-15mg/mL、优选10mg/mL的水溶液形式加入；
    优选地，所述抗体为IgG，优选IgG1；
    优选地，所述抗体的浓度为10-15mg/mL、优选12mg/mL；
    优选地，所述缓冲液为pH 6.5-7.6的磷酸盐缓冲液。
  8. 根据权利要求1至7中任一项所述的制备方法，其特征在于，在所述步骤b中：
    优选地，所述缓冲液为pH 7.2-7.4，优选pH 7.2-7.4的磷酸盐缓冲液或磷酸盐-EDTA缓冲液；
    优选地，置换时将抗体稀释到3.5-10mg/mL浓度；
    优选地，所述式I所示的化合物为5.0-6.0倍抗体摩尔当量；
    优选地，所述有机共溶剂为DMA、DMSO或ACN；
    优选地，使得所述有机共溶剂占反应体系体积的6％-30％；
    优选地，在15-35℃下搅拌反应0.5-1h。
  9. 根据权利要求1至8中任一项所述的制备方法，其特征在于，在所述步骤c中：固定相使用以硫酸铵作为层析盐的缓冲液，硫酸铵浓度优选为0.45mol/L；
    优选地，固定相使用以硫酸铵作为层析盐的磷酸盐缓冲液，洗脱相为磷酸盐缓冲液；
    更优选地，磷酸盐为0-100mM磷酸氢二钠-磷酸二氢钠缓冲溶液，pH  7.4-8.0。
  10. 根据权利要求1至9中任一项所述的制备方法，其特征在于，在所述步骤d中：
    所述抗体偶联产物在水解缓冲液中的浓度为2-30mg/mL、优选5-20mg/mL；
    优选地，所述水解缓冲液为磷酸二氢钠-磷酸氢二钠缓冲液、磷酸二氢钾-磷酸氢二钾缓冲液或氨基丁三醇缓冲液；
    优选地，所述水解包括：将抗体偶联产物在所述水解缓冲液中在25-35℃下加热1-6h、优选1-3h；
    优选地，所述水解缓冲液为pH 7.5-8.5，优选pH 7.8。
  11. 通过权利要求1至10中任一项方法得到的抗体偶联产物。
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