WO2017107817A1 - 一种抗体药物偶联物的制备方法 - Google Patents

一种抗体药物偶联物的制备方法 Download PDF

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WO2017107817A1
WO2017107817A1 PCT/CN2016/109807 CN2016109807W WO2017107817A1 WO 2017107817 A1 WO2017107817 A1 WO 2017107817A1 CN 2016109807 W CN2016109807 W CN 2016109807W WO 2017107817 A1 WO2017107817 A1 WO 2017107817A1
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antibody
drug conjugate
antibody drug
reaction
drug
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PCT/CN2016/109807
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English (en)
French (fr)
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梁金栋
蒋贵阳
李昂
叶鑫
许建烟
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江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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Application filed by 江苏恒瑞医药股份有限公司, 上海恒瑞医药有限公司 filed Critical 江苏恒瑞医药股份有限公司
Priority to CN201680011806.8A priority Critical patent/CN107405408B/zh
Publication of WO2017107817A1 publication Critical patent/WO2017107817A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells

Definitions

  • the invention relates to a method for preparing a class of antibody drug conjugates, in particular to a purification step of a method for preparing a class of antibody drug conjugates. More specifically, the present invention relates to a method of preparing antibody drug conjugates T-DM1 and P-mab-linker-MC-MMAF.
  • Chemotherapy remains one of the most important anti-cancer methods including surgery, radiation therapy, and targeted therapy. Although there are many types of highly effective cytotoxic drugs, the difference between tumor cells and normal cells is small, which limits the clinical application of these antitumor compounds due to toxic side effects. While anti-tumor monoclonal antibodies are specific for tumor cell surface antigens, antibody drugs have become a front-line drug for anti-tumor therapy, but when antibodies are used as anti-tumor drugs alone, the efficacy is often unsatisfactory.
  • Antibody drug conjugates link monoclonal antibodies or antibody fragments to biologically active cytotoxic drugs via stable chemical linker compounds, making full use of the specificity of antibodies for normal cell and tumor cell surface antigen binding.
  • ADCs Antibody drug conjugates
  • the preparation method of the antibody drug conjugate mainly includes two steps of preparation and purification of the conjugate.
  • the purification step can remove impurities such as small molecules and multimers in the initial preparation solution of the antibody drug conjugate, and improve the purity.
  • the purification step uses ion exchange chromatography (Ion Exchange Chromatography for short) or tangential flow ultrafiltration (TFF, ultrafiltration).
  • Ion exchange chromatography is a chromatographic method in which an ion exchanger is used as a stationary phase, and the difference in the binding force when the component ions in the mobile phase are reversibly exchanged with the counter ion on the exchanger.
  • ion exchange chromatography is employed, the charge distribution of the antibody drug conjugate of different drug loading amounts is affected due to the difference in the amount of antibody molecule-coupled drug in the antibody drug conjugate sample. Therefore, when using an ion column for chromatographic separation, the charge distribution caused by the drug loading will affect the chromatographic results, such as affecting the purity and average drug loading of the final chromatographic product.
  • Tangential flow ultrafiltration refers to the vertical flow direction of the liquid flow direction and the filtration direction.
  • the flow direction of the liquid to be filtered is parallel to the direction of the plane of the filter membrane, and the liquid passes through the membrane surface perpendicularly.
  • Membrane hole The tangential flow ultrafiltration method can easily remove small molecular impurities in the system, but the disadvantage is that some impurities having a relatively large molecular weight cannot be removed.
  • Size exclusion chromatography also known as space exclusion chromatography (SEC) or gel chromatography
  • SEC space exclusion chromatography
  • gel chromatography is the uniqueness of using a porous gel stationary phase, the pore size of the gel pores, and the relative size of the molecular mass of the polymer sample molecules. Relationship, and the method of separating the solute. The separation mechanism depends mainly on the pore size of the gel and its separation. The relationship between the molecular dimensions of the components is not directly related to the nature of the mobile phase; there is no interaction between the sample molecules and the stationary phase.
  • the chromatographic stationary phase is a porous gel that allows only the entry of components smaller than the pore size to be entered. These pores are quite large for the solvent molecules so that the solvent molecules can diffuse freely.
  • the macromolecules in the sample cannot enter the gel pores and are completely excluded. They can only pass through the column between the porous gel particles, and are first eluted from the column by the mobile phase; medium-sized molecules can enter the gel. Some of the appropriate holes, but not into smaller pores, are trapped in the column and elute slowly from the column; small molecules can enter most of the pores in the gel and are stronger in the column The retention is eluted more slowly; the solvent molecules that dissolve the sample have the smallest molecular weight, can enter all the pores of the gel, and finally flow out of the column, thereby achieving complete separation of samples of different molecular sizes.
  • the molecular exclusion method can distinguish components of different molecular weights by different retention times in the gel by different molecular weight components. Moreover, this method does not require different charge distributions of the components. Therefore, size exclusion chromatography is not affected by the charge distribution of the molecule when it is purified.
  • T-DM1 is an antibody-drug conjugate (ADC) conjugated with Herceptin (trastuzumab) and small molecule microtubule drug DM1.
  • ADC antibody-drug conjugate
  • Herceptin tacuzumab
  • small molecule microtubule drug DM1 small molecule microtubule drug DM1.
  • CN101267841B discloses a process for the preparation of purified T-DM1 in which both methods of purification use tangential flow ultrafiltration.
  • both methods of purification use tangential flow ultrafiltration.
  • tangential flow ultrafiltration cannot remove some impurities with a large molecular weight, the use of this method does not stably obtain a high-purity final product, and sometimes it is necessary to increase the purity of the product by adding other purification steps. Processing.
  • WO2015113476 discloses a novel antibody-drug conjugate pertuzumab-propyl-1-sulfate-MC-MMAF (P-mab-linker-MC-MMAF), which can effectively inhibit the growth of tumor cells .
  • P-mab-linker-MC-MMAF pertuzumab-propyl-1-sulfate-MC-MMAF
  • the present invention provides an improved preparation method of antibody drug conjugates, comprising an antibody-linker coupling reaction with a cytotoxic drug to prepare an antibody drug conjugate and antibody drug coupling.
  • a purification step of the material comprising a molecular exclusion step.
  • the antibody linker is prepared by reacting a bifunctional cross-linking reagent with an antibody, thereby causing a linker molecule to be covalently linked to the antibody to obtain a modified antibody.
  • the "bifunctional crosslinking reagent” is preferably 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC) or 3-acetyl decylpropionaldehyde.
  • the antibody stock solution has a concentration of 5-15 mg/mL, preferably 10 mg/mL.
  • the antibody linker is present in a concentration of 5-15 mg/mL, preferably 10 mg/mL.
  • the step of catalyzing comprises using a gel having a molecular weight in the range of from 5,000 to 5,000,000, preferably from 10,000 to 600,000, as a filler for the purification column.
  • the gel filler having a molecular weight ranging from 10,000 to 600,000 comprising a G200 gel.
  • G200 gel capable of separating a protein molecular weight range between 10000-600000 commercially available products Sephadex G200, Superdex G200, Superdex TM 200pg like.
  • the present invention is preferably Superdex TM 200pg. More in line with production purposes.
  • the antibody in the method of preparing an antibody drug conjugate, is trastuzumab.
  • the antibody drug conjugate in the method of preparing an antibody drug conjugate, is T-DM1.
  • the antibody in the method of preparing an antibody drug conjugate, is pertuzumab.
  • the antibody drug conjugate in the method of preparing an antibody drug conjugate, is P-mab-linker-MC-MMAF.
  • the method of purifying the antibody linker can use a small molecule filtration method, preferably a tangential flow ultrafiltration.
  • the ultrafiltration membrane has a pore size of from 10 to 50 KD, preferably 30 KD.
  • the preparation and purification steps of the antibody-linker may also be included prior to the coupling reaction of the antibody linker with the cytotoxic drug.
  • the invention extracts the antibody-drug conjugate by the molecular exclusion method, can effectively remove the polymer in the antibody, and has little influence on the drug load of the sample, and improves the purity of the coupled drug while ensuring the activity.
  • the method has the advantages that the polymer produced in the coupling process and the unreacted small molecule toxin in the system can be removed in one step, thereby obtaining a high-purity conjugate, reducing the purification step, improving the purification efficiency, and facilitating industrialization. produce.
  • the applicant intends to include a formulation of the trade name product, a generic drug of the trade name product, and an active drug moiety.
  • cytotoxic drug refers to a chemical molecule that has a strong disruption to its normal growth in tumor cells.
  • cytotoxic drugs can kill tumor cells at a sufficiently high concentration, but due to lack of specificity, while killing tumor cells, it also causes apoptosis of normal cells, leading to serious side effects.
  • non-limiting examples thereof include tubulin inhibitors, DNA alkylating agents, tyrosine kinase inhibitors, topoisomerase inhibitors or DNA synthesis inhibitors.
  • antibody refers to any form of antibody that exhibits the desired biological activity. Thus, it is used in the broadest sense and specifically includes, but is not limited to, full length antibodies, antibody binding fragments or derivatives. Sources of antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, genetically engineered antibodies (eg, bispecific antibodies).
  • antibody stock solution refers to an antibody solution formed by preserving an antibody obtained after purification in a suitable buffer system conventionally used by those skilled in the art.
  • the buffer system is, for example, an acetic acid-sodium acetate buffer system or a phosphate buffer system (PBS) used in the present invention.
  • PBS phosphate buffer system
  • the linker (SMCC) described in the present invention may be commercially available or may be prepared and purified by reference to Facile Synthesis of Reagents Containing a Terminal Maleimido Ligand Linked to an Active Ester, Synthesis 1991 (10), 819-821. .
  • the antibody-linker described in the present invention refers to a trastuzumab-linker obtained in the second step of the reaction of the antibody and SMCC after the coupling reaction.
  • the reaction substrate equivalent ratio of the coupling reaction described herein, i.e., the SMCC:antibody range is from 2 to 10:1; in the present invention, it is preferably 8:1.
  • the reaction time is 1-5 hours, preferably 2 hours.
  • the coupling reaction of the antibody-linking agent described in the present invention with a cytotoxic drug means that the antibody-linking agent is subjected to a C-S coupling reaction with a cytotoxic drug.
  • the reaction substrate equivalent ratio of the coupling reaction described herein, that is, the L-DM1:antibody-linking agent ranges from 2 to 15:1; in the present invention, it is preferably 6:1.
  • the reaction solution used in the coupling reaction is EDTA-containing phosphate buffered saline (PBS) having a pH of 5.5 to 7.5, preferably 6.5.
  • the reaction time is 2-18 hours, preferably 4 hours.
  • the reaction temperature is 4-37 ° C, preferably 25 ° C.
  • the reaction solution further comprises one or more solvents selected from the group consisting of acetonitrile, ethanol, DMSO, DMA, DMF, and the content of the organic solvent is 2-20%, preferably 5-10%; in the present invention, 10% ethanol and 2% are preferred.
  • acetonitrile Reference is also made to the description of US 2005/0169933 A1.
  • the G200 gel described in the present invention represents a gel model, the G value represents the degree of cross-linking, and the number represents 10 times the water absorption per gram of dry rubber, for example, Superdex G200 means that water can be absorbed per gram of dry rubber. 20 grams.
  • Sephadex is a trade name for a dextran gel;
  • Superdex is a trade name for a composite gel composed of agarose and dextran, which is a modified form of Sephadex.
  • Superdex TM 200pg commercially available gel preparative scale model.
  • trastuzumab monoclonal antibody trastuzum antibody stock solution (Trastuzumab, antibody disclosed in US 6165464A) in a buffer (pH 6.5) containing 50 mM NaCl, 2 mM EDTA and 50 mM potassium phosphate and 10% ethanol and 5% acetonitrile at 25 °C.
  • the resulting Trastuzumab-MCC was replaced with 50 mM NaCl, 2 mM EDTA and 50 mM potassium phosphate buffer solution (pH 6.5) to Trastuzumab-MCC in 50 mM NaCl, 2 mM EDTA and 50 mM potassium phosphate, and a buffer solution of 10% ethanol and 2% acetonitrile ( In pH 6.5), the adjusted concentration was 10 mg/mL, and then 300 mg of an L-DM1 ethanol solution was added to the above reaction system. The reaction was carried out for 4 hours on a reaction shaker to form a reaction solution of Trastuzumab-MCC-DM1. The sample was taken for drug loading test and SEC-HPLC purity test.
  • the SEC-HPLC result was 94.59%; the resulting Trastuzumab-MCC- the reaction liquid was DM1 filler molecular exclusion column Superdex TM 200pg of a purified sample, the sample in order to remove aggregates and other small molecule impurities, monomer was recovered, samples were taken and tested for drug loading SEC-HPLC purity test, the SEC - HPLC result was 99.11%. Calculated according to the drug load sampled by the purification process (UV method), the results are shown in Table 1.
  • Methods for determining the drug loading of Trastuzumab-MCC-DM1 include ultraviolet-visible spectrophotometry and LC/MS.
  • the principle of the ultraviolet-visible spectrophotometry is based on the absorption of the absorbance at 280 nm of the drug and the antibody contribute to the total absorbance (A 280 nm ) at 280 nm .
  • the calculation process is as follows :
  • a 280nm ⁇ 1 bc 1 + ⁇ 2 bc 2 ( 1)
  • ⁇ 1 extinction coefficient of the drug at 280 nm
  • ⁇ 2 extinction coefficient of monoclonal antibody at 280 nm
  • P-mab antibody disclosed in US7041292B1
  • the compound MC-MMAF (compound disclosed in WO2005081711, 125 mg, 13.5 mmol) was dissolved in 20 mL of acetonitrile, and the solution was added to the pertuzumab-propanol solution (prepared in the second step), and the reaction was stirred at 25 ° C for 4 hours.
  • the ADC sample of P-mab-linker-MC-MMAF was obtained, and the sample was taken for complete molecular weight test and SEC-HPLC purity test.
  • the SEC-HPLC result was 94.70% and the drug load was 2.1.
  • the ADC samples obtained P-mab-linker-MC- MMAF reaction solution was purified sample of filler molecular exclusion column Superdex TM 200pg to sample the residual polymer is removed, and other small molecules, can be purified Samples were taken for complete molecular weight testing and SEC-HPLC purity testing with SEC-HPLC results of 99.39% and drug loading of 2.08.
  • the resulting Trastuzumab-MCC was replaced with 50 mM NaCl, 2 mM EDTA and 50 mM potassium phosphate buffer solution (pH 6.5) to Trastuzumab-MCC in 50 mM NaCl, 2 mM EDTA and 50 mM potassium phosphate, and a buffer solution of 10% ethanol and 2% acetonitrile ( In pH 6.5), the adjusted concentration was 10 mg/mL, and then 1500 mg of an L-DM1 ethanol solution was added to the above reaction system. The reaction was carried out for 4 hours on a reaction shaker to obtain a Trastuzumab-MCC-DM1 reaction solution, and the purity and drug load were tested. The SEC-HPLC purity was 95.25% and the drug loading was 3.43.
  • Trastuzumab-MCC-DM1 reaction solution was purified sample molecular exclusion column Superdex TM 200pg of filler, the polymer to remove the residual sample and other small molecules. Finally, a purity test and a drug load test were performed with a SEC-HPLC purity of 99.26% and a drug loading of 3.40. The sample recovery rate is 95%.
  • Trastuzumab-MCC was replaced with 50 mM NaCl, 2 mM EDTA and 50 mM potassium phosphate buffer solution (pH 6.5) to Trastuzumab-MCC 50 mM NaCl, 2 mM EDTA and 50 mM potassium phosphate, and a buffer solution of 10% ethanol and 2% acetonitrile. (pH 6.5), the adjusted concentration was 10 mg/ml, and 300 mg of an L-DM1 ethanol solution was added to the above reaction system.
  • the reaction was carried out on a reaction shaker for 4 hours to form a reaction solution of Trastuzumab-MCC-DM1; after the reaction was completed, the reaction product of the resulting Trastuzumab-MCC-DM1 was desalted and purified by a Sephadex G25 gel column (elution phase: pH was 6.5 buffer containing 50 mM NaCl, 2 mM EDTA and 50 mM potassium phosphate) to remove unreacted L-DM1 and other small molecular impurities in the reaction solution, and finally obtain 50 mM NaCl, 2 mM EDTA and 50 mM phosphoric acid of Trastuzumab-MCC-DM1. Potassium buffer solution (pH 6.5). Finally, a purity test and a drug load test were performed with a SEC-HPLC purity of 94.45% and a drug loading of 3.45.
  • trastuzole (Trastuzumab final concentration 10 mg/ml) and 181 in a buffer (pH 6.5) containing 50 mM NaCl, 2 mM EDTA and 50 mM potassium phosphate and 10% ethanol and 5% acetonitrile at 25 °C.
  • the package uses a 30kd polycellulose membrane package) to remove unreacted SMCC and other small molecular impurities in the reaction solution, and finally an equal volume of 5 times volume with 50 mM NaCl, 2 mM EDTA and 50 mM potassium phosphate buffer (pH 6.5).
  • the obtained Trastuzumab-MCC was buffered in 50 mM NaCl, 2 mM EDTA and 50 mM potassium phosphate.
  • the liquid solution (pH 6.5) was replaced with Travisuzumab-MCC in 50 mM NaCl, 2 mM EDTA and 50 mM potassium phosphate, and a buffer solution (pH 6.5) of 10% ethanol and 2% acetonitrile, adjusted to a concentration of 10 mg/ml, and then 300 mg of L- An ethanol solution of DM1 and the above reaction system.
  • the reaction was carried out on a reaction shaker for 4 hours to form a reaction solution of Trastuzumab-MCC-DM1 at a temperature of 25 ° C with 50 mM NaCl, 2 mM EDTA and 50 mM potassium phosphate, and a buffer of 10% ethanol and 2% acetonitrile (pH 6.5).

Abstract

一种抗体药物偶联物的制备方法。具体而言，涉及抗体药物偶联物的合成和纯化步骤，所述的纯化步骤包括分子排阻步骤。

Description

一种抗体药物偶联物的制备方法 技术领域
本发明涉及一类抗体药物偶联物的制备方法，尤其涉及一类抗体药物偶联物的制备方法的纯化步骤。更具体地说，本发明涉及抗体药物偶联物T-DM1及P-mab-linker-MC-MMAF的制备方法。
背景技术
化疗依然是包括手术、放疗、以及靶向治疗法在内的最重要的抗癌手段之一。尽管高效细胞毒性药物的种类很多，但是肿瘤细胞和正常细胞之间差别很小，限制了这些抗肿瘤化合物由于毒副作用在临床上的广泛应用。而抗肿瘤单克隆抗体对于肿瘤细胞表面抗原的特异性，抗体药物已成为抗肿瘤治疗的前线药物，但单独使用抗体作为抗肿瘤药物时，疗效经常不尽人意。
抗体药物偶联物(antibody drug conjugate，ADC)把单克隆抗体或者抗体片段通过稳定的化学接头化合物与具有生物活性的细胞毒性药物相连，充分利用了抗体对正常细胞和肿瘤细胞表面抗原结合的特异性和细胞毒性药物的高效性，同时又避免了前者疗效偏低和后者毒副作用过大等缺陷。这也就意味着，与以往传统的化疗药物相比，抗体药物偶联物能精准地结合肿瘤细胞并降低将对正常细胞的影响(Mullard A,(2013)Nature Reviews Drug Discovery,12:329–332；DiJoseph JF,Armellino DC,(2004)Blood,103:1807-1814)。
抗体药物偶联物的制备方法主要包括偶联物的制备和纯化两个步骤。纯化步骤可以去除抗体药物偶联物初始制备液中的小分子及多聚体等杂质，提高纯度。
通常纯化步骤会用到离子交换层析(Ion Exchange Chromatography简称为IEC)方法或者切向流超滤(TFF，超滤)。离子交换层析是以离子交换剂为固定相，依据流动相中的组分离子与交换剂上的平衡离子进行可逆交换时的结合力大小的差别，而进行分离的一种层析方法。采用离子交换层析时，由于抗体药物偶联物样品中抗体分子偶联药物的数量不同，从而影响不同载药量的抗体药物偶联物的电荷分布。因此在采用离子柱进行层析分离时，药物载量引起的电荷分布会比较大的影响层析结果，比如影响最终层析产物的纯度及平均载药量。
切向流超滤是指液体流动方向与过滤方向呈垂直方向的过滤形式，切向流过滤时，待过滤的液体的流动方向和过滤膜平面的方向平行，液体就会垂直于膜表面穿过膜孔。切向流超滤方法可以很方便除去体系中的小分子杂质，但缺点是无法除去分子量比较大的一些杂质。
分子排阻色谱法又称空间排阻色谱法(SEC)或凝胶色谱法，是利用多孔凝胶固定相的独特性，凝胶孔隙的孔径大小，高分子样品分子的线团尺寸间的相对关系，而对溶质进行分离的方法。其分离机理主要取决于凝胶的孔径大小与被分离 组分分子尺寸之间的关系，与流动相的性质没有直接的关系；样品分子与固定相之间不存在相互作用。色谱固定相是多孔性凝胶，仅允许直径小于孔径的组分进入，这些孔对于溶剂分子来说是相当大的，以致溶剂分子可以自由的扩散出入。样品中的大分子不能进入凝胶孔洞而完全被排阻，只能沿多孔凝胶粒子之间的空隙通过色谱柱，首先从柱中被流动相洗脱出来；中等大小的分子能进入凝胶中一些适当的孔洞中，但不能进入更小的微孔，在柱中受到滞留，较慢地从色谱柱洗脱出来；小分子可进入凝胶中绝大部分孔洞，在柱中受到更强的滞留，会更慢的被洗脱出；溶解样品的溶剂分子，其分子量最小，可进入凝胶的所有孔洞，最后从柱中流出，从而实现具有不同分子大小样品的完全分离。分子排阻法可以通过不同分子量的组分在在凝胶中的保留时间差异不同，从而区分出不同分子量的组分。而且该方法对组分的不同电荷分布没有要求。因此分子排阻色谱法在进行纯化时，不受分子的电荷分布情况的影响。
T-DM1是一种抗体-药物偶联物(ADC)，由赫赛汀(曲妥珠单抗)和小分子微管类药物DM1偶联而成。我们发现在用常规方法制备T-DM1时，无法得到同时符合载药量和SEC纯度要求以及回收率的产物。
CN101267841B公开了一种制备纯化T-DM1的方法，其中两步纯化方法都使用了切向流超滤。然而，由于切向流超滤无法除去一些分子量较大的杂质，使用该方法并不能稳定地得到高纯度的终产品，有时候还需要通过增加其他纯化步骤才可以获得纯度合格的产品，延长后处理过程。
WO2015113476公开了一种新型的抗体-药物偶联物帕妥珠单抗-丙基-1-硫-MC-MMAF(P-mab-linker-MC-MMAF)，其能够有效地抑制肿瘤细胞的生长。在制备P-mab-linker-MC-MMAF时，我们同样发现使用常规方法制备纯化无法得到同时符合载药量和SEC纯度以及回收率的产物。
通过研究，我们发现了一种在保证载药量的同时，即能保持活性，又能提高回收率的方法，使抗体偶联物的制备效率得到有效的提高。这种方法也同样适合于其他抗体偶联物的制备，并取得优良的效果。
发明内容
为了改进ADC药物制备效果，本发明提供了一种改进的抗体药物偶联物的制备方法，包括抗体-连接剂与细胞毒性药物进行偶联反应，以制备抗体药物偶联物以及抗体药物偶联物的纯化步骤，所述的纯化步骤包括分子排阻步骤。
其中，抗体连接剂的制备是通过使双功能交联试剂与抗体反应，从而导致连接体分子与抗体共价连接，得到修饰的抗体。所述的“双功能交联试剂”优选4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯(SMCC)、3-乙酰巯基丙醛。
在本发明的一个优选实施例方案中，所述的抗体原液浓度为5-15mg/mL，优选10mg/mL。
在本发明的一个优选实施例方案中，所述的抗体连接剂的浓度为5-15mg/mL，优选10mg/mL。
在本发明的一个优选实施例方案中，分子排阻步骤包括使用分离分子量范围在5000～5000000，优选10000～600000的凝胶作为纯化柱的填充料。
在本发明的一个优选实施例方案中，分离分子量范围10000～600000的凝胶填充料包括G200凝胶。G200凝胶能够分离的蛋白分子量范围在10000-600000之间，市售产品有Sephadex G200、Superdex G200、SuperdexTM 200pg等。本发明中优选SuperdexTM 200pg。更符合生产目的。
在本发明的一个优选实施例方案中，在抗体药物偶联物的制备方法中，所述的抗体为曲妥珠单抗。
在本发明的一个优选实施例方案中，在抗体药物偶联物的制备方法中，所述的抗体药物偶联物为T-DM1。
在本发明的一个优选实施例方案中，在抗体药物偶联物的制备方法中，所述的抗体为帕妥珠单抗。
在本发明的一个优选实施例方案中，在抗体药物偶联物的制备方法中，所述的抗体药物偶联物为P-mab-linker-MC-MMAF。
在本发明的一个优选实施例方案中，抗体连接剂的纯化方法可使用小分子过滤法，优选切向流超滤。在本发明的一个优选实施例方案中，超滤的滤膜孔径为10-50KD，优选30KD。
在抗体连-接剂与细胞毒性药物进行偶联反应前，还可以包括抗体-连接剂的制备和纯化步骤。
本发明用分子排阻法纯化抗体‐药物偶联物，可以有效除去抗体中的多聚体，并且对样品的药物载量几乎没有影响，在保证活性的同时，提高偶联药物纯度。该方法的优点是可以一步法除去偶联过程中产生的聚体以及体系中未反应的小分子毒素，得到高纯度的偶联物，减少了纯化的步骤，提高了纯化的效率，有利于工业化生产。
除非另有限定，本文所用的所有技术和科学术语均与本发明所属领域普通技术人员的通常理解一致。虽然也可采用与本文所述相似或等同的任何方法和材料实施或测试本发明，但本文描述了优选的方法和材料。描述和要求保护本发明时，依据以下定义使用下列术语。
当本发明中使用商品名时，申请人旨在包括该商品名产品的制剂、该商品名产品的非专利药和活性药物部分。
除非有相反陈述，在说明书和权利要求书中使用的术语具有下述含义。
术语“细胞毒性药物”是指在肿瘤细胞内具有较强破坏其正常生长的化学分子。细胞毒性药物原则上在足够高的浓度下都可以杀死肿瘤细胞，但是由于缺乏特异性，在杀伤肿瘤细胞的同时，也会导致正常细胞的凋亡，导致严重的副作用。 在本发明的实施方式中，其非限制性实例包括微管蛋白抑制剂、DNA烷化剂、酪氨酸激酶抑制剂、拓扑异构酶抑制剂或DNA合成抑制剂。
术语“抗体”是指表现出所需生物学活性的任何形式的抗体。因此，它以最广义使用，具体地说，包括但不限于全长抗体，抗体结合片段或衍生物。抗体的来源包括但不限于单克隆抗体、多克隆抗体、基因工程抗体(例如双特异性抗体)。
术语“抗体原液”是指以合适的，所属领域技术人员常规使用的缓冲液体系，对纯化后得到的抗体进行保存所形成的抗体溶液。所述缓冲液体系如本发明中所用的醋酸-醋酸钠缓冲液体系或磷酸盐缓冲液体系(PBS)。
本发明中所述的连接剂(SMCC)可以是市售的，也可以参考文献Facile Synthesis of Reagents Containing a Terminal Maleimido Ligand Linked to an Active Ester，Synthesis 1991(10)，819‐821，进行制备和纯化。
本发明中所述的抗体-连接剂指的是抗体和SMCC进行偶联反应后得到的，如实施例1步骤2中的曲妥珠单抗-连接剂。此处所述的偶联反应的反应底物当量比，即SMCC：抗体范围为2-10:1；本发明中优选8:1。反应时间为1-5小时，优选2小时。
本发明中所述的抗体-连接剂与细胞毒性药物进行偶联反应指抗体-连接剂与细胞毒性药物进行C-S偶联反应。此处所述的偶联反应的反应底物当量比，即L-DM1：抗体-连接剂的范围为2-15:1；本发明中优选6:1。偶联反应所用的反应液为含EDTA的磷酸缓冲盐溶液(PBS)，pH为5.5-7.5，优选6.5。反应时间为2-18小时，优选4小时。反应温度4-37℃，优选25℃。反应液中还包含一种或多种选自以下的溶剂乙腈、乙醇、DMSO、DMA、DMF，有机溶剂的含量2-20％，优选5-10％；本发明中优选10％乙醇和2％乙腈。也可参考US 2005/0169933A1的描述。
本发明中所述的G200凝胶表示凝胶型号，G值表示交联度大小，数字表示每克干胶膨胶时吸水量的10倍，如Superdex G200表示每克干胶膨胶时可以吸水20克。Sephadex为一种葡聚糖凝胶(dextran gel)的商品名；Superdex为一种琼脂糖及葡聚糖组成的复合凝胶的商品名，其为Sephadex的改进形式。SuperdexTM 200pg，为市售制备级的凝胶型号。
具体实施方式
以下结合实施例进一步描述解释本发明，但这些实施例并非意味着限制本发明的范围。
本发明实施例中未注明具体条件的实验方法，通常按照常规条件，或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1：抗体药物偶联物T-DM1的合成与纯化
步骤一、L-DM1合成工艺路线
Figure PCTCN2016109807-appb-000001
合成方法参考Semisynthetic Maytansine Analogues for the Targeted Treatment of Cancer，J.Med.Chem.2006,49,4392-4408。
步骤二、T-DM1合成工艺路线
Figure PCTCN2016109807-appb-000002
在25℃，在含有50mM NaCl、2mM EDTA和50mM磷酸钾以及10％乙醇和5％乙腈的缓冲液(pH 6.5)中，10.0克单克隆抗体曲妥珠抗体原液(Trastuzumab，抗体公开于US6165464A)(用50mM,，pH 6.5的磷酸盐缓冲液稀释Trastuzumab至抗体终浓度10mg/mL)与181毫克的4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥 珀酰亚胺酯(连接剂SMCC，上海鸿翰生科科技有限公司)在反应摇床上反应进行2小时，生成Trastuzumab-MCC的反应液；将反应生成得到的Trastuzumab-MCC反应液，在25℃温度下，用50mM NaCl、2mM EDTA和50mM磷酸钾以及10％乙醇和5％乙腈的缓冲液(pH 6.5)10倍体积进行等体积超滤(超滤膜包采用30kd的 聚纤维素膜包)，以除去反应液中未反应的SMCC以及其他小分子杂质，最后用50mM NaCl、2mM EDTA和50mM磷酸钾缓冲液(pH 6.5)5倍体积进行等体积超滤得到Trastuzumab-MCC的50mM NaCl、2mM EDTA和50mM磷酸钾缓冲液溶液(pH 6.5)。
得到的Trastuzumab-MCC在50mM NaCl、2mM EDTA和50mM磷酸钾缓冲液溶液(pH 6.5)置换到Trastuzumab-MCC的50mM NaCl、2mM EDTA和50mM磷酸钾以及10％乙醇和2％乙腈的缓冲液溶液(pH 6.5)中，调整浓度为10mg/mL，再加入300mgL-DM1的乙醇溶液与上述反应体系。在反应摇床上反应进行4小时，生成Trastuzumab-MCC-DM1的反应液，取样品进行药物载量测试以及SEC-HPLC纯度测试，SEC-HPLC结果是94.59％；将反应生成得到的Trastuzumab-MCC-DM1反应液用填料为SuperdexTM 200pg的分子排阻柱进行纯化样品，以除去样品中的聚合体和其他小分子杂质，回收单体，取样品进行药物载量测试以及SEC-HPLC纯度测试，SEC-HPLC结果是99.11％。根据纯化过程取样的药物载量计算(UV法)，结果见表1。
表1.根据纯化过程取样的药物载量计算(UV法)
样品 纯化前 纯化后
载量 3.40 3.34
附：Trastuzumab-MCC-DM1药物载量的测定方法：
Trastuzumab-MCC-DM1药物载量的测定方法包括紫外-可见分光光度法和LC/MS法。
紫外-可见分光光度法的原理是基于在280nm下药物和单克隆抗体共同对总吸光值产生作用(The absorbance at 280nm of the drug and the antibody contribute to the total absorbance(A280nm))，计算过程如下：
A280nm＝ε1bc1+ε2bc2         ⑴
其中：
ε1：药物在280nm下的消光系数；
ε2：单克隆抗体在280nm下的消光系数；
c1：药物的浓度；
c2：单克隆抗体的浓度；
b：光程长度(detector path length)；
同理可以得到样品在252nm下的总吸光值方程：
A252nm＝ε3bc3+ε4bc4         ⑵
由⑴和⑵两种方程结合单克隆抗体和药物在两个检测波长下的消光系数和浓 度数据可以计算出药物的载量。在该工艺中采用紫外-可见分光光度法来测量计算原液最后的载量。
实施例2：抗体药物偶联物P-mab-linker-MC-MMAF的合成
Figure PCTCN2016109807-appb-000003
取帕妥珠单抗(P-mab，抗体公开于US7041292B1)原液(pH＝4.3-4.5的100mM的醋酸-醋酸钠缓冲液中，并浓缩至浓度为10.0mg/mL左右)，得P-mAb的醋酸-醋酸钠缓冲液200mL(13.5mmol)，将3-乙酰巯基丙醛(14.3mg，0.108mmol)，溶解于20mL乙腈后滴加入上述缓冲液中，将氰基硼氢化钠(173mg,2.7mmol)溶于10mL水中滴加入上述反应液中，25±2℃下搅拌反应2-3小时。反应结束后用50mM NaCl、2mM EDTA和50mM磷酸钾以及10％乙腈的缓冲液(pH 6.5)5倍体积进行等体积超滤，以除去反应液中未反应的其他小分子杂质，最后用50mM NaCl、2mM EDTA和50mM磷酸钾缓冲液(pH 6.5)5倍体积进行等体积超滤后得200mL帕妥珠单抗-丙硫醇乙酯溶液，浓度约为10mg/mL。
向帕妥珠单抗-丙硫醇乙酯溶液(第一步制得)加入2M盐酸羟胺4.0mL，25℃下搅拌反应1小时后将反应液用50mM NaCl、2mM EDTA和50mM磷酸钾缓冲液(pH 6.5)5倍体积进行等体积超滤后得200mL帕妥珠单抗-丙硫醇溶液，浓度10mg/mL。
取化合物MC-MMAF(化合物公开于WO2005081711，125mg，13.5mmol)溶解于20mL乙腈中，加入帕妥珠单抗-丙硫醇溶液(第二步制得)中，25℃下搅拌反应4小时后，得到P-mab-linker-MC-MMAF的ADC样品，取样品进行完整分子量测试以及SEC-HPLC纯度测试，SEC-HPLC结果详是94.70％以及药物载量是2.1。将得到的P-mab-linker-MC-MMAF的ADC样品反应液用填料为SuperdexTM 200pg的分子排阻柱进行纯化样品，以除去样品中的聚体以及其他的小分子残余，可以得到纯化的样品，取样品进行完整分子量测试以及SEC-HPLC纯度测试，SEC-HPLC结果是99.39％以及药物载量是2.08。
附：测试单抗类样品P-mab-linker-MC-MMAF的完整分子量实验条件：
色谱条件：
色谱柱：Poroshell 300SB-C8 5um 2.1*75mm
流动相：A：0.1％HCOOH/H2O B:0.1％HCOOH/ACN
梯度：
RT(min) 0 5 8 12 12.1 15
B％ 5 5 95 95 5 5
流速：0.4mL/min
柱温：75℃
质谱条件：
质谱：Agilent 6530Q-TOF LC-MS
模式：1GHz
毛细管电压：3500V
去溶剂气体温度：325℃
气体流速：10L/min
Fragmentor电压：200V
MS采集范围：500m/z-5000m/z
实施例3：抗体药物偶联物T-DM1的合成与纯化
在25℃，在含有50mM NaCl、2mM EDTA和50mM磷酸钾以及10％乙醇和5％乙腈的缓冲液(pH 6.5)中，50.0克单克隆抗体曲妥珠(Trastuzumab终浓度10mg/mL)与905毫克的4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯在反应摇床上反应进行2小时，生成Trastuzumab-MCC的反应液；将反应生成得到的Trastuzumab-MCC反应液，在25℃温度下，用50mM NaCl、2mM EDTA和50mM磷酸钾以及10％乙醇和5％乙腈的缓冲液(pH 6.5)10倍体积进行等体积超滤，以除去反应液中未反应的SMCC以及其他小分子杂质，最后用50mM NaCl、2mM EDTA和50mM磷酸钾缓冲液(pH 6.5)5倍体积进行等体积超滤得到Trastuzumab-MCC的50mM NaCl、2mM EDTA和50mM磷酸钾缓冲液溶液(pH 6.5)。
得到的Trastuzumab-MCC在50mM NaCl、2mM EDTA和50mM磷酸钾缓冲液溶液(pH 6.5)置换到Trastuzumab-MCC的50mM NaCl、2mM EDTA和50mM磷酸钾以及10％乙醇和2％乙腈的缓冲液溶液(pH 6.5)中，调整浓度为10mg/mL，再加入1500mgL-DM1的乙醇溶液与上述反应体系。在反应摇床上反应进行4小时，反应得到Trastuzumab-MCC-DM1反应液，测试其纯度以及药物载量。SEC-HPLC纯度是95.25％，药物载量是3.43。
将得到的Trastuzumab-MCC-DM1反应液用填料为SuperdexTM 200pg的分子排阻柱进行纯化样品，以除去样品中的聚合体和残余其他小分子物质。最后在进行纯度测试以及药物载量测试，其SEC-HPLC纯度为99.26％，药物载量是3.40。样品回收率是95％。
表2.根据纯化过程取样的药物载量计算(UV法)
样品 纯化前 纯化后
载量 3.43 3.40
比较实施例1：
在25℃，在含有50mM NaCl、2mM EDTA和50mM磷酸钾以及10％乙醇和5％乙腈的缓冲液(pH 6.5)中，10.0克单克隆抗体曲妥珠(Trastuzumab终浓度10mg/ml)与180毫克的4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯在反应摇床上反应进行2小时，生成Trastuzumab-MCC的反应液；反应结束后，将反应生成得到的Trastuzumab-MCC反应液，用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的含有50mM NaCl、2mM EDTA和50mM磷酸钾的缓冲液)，以除去反应液中未反应的SMCC以及其他小分子杂质，最后得到Trastuzumab-MCC的50mM NaCl、2mM EDTA和50mM磷酸钾缓冲液溶液(pH 6.5)。
将得到的Trastuzumab-MCC在50mM NaCl、2mM EDTA和50mM磷酸钾缓冲液溶液(pH 6.5)置换到Trastuzumab-MCC的50mM NaCl、2mM EDTA和50mM磷酸钾以及10％乙醇和2％乙腈的缓冲液溶液(pH 6.5)中，调整浓度为10mg/ml，再加入300mg L-DM1的乙醇溶液与上述反应体系。在反应摇床上反应进行4小时，生成Trastuzumab-MCC-DM1的反应液；反应结束后，将反应生成得到的Trastuzumab-MCC-DM1反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的含有50mM NaCl、2mM EDTA和50mM磷酸钾的缓冲液)，以除去反应液中未反应的L-DM1以及其他小分子杂质，最后得到Trastuzumab-MCC-DM1的50mM NaCl、2mM EDTA和50mM磷酸钾缓冲液溶液(pH 6.5)。最后在进行纯度测试以及药物载量测试，其SEC-HPLC纯度为94.45％，药物载量是3.45。
比较实施例2：
在25℃，在含有50mM NaCl、2mM EDTA和50mM磷酸钾以及10％乙醇和5％乙腈的缓冲液(pH 6.5)中，10.0克单克隆抗体曲妥珠(Trastuzumab终浓度10mg/ml)与181毫克的4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯在反应摇床上反应进行2小时，生成Trastuzumab-MCC的反应液；将反应生成得到的Trastuzumab-MCC反应液，在25℃温度下，用50mM NaCl、2mM EDTA和50mM磷酸钾以及10％乙醇和5％乙腈的缓冲液(pH 6.5)5倍体积进行等体积超滤(超滤膜包采用30kd的聚纤维素膜包)，以除去反应液中未反应的SMCC以及其他小分子杂质，最后用50mM NaCl、2mM EDTA和50mM磷酸钾缓冲液(pH 6.5)5倍体积进行等体积超滤得到Trastuzumab-MCC的50mM NaCl、2mM EDTA和50mM磷酸钾缓冲液溶液(pH 6.5)。
得到的Trastuzumab-MCC在50mM NaCl、2mM EDTA和50mM磷酸钾缓冲 液溶液(pH 6.5)置换到Trastuzumab-MCC的50mM NaCl、2mM EDTA和50mM磷酸钾以及10％乙醇和2％乙腈的缓冲液溶液(pH 6.5)中，调整浓度为10mg/ml，再加入300mgL-DM1的乙醇溶液与上述反应体系。在反应摇床上反应进行4小时，生成Trastuzumab-MCC-DM1的反应液，在25℃温度下，用50mM NaCl、2mM EDTA和50mM磷酸钾以及10％乙醇和2％乙腈的缓冲液(pH 6.5)5倍体积进行等体积超滤(超滤膜包采用30kd的聚纤维素膜包)，以除去反应液中未反应的L-DM1以及其他小分子杂质，最后用50mM NaCl、2mM EDTA和50mM磷酸钾缓冲液(pH6.5)5倍体积进行等体积超滤得到Trastuzumab-MCC-DM1(T-DM1)的50mM NaCl、2mM EDTA和50mM磷酸钾缓冲液溶液(pH 6.5)。最后在进行纯度测试以及药物载量测试，其SEC-HPLC纯度为94.61％，药物载量是3.51。

Claims (12)

  1. 一种抗体药物偶联物的制备方法，包括抗体-连接剂与细胞毒性药物进行偶联反应，以制备抗体药物偶联物，还包括抗体药物偶联物的纯化步骤，所述的纯化步骤包括分子排阻步骤。
  2. 如权利要求1所述的抗体药物偶联物的制备方法，其特征在于，分子排阻步骤包括使用分离分子量范围在5000～5000000，优选10000～600000的凝胶作为纯化柱的填充料。
  3. 如权利要求2所述的抗体药物偶联物的制备方法，其特征在于，分子排阻步骤包括使用G200凝胶作为纯化柱的填充料，优选SuperdexTM200pg凝胶作为纯化柱的填充料。
  4. 如权利要求1-3任一项所述的抗体药物偶联物的制备方法，其特征在于，所述的抗体连接剂的纯化的方法为超滤。
  5. 如权利要求4所述的抗体药物偶联物的制备方法，其特征在于，所述超滤的滤膜孔径为10-50KD，优选30KD。
  6. 如权利要求1-3任一项所述的抗体药物偶联物的制备方法，其特征在于，所述的抗体原液浓度为5-15mg/mL，优选10mg/mL。
  7. 如权利要求1-3任一项所述的抗体药物偶联物的制备方法，其特征在于，所述的抗体连接剂的浓度为5-15mg/mL，优选10mg/mL。
  8. 如权利要求1-3任一项所述的抗体药物偶联物的制备方法，其特征在于，所述的抗体为曲妥珠单抗。
  9. 如权利要求8所述的抗体药物偶联物的制备方法，其特征在于，所述的抗体药物偶联物为T-DM1。
  10. 如权利要求1-3任一项所述的抗体药物偶联物的制备方法，其特征在于，所述的抗体为帕妥珠单抗。
  11. 如权利要求10所述的抗体药物偶联物的制备方法，其特征在于，所述的抗体药物偶联物为P-mab-linker-MC-MMAF。
  12. 如权利要求1至11任意一项所述的制备方法，其特征在于，在抗体-连接剂与细胞毒性药物进行偶联反应前，还包括抗体-连接剂的制备和纯化步骤。
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