Immunoconjugates exploit the high affinity of monoclonal antibodies for a recognized antigen to selectively deliver a cytotoxic payload, such as drugs or radioactive nuclides, at the site of disease. Despite numerous techniques have been recently developed for site-selective bioconjugations of protein structures, reaction of ε-amine group of lysine residues with electrophilic reactants, such as activated esters (NHS), is the main method reported in the literature as it maintains proteins in their native conformation. Since antibodies hold a high number of lysine residues, a heterogeneous mixture of conjugates will be generated, which can result in decreased target affinity. Here, we report an intradomain regioselective bioconjugation between the monoclonal antibody Trastuzumab and the N-hydroxysuccinimide ester of the chelator 2,2′,2″,2‴-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid (DOTA) by a kinetically controlled reaction adding substoichiometric quantities of the activated ester to the mAb working at slightly basic pH. Liquid chromatography–mass spectrometry (LC–MS) analyses were carried out to assess the chelator-antibody ratio (CAR) and the number of chelating moieties linked to the mAb chains. Proteolysis experiments showed four lysine residues mainly involved in bioconjugation (K188 for the light chain and K30, K293, and K417 for the heavy chain), each of which was located in a different domain. Since the displayed intradomain regioselectivity, a domain mapping MS-workflow, based on a selective domain denaturation, was developed to quantify the percentage of chelator linked to each mAb domain. The resulting immunoconjugate mixture showed an average CAR of 0.9. About a third of the heavy chains were found as monoconjugated, whereas conjugation of the chelator in the light chain was negligible. Domain mapping showed the CH3 domain bearing 13% of conjugated DOTA, followed by CH2 and VH respectively bearing 12.5 and 11% of bonded chelator. Bioconjugation was not found in the CH1 domain, whereas for the light chain, only the CL domain was conjugated (6%). Data analysis based on LC–MS quantification of different analytical levels (intact, reduced chains, and domains) provided the immunoconjugate formulation. A mixture of immunoconjugates restricted to 15 species was obtained, and the percentage of each one within the mixture was calculated. In particular, species bearing 1 DOTA with a relative abundance ranging from 4 to 20-fold, in comparison to species bearing 2DOTA, were observed. Pairing of bioconjugation under kinetic control with the developed domain mapping MS-workflow could raise the standard of chemical quality for immunoconjugates obtained with commercially available reactants.

中文翻译：

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用于合成放射免疫缀合物的动力学控制生物缀合方法及其表征的域映射 MS 工作流程的开发

免疫缀合物利用单克隆抗体对识别抗原的高亲和力，选择性地在疾病部位递送细胞毒性有效负载，例如药物或放射性核素。尽管最近开发了许多用于蛋白质结构的位点选择性生物共轭的技术，但赖氨酸残基的ε-胺基与亲电子反应物（例如活化酯（NHS））的反应是文献中报道的主要方法，因为它使蛋白质保持在他们的天然构象。由于抗体含有大量赖氨酸残基，因此会产生缀合物的异质混合物，这可能导致靶标亲和力降低。在这里，我们报告了单克隆抗体曲妥珠单抗和螯合剂2,2 ',2",2‴-(1,4,7,10-tetraazacyclododecane-1,4,7, 10-四基）四乙酸 (DOTA)，通过动力学控制反应将亚化学计量的活化酯添加到在弱碱性 pH 下工作的 mAb。进行液相色谱-质谱 (LC-MS) 分析以评估螯合剂-抗体比率 (CAR) 以及与 mAb 链连接的螯合部分的数量。蛋白水解实验显示，四个赖氨酸残基主要参与生物缀合（轻链为 K188，重链为 K30、K293 和 K417），每个赖氨酸残基位于不同的结构域中。由于显示了域内区域选择性，因此开发了基于选择性域变性的域映射 MS 工作流程，以量化与每个 mAb 域连接的螯合剂的百分比。所得免疫缀合物混合物的平均 CAR 为 0.9。大约三分之一的重链被发现是单缀合的，而轻链中螯合剂的缀合可以忽略不计。结构域作图显示CH3结构域含有13%的缀合DOTA，其次是CH2和VH，分别含有12.5%和11%的键合螯合剂。 CH1 结构域中未发现生物缀合，而对于轻链，仅 CL 结构域发生缀合 (6%)。基于不同分析水平（完整链、还原链和结构域）的 LC-MS 定量的数据分析提供了免疫偶联物配方。获得了仅限于 15 种的免疫缀合物混合物，并计算了混合物中每种免疫缀合物的百分比。特别是，与携带 2DOTA 的物种相比，携带 1 DOTA 的物种的相对丰度范围为 4 至 20 倍。将动力学控制下的生物偶联与开发的域映射 MS 工作流程配对可以提高用市售反应物获得的免疫偶联物的化学质量标准。