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Abstract：
A sensitive and specific bioanalytical method was required to measure the exposure of a LAGA-mutated surrogate mouse IgG2a monoclonal antibody in mouse plasma, but the lack of highly specific reagents for the LAGA mutant hindered the development of a ligand-binding assay. Equally problematic is that no sensitive unique tryptic peptides suitable for quantitative mass spectrometric analysis could be identified in the mIgG2a complementarity-determining regions. To overcome these challenges, a trypsin alternative pepsin, an aspartic protease, was systematically investigated for its use in digesting the mutated mIgG2a antibody to allow generation of signature peptides for the bioanalytical quantification purpose. After a series of evaluations, a rapid one-hour pepsin digestion protocol was established for the mutated Fc backbone. Consequently, a new pepsin digestion-based liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was successfully developed to support the mouse pharmacokinetic (PK) sample analysis. In brief, robust and reproducible C-terminal cleavage of both leucine and phenylalanine near the double mutation site of the mutated mIgG2a was accomplished at pH ≤2 and 37°C. Combined with a commercially available rat anti-mIgG2a heavy-chain antibody, the established immunoaffinity LC/MS/MS assay achieved a limit of quantitation of 20 ng/mL in the dynamic range of interest with satisfactory assay precision and accuracy. The successful implementation of this novel approach in discovery PK studies eliminates the need for tedious and costly generation of specific immunocapturing reagents for the LAGA mutants. The approach should be widely applicable for developing popular LAGA mutant-based biological therapeutics.
摘要：
需要一种灵敏和特异的生物分析方法来测量小鼠血浆中LAGA突变的替代小鼠IgG2a单克隆抗体的暴露，但是缺乏针对LAGA突变体的高度特异性试剂阻碍了配体结合测定的发展.同样有问题的是,在mIgG2a互补决定区中没有鉴定出适合于定量质谱分析的敏感的独特胰蛋白酶肽。为了克服这些挑战，胰蛋白酶替代胃蛋白酶，一种天冬氨酸蛋白酶,系统地研究了其在消化突变的mIgG2a抗体中的用途,以允许产生用于生物分析定量目的的特征肽。经过一系列的评估,为突变的Fc主链建立了快速的一小时胃蛋白酶消化方案。因此,成功开发了一种新的基于胃蛋白酶消化的液相色谱-串联质谱（LC/MS/MS）方法，以支持小鼠药代动力学（PK）样品分析。简而言之,在pH≤2和37°C下完成突变的mIgG2a的双突变位点附近亮氨酸和苯丙氨酸的稳健且可重复的C端切割。结合市售大鼠抗mIgG2a重链抗体，已建立的免疫亲和LC/MS/MS测定在感兴趣的动态范围内达到20ng/mL的定量限，具有令人满意的测定精度和准确性。这种新方法在发现PK研究中的成功实施消除了对用于LAGA突变体的特异性免疫捕获试剂的冗长且昂贵的产生的需要。该方法应广泛适用于开发流行的基于LAGA突变体的生物治疗剂。
