Department of Drug Metabolism, Pharmacokinetics & Modeling, Takeda Development Center Americas, Inc., Cambridge, MA, USA.
MAbs. 2024 Jan-Dec;16(1):2379903. doi: 10.1080/19420862.2024.2379903. Epub 2024 Jul 30.
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.
需要一种灵敏且特异的生物分析方法来测量 LAGA 突变型模拟鼠 IgG2a 单克隆抗体在鼠血浆中的暴露情况，但缺乏针对 LAGA 突变体的高度特异性试剂阻碍了配体结合测定法的开发。同样存在问题的是，在 mIgG2a 互补决定区中，无法鉴定出适合定量质谱分析的灵敏独特的胰酶肽。为了克服这些挑战，系统性地研究了一种替代胰蛋白酶的胃蛋白酶（一种天冬氨酸蛋白酶），用于消化突变的 mIgG2a 抗体，以产生用于生物分析定量目的的特征肽。经过一系列评估，建立了一种用于突变 Fc 骨架的快速一小时胃蛋白酶消化方案。因此，成功开发了一种基于胃蛋白酶消化的新型液相色谱-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，能够在突变 mIgG2a 的双突变位点附近实现对亮氨酸和苯丙氨酸的 C 端切割，且具有稳健性和重现性。与市售的抗鼠 IgG2a 重链抗体结合使用，所建立的免疫亲和 LC/MS/MS 测定法在感兴趣的动态范围内达到了 20ng/mL 的定量下限，具有令人满意的测定精密度和准确度。在发现性 PK 研究中成功实施这种新方法，消除了对 LAGA 突变体进行繁琐和昂贵的特异性免疫捕获试剂生成的需求。该方法应该广泛适用于开发基于 LAGA 突变体的热门生物治疗药物。
