Linlin Dong, Susan Chen, Konstantin Piatkov, Dong Wei, Mark G Qian
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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. 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引用次数: 0
摘要
我们需要一种灵敏而特异的生物分析方法来测量小鼠血浆中 LAGA 突变代用品小鼠 IgG2a 单克隆抗体的暴露量,但由于缺乏针对 LAGA 突变体的高度特异性试剂,配体结合检测方法的开发受到了阻碍。同样存在问题的是,在 mIgG2a 互补性决定区无法鉴定出适用于定量质谱分析的敏感的独特胰蛋白酶肽。为了克服这些难题,我们系统地研究了一种胰蛋白酶替代品胃蛋白酶(一种天冬氨酸蛋白酶),研究它在消化变异 mIgG2a 抗体时的用途,以便生成用于生物分析定量目的的特征肽。经过一系列评估后,针对突变的 Fc 骨架建立了一小时快速胃蛋白酶消化方案。因此,成功开发了一种基于胃蛋白酶消化的液相色谱-串联质谱(LC/MS/MS)新方法,以支持小鼠药代动力学(PK)样本分析。简而言之,在pH≤2和37°C条件下,对突变mIgG2a双突变位点附近的亮氨酸和苯丙氨酸进行了稳健且可重复的C端裂解。结合市售的大鼠抗 mIgG2a 重链抗体,所建立的免疫亲和 LC/MS/MS 检测方法在相关动态范围内的定量限为 20 ng/mL,检测精度和准确度令人满意。在发现 PK 研究中成功实施这种新方法后,就不再需要为 LAGA 突变体制作繁琐而昂贵的特异性免疫捕获试剂了。这种方法应广泛适用于开发基于 LAGA 突变体的流行生物疗法。
Quantifying LAGA mutated mouse IgG2a monoclonal antibody with a rapid pepsin digestion enabled immunoaffinity LC/MS/MS assay.
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.
期刊介绍:
mAbs is a multi-disciplinary journal dedicated to the art and science of antibody research and development. The journal has a strong scientific and medical focus, but also strives to serve a broader readership. The articles are thus of interest to scientists, clinical researchers, and physicians, as well as the wider mAb community, including our readers involved in technology transfer, legal issues, investment, strategic planning and the regulation of therapeutics.