General LC-MS/MS Method Approach to Quantify Therapeutic Monoclonal Antibodies Using a Common Whole Antibody Internal Standard with Application to Preclinical Studies
General LC-MS/MS Method Approach to Quantify Therapeutic Monoclonal Antibodies Using a Common Whole Antibody Internal Standard with Application to Preclinical Studies
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DOI：
10.1021/ac202792n
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发表时间：
2012-02-07
影响因子：
7.4
通讯作者：
Lee, Jean W.
中科院分区：
文献类型：
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作者：
Li, Hongyan;Ortiz, Robert;Lee, Jean W.
配基结合分析(LBA)被广泛用于生物样品中治疗性单抗的定量。主要的限制是方法开发时间长、试剂采购和基质效应。使用特征多肽的LC-MS/MS方法正在成为一种替代方法，它通常使用稳定的同位素标记的特征多肽作为内标(IS)。然而，必须为每个候选生成新的IS，并且IS可能不会针对所有处理步骤中的变化进行校正。我们建立了一种通用的LC-MS/MS方法，使用统一的重同位素标记的通用全单抗和通用的免疫捕捉器进行样品处理。该方法经过了简化，实现了一致性和吞吐量的自动化。方法4株Ig G(2)和4株Ig G(1)单抗的灵敏度分别为0.1µg/m L和0.1~1 5 m u g/m L。这8株单抗的质控数据准确、准确。标记IS全分子的质控性能优于人工合成的标记IS多肽。两种单抗在大鼠体内的药代动力学结果与LBA相当。一般的LC-MS/MS方法克服了当前方法的局限性，以减少临床前研究所需的时间和资源。
Ligand binding assays (LBAs) are widely used for therapeutic monoclonal antibody (mAb) quantification in biological samples. Major limitations are long method development times, reagent procurement, and matrix effects. LC-MS/MS methods using signature peptides are emerging as an alternative approach, which typically use a stable isotope labeled signature peptide as the internal standard (IS). However, a new IS has to be generated for every candidate, and the IS may not correct for variations at all processing steps. We have developed a general LC-MS/MS method approach employing a uniformly heavy-isotope labeled common whole mAb IS and a common immunocapture for sample processing. The method was streamlined with automation for consistency and throughput. Method qualification of four IgG(2) and four IgG(1) mAbs showed sensitivity of 0.1 mu g/mL and linearity of 0.1-15 mu g/mL. Quality control (QC) data of these eight mAbs were accurate and precise. The QC performance of the whole molecule labeled IS was better than those of synthetic labeled IS peptides tested. The pharmacokinetic results of two mAbs (an IgG(2) and IgG(1) candidate) dosed in rats were comparable to those of LBA. The general LC-MS/MS method approach overcomes the limitations of current methods to reduce time and resources required for preclinical studies.