Liu Luna, Xu Keyang, Li Jenny, Maia Mauricio, Mathieu Mary, Elliott Rebecca, Yang Jihong, Nijem Ihsan, Kaur Surinder
Department of BioAnalytical Sciences, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA.
Department of Antibody Engineering, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA.
Bioanalysis. 2018 Nov 1;10(22):1819-1831. doi: 10.4155/bio-2018-0196. Epub 2018 Oct 16.
Hybrid ligand-binding (LB) LC-MS/MS protein quantitative assays involve a LB step for analyte enrichment that has less stringent requirements than the conventional LB assays. Herceptin™(trastuzumab) binding to HER2 extracellular domain was evaluated using on-bead and off-bead capture formats. The two formats yielded significantly different trastuzumab concentrations in human and monkey serum pharmacokinetic samples. Biotransformations, including deamidation of asparagine and isomerization of aspartic acid near the complementarity-determining regions of trastuzumab, had a profound impact on the LB step for analyte enrichment and trastuzumab quantification. Quantitative measurements were profoundly impacted by LB conditions in a hybrid LB LC-MS/MS protein assay due to biotransformations. Therefore, similar to conventional LB assays, binding conditions should be carefully evaluated during assay development.
混合配体结合（LB）液相色谱-串联质谱（LC-MS/MS）蛋白质定量分析涉及一个用于分析物富集的LB步骤，该步骤的要求不如传统LB分析严格。使用珠上和珠外捕获形式评估了赫赛汀™（曲妥珠单抗）与HER2细胞外结构域的结合。这两种形式在人和猴血清药代动力学样品中产生了显著不同的曲妥珠单抗浓度。生物转化，包括曲妥珠单抗互补决定区附近天冬酰胺的脱酰胺化和天冬氨酸的异构化，对用于分析物富集和曲妥珠单抗定量的LB步骤产生了深远影响。在混合LB LC-MS/MS蛋白质分析中，由于生物转化，定量测量受到LB条件的深刻影响。因此，与传统LB分析类似，在分析方法开发过程中应仔细评估结合条件。
