کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
7610905 1493502 2015 25 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Post-pellet-digestion precipitation and solid phase extraction: A practical and efficient workflow to extract surrogate peptides for ultra-high performance liquid chromatography - tandem mass spectrometry bioanalysis of a therapeutic antibody in the low n
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
Post-pellet-digestion precipitation and solid phase extraction: A practical and efficient workflow to extract surrogate peptides for ultra-high performance liquid chromatography - tandem mass spectrometry bioanalysis of a therapeutic antibody in the low n
چکیده انگلیسی
The current LC-MS/MS approach for bioanalysis of protein therapeutics requires generating peptides from protein molecules via trypsin digestion, followed by sensitive detection of these surrogate peptides by multiple reaction monitoring (MRM). However, the presence of huge amount of matrix-related interference peptides generated from trypsin digestion often causes substantial matrix effect or isobaric interferences during LC-MS/MS analysis. Solid phase extraction (SPE) exhibits great potential in sample extraction to overcome these challenges due to its characteristic features of high selectivity, reproducibility, cost-effectiveness and potential to be automated. Here, we report an effective SPE methodology for the bioanalysis of protein therapeutics involving post-pellet-digestion precipitation and SPE cleanup prior to UHPLC-MS/MS analysis. Specifically, proteins in serum samples were first precipitated with methanol to enrich the protein analyte in the pellet prior to trypsin digestion of the pellet (pellet-digestion). The trypsin digest was further processed by a post-pellet-digestion precipitation (second precipitation) to remove matrix-related clog-causing components prior to SPE on OASIS™ MAX (Mixed-Mode Anion-Exchange) SPE plate. This methodology successfully overcame SPE clogging issue and enabled extraction of 100 μL of monkey serum on SPE with significant sensitivity improvement to achieve a lower limit of quantitation (LLOQ) of 50 ng/mL for a human monoclonal antibody of the IgG4 subclass. This UHPLC-MS/MS assay was validated in a concentration range of 50-5000 ng/mL with intra- and inter-assay precisions of within 9.6% CV, and assay accuracy of within ±2.9% Dev of their nominal concentrations. To our best knowledge, this is the pellet digestion with SPE method for LC-MS/MS bioanalysis of a monoclonal antibody for the first time to achieve a LLOQ in the low ng/mL concentration range.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Chromatography A - Volume 1424, 11 December 2015, Pages 27-36
نویسندگان
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