Challenges and Advanced Solutions in Large Molecule Quantification by LC-MS

Algorithme Pharma | December 2012 e-Bulletin
e-Bulletin / December 2012
Challenges and Advanced Solutions in
Large Molecule Quantification by LC-MS: "Glucagon Case Study"
Traditionally, large molecules have been quantified using Ligand Binding Assays (LBA). Recently LC-MS has also been shown to be highly successful in the bioanalysis of large peptides, proteins and biomarkers. The quantification of such compounds using LC-MS based bioanalytical methods can however be extremely challenging.
Large molecules can interact/adsorb strongly with both plastic and glass surfaces and generate aggregates. Enzymatic and chemical degradation can also occur and affect sample integrity. Finally, sensitivity and selectivity could also be difficult to achieve even when using the most advanced mass spectrometers. Glucagon is an interesting case study which demonstrates how our scientists were able to overcome these specific challenges.
Stability: Glucagon was found to be very unstable in plasma even in presence of aprotinin, the traditional stabilizer previously used for this large molecule. We were able to prevent degradation by the addition of an innovative protease inhibitor cocktail.
Adsorption & Aggregation: Both adsorption and aggregation were found to cause major issues for precision and accuracy of Glucagon quantification. We eliminated both by acidifying and sonicating the samples.
Sensitivity & Selectivity: The choice of a charge state that was able to generate a sensitive and selective fragment allowed us to increase the S/N 7-fold versus the most commonly used transitions.
Solving the issues described above allowed for the development and validation of a rugged and sensitive assay for the quantification of Glucagon in plasma.
Algorithme Pharma is a leader in the quantification of large molecules by LC-MS. Our scientists have worked extensively on the analysis of large molecules by LC-MS in our laboratory which is equipped with all the necessary instrumentation (QTRAP, QTOF and immunocapturing capabilities). Our expertise is demonstrated by methods for New Molecular Entities and non-proprietary large molecules such as Lysozyme, MUC5AC13, Hepcidin, Calcitonin, Desmopressin and Exenatide. Presently, we are working on the LC-MS quantification of Fusion proteins and PEGylated therapeutic proteins.
To learn more about our Bioanalysis services, please contact us at contact@algopharm.com
Contact us at 1 (450) 973-6077, 1 (888) 267-7449, or contact@algopharm.com
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