Liquid chromatography coupled with tandem mass spectrometry for the bioanalysis of proteins in drug development: practical considerations in assay development and validation.

In recent years, there has been increasing interest in using LC-MS/MS to measure protein drugs (biotherapeutics) in plasma/serum to support pharmacokinetic/toxicokinetic (PK/TK) studies. A number of strategies have been proposed to quantify different types of protein drug candidates in biological matrices. However, the application of LC-MS/MS in biotherapeutics is still very limited in regulated bioanalysis due to practical challenges. In this manuscript, we propose a "data-dependent" approach for the LC-MS/MS support of protein drug candidates, focusing on operational aspects. We have developed and validated a fast, simple and reliable LC-MS/MS method to quantify a protein drug candidate in mouse serum. This method can fit into our current workflow for routine small molecule LC-MS/MS assays with an overall sample preparation time of less than two hours. The method has been demonstrated to be accurate, precise and rugged, and it has been successfully applied to analyze samples from toxicology studies. The results were verified using a ligand binding assay and tested by standard-addition sample reanalysis. In addition, general recommendations for developing and validating an LC-MS/MS assay based on surrogate peptide(s) of protein drug candidates are also proposed for regulated bioanalysis.