Quantitative analysis of protein-biotherapeutics: Recent advances in LC-MS/MS technology for improved selectivity and sensitivity
With an ever increasing flow of protein-based biopharmaceutical candidates entering the drug development funnel, reliable bioanalytical techniques are required to support quantitation in various biological fluids in order to generate toxicokinetic, pharmacokinetic, and bioavailability data. Trends in the scientific literature clearly indicate that the coupling of liquid chromatography with tandem mass spectrometry (LC-MS/MS) is replacing traditional ligand binding assays due to improved selectivity and linear dynamic range, and that the mainstream MS approach for quantitation relies upon targeted proteomics techniques involving internal standardization, proteolytic digestion, surrogate peptide selection, peptide and/or protein purification, LC separation and MS/MS detection. This review focuses primarily on recently reported advancements in MS workflows that have resulted in improved sensitivity through the effective elimination of interference from matrix components. Further, case studies are presented illustrating advantages of an immunocapture pre-digestion approach when LC-MS/MS alone fails to provide suitable required assay performance.
The biologics market continues to experience rapid rates of growth, with > 50% of approved drugs expected to be protein-based biotherapeutics by 2019 (1). Further, patents for several existing biologic blockbusters are set to expire within the next two years, which predictably shall lead to a surge in biosimilars. Therefore, the increased importance of protein therapeutics necessitates performance improvements in bioanalytical techniques, with LC-MS/MS emerging as the most attractive and flexible technology able to cope with the high demands of throughput and complexity of analysis. While ligand-binding assays (LBAs) have historically been the most popular technique for protein quantitation, they demonstrate lower selectivity, are often unable to distinguish catabolites from parent drug, provide limited dynamic range, and are unsuitable for high-throughput early development work due to the time-consuming task of raising antibodies (2). In contrast, the increasingly mainstream workflow to quantify protein therapeutics using a surrogate peptide following proteolytic digestion with subsequent LC-MS/MS detection offers rapid method development, a wide ...