Validation of LC-MS/MS bioanalytical methods for protein therapeutics.

Background There are more than 100 protein and peptide drugs currently approved by the US FDA and now over 100 more are currently in active development by pharmaceutical companies worldwide [1]. The quantification of protein therapeutics has traditionally relied upon ligand-binding assays (LBAs). Such assays can be very sensitive and selective if properly developed using appropriate reagents, but are nevertheless susceptible to interferences due to circulating ligands, antidrug antibodies (ADAs), cross reactions and various forms of non-specific binding. LC– MS/MS analysis has emerged as an effective quantitative tool for protein bioanalysis. Of particular interest is LC–MS/MS analysis of therapeutic proteins via quantification of unique surrogate peptides that are produced by enzymatic digestion, a technique that is being widely used [1–4]. For simplicity, we will call this type of analysis protein digestion LC–MS/MS (PrD-LC–MS). PrD-LC– MS can be used as an alternative and supplemental approach to LBAs, and this has been extensively discussed in recent years [1–4]. Such LC–MS/MS methods, where the protein biotherapeutic is digested directly in the biomatrix prior to LC–MS/MS, can provide bioanalytical data with low interference from ADAs and other circulating ligands [5]. These methods can yield more representative quantification of total drug, but they may have limited sensitivity. Where higher sensitivity has been required, analyte enrichment or affinity capture concentration steps can be added either before [6] or after [7] proteolytic digestion. These enrichment methods can improve sensitivity by eliminating background signals either from the biomatrix itself or from the digest prior to peptide LC–MS/MS. As the methodology for PrD-LC–MS matures, there will be an increased need to use these assays in a regulated environment, both for good laboratory practice (GLP) toxicology studies and for regulated sample bioanalysis in clinical trials. Neither the recent EMA guidance [8], nor the FDA draft Bioanalytical Method Validation (BMV) guidance [9] has specifically discussed the validation of PrD-LC–MS or its application to regulated bioanalysis. Several questions need to be addressed for any regulated PrD-LC–MS assay validation: