We are witnessing a major shift in pharmaceutical industry from small molecule drugs toward biotherapeutics such as antibody or Fc-protein based therapies. Biotherapeutics can have similar properties compared to some small molecule drugs, thus motivating their misuse in performance enhancing. The available methods developed for the detection of these substances in plasma are in vast majority based on liquid chromatography coupled to mass spectrometry. They are mainly targeted LC-HRMS methods that aim to detect biotherapeutic specific peptides in horse plasma. Our laboratory previously published a targeted method based on the analysis of a set of 10 tryptic peptides shared by 95% of available biotherapeutics and absent from horse proteome1. These methods are efficient for routine analysis. They can be improved with validation methods designed to increase amino-acid sequence coverage of biotherapeutics.
In this study we propose a protocol of therapeutic antibody analysis that includes a targeted LC-HRMS analysis of selected tryptic peptides. This first analysis is followed by untargeted LC-HRMS analysis of all generated peptides by Immunoglobulins tryptic digestion. LCMS data from untargeted analysis will be interrogated against amino-acid sequence database containing characterized immunoglobulin peptides that are endogenous to equine plasma (developed in our lab by analyzing 49 horse plasmas including males, females and gelding from 3 horse breeds) and supplemented with available amino-acid sequence of approved biotherapeutics.
Denosumab is a human IgG2 monoclonal antibody approved for osteoporosis treatment. It generates 3 tryptic peptides among the selected 10 targeted tryptic sequences in our routine method. Results obtained for the analysis of Denosumab in supplemented plasmas of after administration to a horse using this protocol are described. We compared the capacity of targeted and untargeted LC-HRMS analysis to detect Denosumab in horse plasma and the detection window provided by each approach.