Discussion

Introduction: The growth of therapeutic monoclonal antibodies (mAb) is expanding with more therapeutics and innovation (eg. Antibody Drug Conjugates). Due to biological complexity, use of mass spectrometry in pre-clinical/clinical development is required. Although MS-based methods can provide improved data content, there are limitations with reproducibility, sensitivity, and automation. Methods: Mass Spectrometric Immunoassay (MSIA), a hybrid affinity-based LC-MS workflow, was applied. Humanized therapeutic mAbs were extracted from rodent plasma using biotinylated anti-human Fc antibodies applied onto MSIA streptavidin microcolumns. Subsequent LC-MS analysis was performed using intact molecule HRAM detection Results: The ability to address a variety of humanized and fully human mAb backbones from animal models was demonstrated. Using Adalimumab as an example, the resultant LC-MS data showed a lack of non-specific binding allowing flow rates to be performed at 200 L/ minute. The study demonstrates a low detection level at 20 ng/mL , a CV <15% and a broad assay dynamic range of intact therapeutic is readily achievable when using MSIA for bioanalysis. Using HRAM, total ion envelope and deconvolved mass spectra clearly showed presence of posttranslational modifications making this approach applicable to in vivo biotransformation and Drug Antibody Ratio (DAR) determination. Further, the MSIA workflow uses automated sample processing meaning a reproducible, push button solution is provided. Conclusions: A MSIA for universal application in the bioanalysis of fully human therapeutic mAbs for pre-clinical research is achievable. The MSIA workflow can provide beneficial improvements to analytical performance, automation and data quality.