Presentation Description: An understanding of analyte recovery and matrix effects is necessary to develop and validate robust bioanalytical methods. An analyte recovery greater than 100% was recently observed during the validation of an LC-MS/MS IgG4 monoclonal antibody (mAB) assay. To understand this phenomenon, a systematic evaluation of various factors affecting recovery was conducted. Those factors included: digestion efficiency, non-specific binding, aggregation and solubility. Addition of organic to the IP eluate aided in denaturing the protein to allow for consistent digestion efficiency. Post-IP spiked mAB adsorbed differently based on the sample preparation plate material affecting MS sensitivity response. The NSB and inconsistent digestion efficiency combined resulted in the greater than theoretical recovery that was initially observed.
Understand that non-specific binding affects mABs differently than peptides even when low binding labware is utilized.
Understand that a post-IP spiked mAB differs from a pre-IP spiked mAB due to the environments seen, resulting in different accessibility of trypsin to arginine and lysine without proper denaturation.
Understand how TFA and organic solvent affect the denaturation of a mAB.