Absolute and Multiplex Quantification of Antibodies in Serum Using Psaq™ Standards and LC-MS/MS

Abstract

The approach presented provides similar accuracy and precision than ELISA, while being multiplex and faster to develop. It has applications at all stages of the pharmaceutical development.

Cutting-edge mass spectrometry methods for the multi-level structural characterization of antibody-drug conjugates

“…Optimized LC-SRM assays offer unequalled sensitivity, high analyte specificity, a high multiplexing capacity and precision, and robust quantitation of analytes down to high ng/ml-low µg/ml concentrations in unfractionated plasma. Most of the methods described above rely on enzymatic digestion of the mAb to yield at least one peptide with a unique sequence to be quantified as a surrogate for the whole mAb [189]. Multidimensional chromatography or immunoenrichment can be used to separate the mAb of interest from those of the endogenous matrix, thereby increasing the relative concentration of the analyte of interest [190,191].…”

Ten years of QconCATs: Application of multiplexed quantification to small medically relevant proteomes

“…This was first developed for the quantification of staphylococcal enterotoxin A in urine, serum, gastric content and food matrices [36,37]. Another notable application of PSAQ was the quantification of monoclonal antibody variants in rat serum for use in pre-clinical studies, which displayed the ability of this method to discriminate different variants/isoforms [38]. PSAQ methodology is sometimes seen as an alternative to the QconCAT, but it is more accurate to regard it as a special example of a QconCAT -one that is very suitable for the detection of single, soluble proteins, but much less appropriate for whole proteomes.…”

An Overall Comparison of Small Molecules and Large Biologics in ADME Testing

“…As demonstrated in recent examples, Xu et al successfully applied a multiplexed hybrid LC-MS/MS pharmacokinetic assay to measure two co-administrated mAbs in a clinical study without requiring stringent affinity capture reagents [79]. Lebert and coworkers also demonstrated absolute and multiplexed LC-MS/MS method combining a strategy so-called PSAQ TM (protein standard absolute quantification or stable isotopically-labeled full-length mAbs) as the ISs for quantification of three mAbs variants with very similar sequences, e.g., IgG1, IgG2 and igG4 isotypes of a lead mAb in rat serum, which can be employed for both preclinical and clinical studies [80]. Moreover, a versatile immunoaffinity LC-MS/MS method was developed to quantify total receptor activator of nuclear factor-kappaB ligand (RANKL) in the presence of denosumab, a humanized monoclonal antibody (mAb) specific to RANKL with an LLOQ of RANKL down to 3.13 ng/mL for mouse plasma PK, which was not able to determine the total RANKL because the interference of denosumab decreased the recovery of RANKL with commercial ELISA kit [81].…”