1 research outputs found
Automated Affinity Capture and On-Tip Digestion to Accurately Quantitate <i>in Vivo</i> Deamidation of Therapeutic Antibodies
Deamidation
of therapeutic antibodies may result in decreased drug
activity and undesirable changes in pharmacokinetics and immunogenicity.
Therefore, it is necessary to monitor the deamidation levels [during
storage] and after <i>in vivo</i> administration. Because
of the complexity of <i>in vivo</i> samples, immuno-affinity
capture is widely used for specific enrichment of the target antibody
prior to LC–MS. However, the conventional use of bead-based
methods requires large sample volumes and extensive processing steps.
Furthermore, with automation difficulties and extended sample preparation
time, bead-based approaches may increase artificial deamidation. To
overcome these challenges, we developed an automated platform to perform
tip-based affinity capture of antibodies from complex matrixes with
rapid digestion and peptide elution into 96-well microtiter plates
followed by LC–MS analysis. Detailed analyses showed that the
new method presents high repeatability and reproducibility with both
intra and inter assay CVs < 8%. Using the automated platform, we
successfully quantified the levels of deamidation of a humanized monoclonal
antibody in cynomolgus monkeys over a time period of 12 weeks after
administration. Moreover, we found that deamidation kinetics between <i>in vivo</i> samples and samples stressed <i>in vitro</i> at neutral pH were consistent, suggesting that the <i>in vitro</i> stress test may be used as a method to predict the liability to
deamidation of therapeutic antibodies <i>in vivo</i>