Chawita Netirojjanakul
Amgen, USA
Title: High throughput optimization and mass spectrometric analysis of covalently labeled proteins and antibody drug conjugates
Biography
Biography: Chawita Netirojjanakul
Abstract
The use of automated high throughput screening in large molecule discovery research still lags behind that of small molecule
discovery. Recently we developed a high-throughput large molecule discovery platform to automate hundreds of bioconjugation
reaction setup in one setting. In addition, given LC-MS is a widespread analytical bottleneck, we also established a high-throughput
mass spectrometry (HT-MS) platform to accurately detect and rapidly quantitate protein conjugates. We showed that our HT-MS
platform can be used to quantitate the extent of covalent inhibitor adducts to a cysteine-containing protein construct (~19 kDa)
and of biotinylated adducts to mAbs and Fc domains (~150 and ~50 kDa, respectively). Sample acquisition time was ~20 seconds
per sample, 10-50x shorter time than traditional LC-MS methods. Site-specific bioconjugation of human Fc domains with cysteine
engineered at different positions were conducted under a matrix of reaction conditions varying equivalents of reductants, oxidants,
and alkylating agents using the high-throughput large molecule discovery platform. Using HT-MS, 4 x 384 well plates were analyzed
in ~8 hours, as opposed to ~11 days using traditional LC-MS. This approach facilitated rapid determination of DAR values for the
reduced and intact huFc domains and selection of optimized conditions for different cysteine-engineered Fc constructs which will be
used in preparation of Fc-peptide conjugates as therapeutic leads.