A sensitive multidimensional method for the detection, characterization, and quantification of trace free drug species in antibody-drug conjugate samples using mass spectral detection.

摘要: Conjugation processes and stability studies associated with the production shelf life of antibody-drug conjugates (ADCs) can result in free (non-conjugated) drug species. These species increase risk to patients reduce efficacy ADC. Despite stringent purification steps, trace levels may be present formulated ADCs, reducing therapeutic window. The reduction sample preparation steps through incorporation multidimensional techniques has afforded analysts more efficient methods assess Multidimensional coupling size-exclusion reversed phase liquid chromatography ultra-violet detection (SEC-RPLC/UV) have been reported, but offer limited sensitivity limit method optimization. current study addresses these challenges a that is specific, sensitive, enables control both dimensions via an on-line solid extraction column RPLC mass spectral (SPE-RPLC/MS). proposed was evaluated using antibody-fluorophore conjugate (AFC) as ADC surrogate brentuximab vedotin its parent maleimide-val-cit-DSEA payload derived N-acetylcysteine adduct formed during conjugation process. Assay found 2 orders sensitive MS comparison UV-based nominal quantitation 0.30 ng/mL (1.5 pg on-column). Free-drug were unadulterated at concentrations below 7 ng/mL, not detectable by UV alone. SPE-RPLC/MS provides high degree specificity assessment offers improved over each dimension, enabling straightforward integration into existing or novel workflows.