Universal Affinity Capture Liquid Chromatography-Mass Spectrometry Assay for Evaluation of Biotransformation of Site-Specific Antibody Drug Conjugates in Preclinical Studies

Abstract

Antibody drug conjugates (ADCs) can undergo in vivo biotransformation (e.g., payload metabolism, deconjugation) leading to reduced or complete loss of activity. The location/site of conjugation of payload-linker can have an effect on ADC stability and hence needs to be carefully optimized. Affinity capture LC–MS of intact ADCs or ADC subfragments has been extensively used to evaluate ADC biotransformation. However, the current methods have certain limitations such as the requirement of specific capture reagents, limited mass resolution of low mass change metabolites, low sensitivity, and use of capillary or nanoflow LC–MS. To address these challenges, we developed a generic affinity capture LC–MS assay that can be utilized to evaluate the biotransformation of any site-specific ADC independent of antibody type and site of conjugation (Fab and Fc) in preclinical studies. The method involves a combination of some or all of these steps: (1) “mono capture” or “dual capture” of ADCs from serum with streptavidin magnetic beads coated with a generic biotinylated antihuman capture reagent, (2) “on-bead” digestion with IdeS and/or PNGase F, and (3) reduction of interchain disulfide bonds to generate ∼25 kDa ADC subfragments, which are finally analyzed by LC–HRMS on a TOF mass spectrometer. The advantages of this method are that it can be performed using commercially available generic reagents and requires sample preparation time of less than 7 h. Furthermore, by reducing the size of intact ADC (∼150 kDa) to subfragments (∼25 kDa), the identification of conjugated payload and its metabolites can be achieved with excellent sensitivity and resolution (hydrolysis and other small mass change metabolites). This method was successfully applied to evaluate the in vitro and in vivo biotransformation of ADCs conjugated at different sites (LC, HC-Fab, and HC-Fc) with various classes of payload-linkers.

Recent Advances in Drug–Antibody Ratio Determination of Antibody–Drug Conjugates

“…The affinity capture approach is considered a promising method to evaluate an antibody's properties. [27][28][29][30] This analysis is conducted under native conditions and has already been applied to ADC analysis and can therefore be expected to be applied for DAR characterization. 31) Hydrophilic interaction chromatography (HILIC) can assess the glycosylation profile of an intact protein 32) and may be applied to evaluate DAR distribution for ADCs based on appropriately glycosylated antibodies.…”

Current approaches for the purification of antibody–drug conjugates

“…Besides the above purification approaches, some other chromatographic techniques have been reported in the field of ADC analysis [61,62]. Affinity chromatography is often used to evaluate the properties of antibodies [63].…”

Bioanalysis in the Age of New Drug Modalities

“…HRMS can measure intact protein with functional information and distinguish small mass shifts of biotransformational variants via its extremely high mass resolving power, hence serving as an important tool for protein characterization (59). In comparison with LBA, a common drawback of LC-MS assay is its lower sensitivity due to limited choices of signature peptide of the intact form.…”