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Custom-Designed Affinity Capture LC-MS F(ab′)2 Assay for Biotransformation Assessment of Site-Specific Antibody Drug Conjugates
Affinity capture
liquid chromatography–mass spectrometry
(LC-MS) intact antibody assay has been widely used for direct drug-to-antibody
ratio (DAR) and catabolite characterization of antibody-drug conjugates
(ADCs). However, the intact mass spectra of new ADCs, which incorporate
new types of linkers and payloads other than maytansines and auristatins,
are more complex than those examined previously. The current method
has showed some limitations in elucidating certain structural modifications.
Herein, we report an alternative analytical approach for ADCs, such
as THIOMAB antibody-drug conjugates (TDCs), where the linker drugs
are site-specifically conjugated in the Fab region. The newly developed
affinity capture LC-MS FÂ(ab′)Â2 assay incorporates affinity
capture of human IgGs via binding to the Fab region, followed by on-bead
IdeS digestion to remove the Fc domain specifically and uniformly.
The resulting FÂ(ab′)Â2 (∼100 kDa) fragments contain the
key ADC biotransformation information, such as drug-to-antibody ratio
and drug metabolism and are more readily analyzed by electrospray
ionization LC-MS than the intact ADC (∼150 kDa). The reduced
size of analytes results in improved mass spectral sensitivity and
resolution. In addition, the reduced and optimized sample preparation
time, for example, rapid removal of the Fc fragment by IdeS digestion,
minimizes assay artifacts of drug metabolism and skewed DAR profiles
that may result from the prolonged incubation times (e.g., overnight
enzymatic treatment for Fc deglycosylation). The affinity capture
LC-MS FÂ(ab′)Â2 assay provides more detailed and accurate information
on ADC biotransformations in vivo, enabling analysis of low-dose,
labile, and complex site-specific ADCs with linker-drug conjugated
in the Fab region