Automated On-tip Affinity Capture Coupled with Mass Spectrometry to Characterize Intact Antibody-Drug Conjugates from Blood

Abstract

Antibody-drug conjugates (ADCs) present unique challenges for ligand-binding assays primarily due to the dynamic changes of the drug-to-antibody ratio (DAR) distribution in vivo and in vitro. Here, an automated on-tip affinity capture platform with subsequent mass spectrometry analysis was developed to accurately characterize the DAR distribution of ADCs from biological matrices. A variety of elution buffers were tested to offer optimal recovery, with trastuzumab serving as a surrogate to the ADCs. High assay repeatability (CV 3%) was achieved for trastuzumab antibody when captured below the maximal binding capacity of 7.5 μg. Efficient on-tip deglycosylation was also demonstrated in 1 h followed by affinity capture. Moreover, this tip-based platform affords higher throughput for DAR characterization when compared with a well-characterized bead-based method. Graphical Abstract ᅟ.

Antibody Subunit LC-MS Analysis for Pharmacokinetic and Biotransformation Determination from In-Life Studies for Complex Biotherapeutics

“…The field of intact antibody quantitation and biotransformation in vivo is evolving, with continued debate around strategies to achieve biotherapeutic stability monitoring from in-life studies. , While there are specific literature reports of LC-MS for biotransformation on antibody subunits and intact ADC, ,, the combination of PK, biotransformation, and DAR demonstrated here by subunit LC-MS provides a wide range of information and possibility for application to multiple end points. As discussed, subunit LC-MS offers benefits and drawbacks compared to intact protein mass LC-MS.…”

“…To demonstrate a simpler approach akin to traditional small molecule LC-MS analysis and applicable to a wide range of biotherapeutic modalities, recent efforts have focused on the use of intact antibody quantitation or subunit quantitation. − Intact protein MS analysis provides the ability to monitor the biotherapeutic as well as mass variant species of the entire molecule. ,, Intact protein MS for PK application requires immunocapture reagents for isolation of the biotherapeutic of interest and has sensitivity limitations, partially due to the challenges of detecting large masses by MS . Other methods have focused on antibody subunit monitoring from in-life studies for drug concentration and mass variants determination by use of disulfide reduction or site-specific enzymes such as IdeS to generate subunit products. − Antibody subunit approaches break the molecule down to smaller masses more amenable to MS while still retaining information about the entire primary amino acid sequence and mass variants.…”

IQ Consortium Perspective: Complementary LBA and LC–MS in Protein Therapeutics Bioanalysis and Biotransformation Assessment

“…Traditional LC-MS methods based on tryptic peptide digestion are routinely implemented, however, for drug quantification purposes, one or few peptides are typically utilized and information about the whole protein is lost in the digestion step. ADC conjugation measurements based on intact masses have been utilized for relative quantification of DAR and other biotransformation species [39,40]. DAR and ADC conjugate information can be obtained at the antibody subunit level as well [41,42].…”

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].…”