Probing Non-Covalent Interactions in Biomatrices by Native Affinity Capture Mass Spectrometry: Biotransformation of Antibodies Conjugated with Small Molecules and siRNA

Protein therapeutic complexes, such as interchain cysteine-conjugated antibody-drug conjugates (ADCs) and siRNA-based antibody-oligonucleotide conjugates (siRNA AOCs) that form non-covalent antibody subunit assemblies, are increasingly prevalent. Biotransformation assessments of protein therapeutics in complex biomatrices are often conducted using affinity capture coupled with reversed phase liquid chromatography–mass spectrometry under denaturing conditions (AC-MS, for clear distinction refer to as AC-denaturing MS), which allows direct measurement of clipping sites and relative quantification. However, AC-denaturing MS is unsuitable for analyzing the biotransformation of non-covalent protein therapeutics as stability information is lost for the non-covalent component. This manuscript presents a novel biotransformation analysis method that preserves non-covalent interactions by combining affinity capture with neutral pH competitive elution and native size exclusion chromatography–mass spectrometry, named as native affinity capture-mass spectrometry (native AC-MS). The successful recovery of non-covalent complexes, including a cysteine-conjugated ADC mimic molecule and siRNA AOCs in various biomatrices, demonstrates the capability of native AC-MS to maintain non-covalent interactions during characterization. Optimized competitive elution exhibits comparable recovery to standard denaturing elution. Native AC-MS offers sufficient sensitivity (low µg/mL limit of detection for antibody) for in vivo studies, as demonstrated by an in vivo plasma siRNA AOC analysis example. To our knowledge, this is one of the first report of in vivo biotransformation analysis of siRNA AOCs. The method provides valuable information, including relative abundance and metabolite identification and can be widely adapted for biotransformation of non-covalent modalities.