LOCALIZING THE CONJUGATION SITES OF CYSTEINE-CONJUGATED ANTIBODY DRUG CONJUGATES BY IMPROVED LC-MS SUBUNIT ANALYSIS FOR ADC POSITIONAL ISOMER IDENTIFICATION

Significance of the topic

Subunit-level structural analysis of cysteine-conjugated antibody–drug conjugates (ADCs) via LC-MS is essential for precise localization of drug attachment sites and for detecting critical modifications such as oxidation or water loss. This information underpins ADC quality control, efficacy, and safety in therapeutic development.

Objectives and study overview

This study compared three proteolytic approaches—IdeS, SpeB, and a limited Lys-C protocol—to optimize hinge-region cleavage of cysteine-linked ADCs with varying drug-to-antibody ratios. The goal was to improve subunit generation for accurate identification of positional isomers across different conjugation loads.

Methodology and instrumentation

ADC samples spanning low to high drug loads were prepared at 1 mg/mL. Enzymatic digestions were carried out at 37 °C for 30 min using:

• IdeS or SpeB (100 U)
• Lys-C under optimized enzyme-to-substrate ratio

Reactions were quenched with acetic acid. Digests were diluted to 0.5 µg/µL for analysis. Instrumentation comprised a Waters ACQUITY H-Class Bio UPLC with a Protein BEH C4 column (300 Å, 1.7 µm, 2.1×50 mm) coupled to a Waters Xevo G2-S or G2-XS QTof MS. Mobile phases included water, acetonitrile/isopropanol (60/40), 1% formic acid, and 1% TFA. ESI settings featured a 3 kV capillary voltage, 150 V cone voltage, source temperature at 500 °C, desolvation at 350 °C, and 800 L/h gas flow.

Main results and discussion

• IdeS and SpeB cleavages were hindered by increased steric bulk at the hinge in high-load ADCs, leading to incomplete subunit release.
• The limited Lys-C protocol achieved consistent cleavage above the hinge across all drug loads, producing clear LC, Fc/2, and Fd fragments.
• Mass spectra revealed oxidation events on Fc fragments and water loss from LC+drug species. Opening of intra-chain disulfides was also detected.
• Targeted middle-down MS/MS of partially reduced subunits enabled unambiguous confirmation of positional isomers.

Benefits and practical applications

• The optimized Lys-C protocol offers a reliable strategy to generate diagnostic subunits regardless of drug loading.
• Enhanced subunit analysis supports robust ADC characterization workflows in both research and QC laboratories.
• Middle-down MS/MS integration enables direct mapping of conjugation sites, improving batch consistency and regulatory compliance.

Future trends and applications

Advancements may include automation of limited proteolysis, integration with higher-resolution MS instruments, and expansion to other conjugation chemistries. Such developments will streamline ADC positional isomer profiling and accelerate biotherapeutic development.

Conclusion

The study demonstrates that a limited Lys-C digestion protocol outperforms IdeS and SpeB for hinge-region cleavage of cysteine-conjugated ADCs, enabling clear subunit production and reliable positional isomer identification. Oxidation and water loss events were characterized, and targeted middle-down MS/MS facilitated definitive mapping of drug attachment sites.