Middle-down approach: A digestion-free analysis of antibody drug conjugates using LC-HRMS

EBF Open Symposium 2025 -- Antibody drug conjugates (ADCs) are a rapidly advancing class of biotherapeutics that integrate the specificity of monoclonal antibodies with the potency of cytotoxic payloads. Structurally, they comprise three key components: antibody, which is designed to target the intended tumor antigen, linker for controlled drug release, and payload, typically a highly potent cytotoxic agent and specific method of operation (MOA). The drug-to-antibody ratio (DAR), a key ADC attribute, reflects the average number of drugs attached per antibody. It influences efficacy, pharmacokinetics, and toxicity; low DAR reduces potency, while high DAR may impair PK and increase side effects. Current lysine or cysteine conjugation typically yields 0-8 drugs per antibody.