Abstract / Summary
Abstract Bioconjugation of linker–payloads (LPs) to antibodies has historically relied on organic cosolvents or surfactants to overcome the poor aqueous solubility of cytotoxic payloads─an assumption that has remained largely unquestioned in antibody–drug conjugate (ADC) manufacturing. Here, we systematically re-evaluate this paradigm by characterizing the aqueous solubility of 13 representative LPs at operationally relevant concentrations (5–50 mM) across water and acidic buffers. Classical hydrophobic LPs, including vedotin, mafodotin, and deruxtecan, were confirmed to be insoluble under all aqueous conditions tested, validating their established dependence on cosolvents. However, LPs incorporating hydrophilic elements, such as PEG chains or ionizable groups, dissolved readily in water at concentrations sufficient for bioconjugation. Most notably, the linker–payload corresponding to sacituzumab govitecan (CL2A-SN-38) achieved concentrations of at least 50 mM in pure water, and two different conjugation reactions were successfully executed in fully aqueous buffered media without any organic solvents or surfactants. Physicochemical analysis revealed that solubility is not governed solely by global hydrophobicity (A log P) but is codetermined by PEG length, charge distribution, pH-dependent ionization, and the continuity of hydrophobic surface topology. Conjugation under fully aqueous conditions yielded ADCs with average DAR values of 1.6–1.9 under target-DAR2 conditions and 6.1–7.0 under target-DAR8 conditions, with minimal aggregation and physicochemical properties comparable to those produced via conventional cosolvent-based workflows. These findings demonstrate that rational hydrophilicity-driven LP design enables cosolvent-free conjugation, thereby simplifying ADC manufacturing, eliminating flammability hazards, removing residual solvent testing requirements, and reducing regulatory complexity.