Hydrophilic Interaction Liquid Chromatography Enables Drug‐to‐Antibody Ratio Measurement of a Heterogeneous Lysine‐Conjugated Antibody Fragment

ABSTRACT

Accurate measurement of drug‐to‐antibody ratio (DAR) is essential for characterizing antibody‐based conjugates, but heterogeneous lysine conjugation and hydrophilic payload structures can limit the selectivity of conventional hydrophobicity‐based separations. Here, we describe an ion‐pairing hydrophilic interaction liquid chromatography (HILIC) method for quantitative DAR measurement of a heterogeneous lysine‐conjugated drug‐Fc conjugate. Column chemistry, sample preparation, and mobile phase additives were systematically investigated. Reduced and deglycosylated samples analyzed on a bare silica column with methanesulfonic acid‐containing mobile phases provided the highest selectivity and resolution. In silico retention modeling was used to rapidly optimize conditions that balanced resolution, robustness, and analysis time. Beyond enabling efficient method optimization, in silico retention modeling can support validation readiness by defining a robust method operating space early in development and strengthening the scientific rationale for assay‐condition selection. The final method resolved DAR‐related species from DAR0 to DAR7 and demonstrated linear detector response from drug‐Fc solutions ranging from 1 to 15 mg/mL in concentration. Consistent average DAR values were observed across concentration levels, with recovery of 99.8%–101.3%. Replicate percent relative standard deviations for average DAR were <0.25%. Together, these results establish HILIC as a practical approach for quality control‐friendly DAR measurement of highly heterogeneous, hydrophilic bioconjugates.