ADC Characterization

ADC characterization covers several critical quality attributes — DAR, drug load distribution, aggregation, free drug/linker, and higher-order structure — each requiring specific analytical techniques.

Drug-to-Antibody Ratio (DAR)

DAR (the average number of payload molecules per antibody) is the single most important quality attribute, since a too-low DAR reduces potency while a too-high DAR can impair stability, antigen binding, and pharmacokinetics. Four main methods are used, each with different trade-offs:

For cysteine-linked ADCs, HIC is considered the gold standard, while RP-HPLC and LC-MS serve as orthogonal confirmation methods, and newer hybrid approaches like native RPLC-MS or HIC×SEC-IM×MS combine chromatographic separation with mass accuracy for resolving even positional isomers within the same DAR class.

Primary Structure and Conjugation Site Verification

Intact, reduced, and deglycosylated mass determination by high-resolution LC-MS (e.g., Orbitrap) confirms that the measured mass matches the theoretical mass, accounting for glycosylation, terminal modifications, and linker-payload conjugation on each chain. Peptide mapping (enzymatic digestion followed by LC-MS/MS) is commonly used to pinpoint exactly which residues carry the payload, which is especially important for confirming site-specificity in engineered-cysteine or non-natural-amino-acid ADCs.

Free Drug and Process-Related Impurities

Residual unconjugated payload or linker-payload must be quantified and controlled, typically via RP-HPLC or LC-MS after removing the ADC by protein precipitation or affinity capture, since free drug is separately regulated and impacts safety assessment. Capillary electrophoresis (CE) is also used to profile drug-load distribution and detect low-abundance species that chromatography might miss.

Biophysical and Higher-Order Structure

Beyond composition, ADC developability requires assessing conformational stability and aggregation propensity, since payload conjugation itself can destabilize the antibody. Common techniques include:

• Thermal/conformational stability: differential scanning calorimetry (DSC), differential scanning fluorimetry (DSF), circular dichroism, and intrinsic fluorescence spectroscopy

• Aggregation/self-association: size-exclusion chromatography with multi-angle light scattering (SEC-MALS), dynamic and static light scattering (DLS/SLS), analytical ultracentrifugation (AUC), and fluorescence correlation spectroscopy (FCS)

• Structural dynamics: hydrogen-deuterium exchange mass spectrometry (HDX-MS), which reveals how conjugation affects protein folding and flexibility

Charge Variants and Potency

Charge heterogeneity (from deamidation, glycation, or conjugation-induced charge changes) is assessed by capillary isoelectric focusing (cIEF) or ion-exchange chromatography, since it can affect immunogenicity and antigen binding. Functional potency and target engagement are confirmed with ligand-binding assays (LBA) and cell-based cytotoxicity assays, which verify that the conjugated ADC still binds its antigen and kills target cells effectively, distinguishing total antibody from actively conjugated ADC.

Practical Testing Sequence

A typical characterization package for a new ADC batch would combine: UV/Vis for a quick DAR estimate, HIC or RP-HPLC for full drug-load distribution, intact/reduced LC-MS for mass confirmation and orthogonal DAR verification, SEC(-MALS) for aggregation, peptide mapping for conjugation-site confirmation, and a cell-based potency assay to confirm biological activity. This multi-orthogonal approach is standard because no single method fully captures all critical quality attributes of these structurally complex molecules.