The success of biotherapeutics is often challenged by the undesirable events of immunogenicity in patients, characterized by the formation of anti-drug antibodies (ADA). Under specific conditions, the ADAs recognizing the biotherapeutic can trigger the formation of immune complexes (ICs), followed by cascades of subsequent effects on various cell types. Hereby, the connection between the characteristics of ICs and their downstream impact is still not well understood. Factors governing the formation of ICs and the characteristics of these IC species were assessed systematically in vitro. Classic analytical methodologies such as SEC-MALS and SV-AUC, and the state-of-the-art technology mass photometry were applied for the characterization. The study demonstrates a clear interplay between (1) the absolute concentration of the involved components, (2) their molar ratios, (3) structural features of the biologic, (4) and of its endogenous target. This surrogate study design and the associated analytical tool-box is readily applicable to most biotherapeutics and provides valuable insights into mechanisms of IC formation prior to FIH studies. The applicability is versatile-from the detection of candidates with immunogenicity risks during developability assessment to evaluation of the impact of degraded or post-translationally modified biotherapeutics on the formation of ICs.
Keywords: SEC-MALS; SV-AUC; aggregation; analytical assessment; anti-drug antibodies; biotherapeutics; immune complexes; immunogenicity; mass photometry; protein complexes.