Development of imaged capillary isoelectric focusing as a platform mispairing byproduct testing method for asymmetric WuXiBody-based bispecific antibodies.

This study explores the application of imaged capillary isoelectric focusing (icIEF) to distinguish and quantify mispairing byproducts in asymmetric WuXiBody-based bispecific antibodies (AsWXbsAbs). Bispecific antibody (BsAb), developed using Knobs-into-Holes (KiH) technology, often result in byproducts such as knob-knob (KK) and hole-hole (HH) homodimers, which share similar sizes with the target BsAb, complicating their separation by traditional methods like size exclusion chromatography-high performance liquid chromatography (SEC-HPLC). Our approach leverages the unique pI differences introduced by substituting the CH1/CL domain with the T cell receptor (TCR) constant domain in AsWXbsAbs. This modification enables icIEF to effectively differentiate between the KK and HH homodimers and the target BsAb. Through the construction and expression of heavy and light chain variants, we validated that the experimental pI values aligned with theoretical predictions, confirming icIEF's capability in distinguishing these entities. Enrichment of in-process K-related and H-related species was achieved, allowing for high-purity samples necessary for icIEF method development. The method was qualified and showed good specificity and linearity, with a quantitation limit of 4 % for K-related species (R = 0.9919) and 1 % for H-related species (R = 0.9805). This method was used effectively as an in-process test and release assay, proving its simple and quick utility in multiple AsWXbsAbs projects.