Impact of Amino Acid Excipients on icIEF Method Development in Protein Therapeutics: A Formulation-Aware Analytical Framework.

Accurate assessment of charge heterogeneity and isoelectric point (pI) is essential for ensuring the quality, stability, and safety of biotherapeutics throughout development and commercialization. Imaged capillary isoelectric focusing (icIEF) is the industry‑standard technique for this purpose, using a stable, linear pH gradient to accurately localize analyte peaks relative to calibrant pI markers. Although amino acids are widely used as formulation excipients for physicochemical stabilization, their potential to interfere with icIEF analyses has not been systematically evaluated. In this study, we investigated the influence of amino acids, with varying pI values and physicochemical properties, on the pH‑gradient linearity, peak focusing, and experimental pI determination of monoclonal antibodies (mAbs). Using a six-pI marker ladder and two representative mAbs (Immunoglobulin-G1: IgG1 and Immunoglobulin-G4: IgG4), our findings demonstrated that amino acids with pI values outside the analyte's focusing region (e.g., arginine, glutamic acid) can shift peak positions within the capillary window without disrupting calibration linearity, thereby preserving the consistent determination of pI values. In contrast, amino acids with pI values within the focusing region (e.g., glycine, histidine, alanine, leucine) induced localized gradient distortion and generated concentration‑dependent pI shifts. Our case studies highlight conditions under which rational pI marker selection mitigated these effects, as well as a scenario with histidine‑containing IgG4 formulations, in which co‑focusing of IgG4 and histidine could not be simply corrected by calibration adjustments. Based on these findings, we propose a formulation design space that quantitatively relates protein concentration, amino acid excipient levels, and assay dilution requirements to minimize analytical interference. This framework provides practical guidance for integrating formulation strategy to support more reliable icIEF method development to ensure high-quality data throughout the biotherapeutic lifecycle.