Development and Validation of an Enzyme-Linked Immunosorbent Assay for Measuring Factor XI in Intravenous Immunoglobulin Products by Mitigating Heterophilic Antibody Interference.

Thromboembolic events associated with intravenous immunoglobulin (IVIG) therapy have been linked to activated coagulation factor XI (FXIa). Therefore, regulatory authorities recommend monitoring FXI levels in IVIG products using quantitative assays, such as sandwich enzyme-linked immunosorbent assays (ELISAs). However, high concentrations of immunoglobulins in IVIG can cause heterophilic antibody interference, leading to false-positive FXI measurements. In this study, we aimed to develop and validate a sandwich ELISA capable of accurately measuring FXI while minimizing interference from high immunoglobin G (IgG) concentrations. We hypothesized that Purified human IgG (Fc) could serve as a heterophilic antibody blocker, thereby neutralizing non-specific interactions and improving assay reliability. The FXI measurements were compared with and without IgG blocker treatment across process intermediates and final IVIG products with varying IgG levels. False-positive signals were observed only in samples with high IgG concentrations and were eliminated after treatment with the IgG blocker. The modified ELISA demonstrated acceptable accuracy, precision, linearity, and quantification limits in accordance with the ICH Q2(R2) guidelines. This validated assay reliably quantified FXI regardless of IgG concentration, providing a robust platform for impurity assessment during IVIG manufacturing.