Reversed‐Phase High‐Performance Liquid Chromatography Method for Titer Estimation of Therapeutic Nanobodies

Nanobodies (NBs) are emerging as next‐generation biologics due to their small size, high stability, and strong antigen‐binding affinity. However, a major gap in the analytical characterization of NBs is the lack of any standardized, validated, and rapid method for their quantification during development or in‐process monitoring. This study presents the validated reversed‐phase high‐performance liquid chromatography (RP‐HPLC) method specifically designed for quantitative analysis of therapeutic NBs, capable of distinguishing and accurately measuring NB titers in both crude and purified samples within 40 min . The method demonstrates linearity (R 2 = 0.99) across 5–80 µg sample loads, with high precision (coefficient of variation < 2%) , specificity, and robustness as per International Council for Harmonisation Q2(R1) guidelines. Coupling the RP‐HPLC workflow with mass spectrometry confirms the molecular identity and purity of the NB, thereby validating the analytical accuracy of the proposed approach. This represents a novel, scalable, and industry‐relevant platform for quantitative and confirmatory analysis of NBs, addressing a critical gap in the analytical characterization of these emerging biologics.