Rapid assessment of target-binding fractions in molecular imaging agents using size-exclusion HPLC

Abstract

Background

The clinical translation of molecularly targeted therapeutics and imaging agents represents a cornerstone of precision oncology, with the global theranostics market projected to exceed $25 billion by 2030. However, the development of theragnostic agents or diagnostic companions remains constrained by analytical bottlenecks in quality control, such as target-binding fraction, which are increasingly required by regulatory agencies as product release criteria during the translation process. Current methods, including enzyme-linked immunosorbent assay (ELISA), which require specialized resources or external CROs, or bead-based assays for radiolabeled compounds, which involve complex multi-step protocols; these limitations and others hamper their practical implementation in clinical manufacturing environments. Assay delays can postpone clinical trial initiation, increase development costs, and delay patient access to these agents.

Results

We have developed and validated a rapid, size-exclusion high-performance liquid chromatography (SE-HPLC) method for the determination of target-binding fractions of labeled biologics. The method separates the unbound biologic from the larger antigen-bound complex, allowing for rapid quantification. We validated the method using a panel of fluorescently labeled antibodies (panitumumab-IRDye800CW, nivolumab-IRDye800CW) and radiolabeled biologics ([18F]GEH200521, [18F]NOTA-ABY-030), assessing linearity, specificity, and concentration independence. The SE-HPLC method achieved excellent separation of bound and unbound species with a resolution (Rs) of 3.2. A strong linear relationship (R² = 0.999) was observed between the antigen-to-antibody ratio and the measured binding fraction. The method demonstrated high specificity, with no binding detected with non-target antigens. The total assay and analysis time was less than 35 min, a significant improvement over traditional methods.

Conclusions

SE-HPLC provides a rapid, specific, and cost-effective alternative to traditional binding fraction assessment methods, reducing quality control timelines from weeks/hours to minutes. The method’s compatibility with both fluorescent and radiolabeled biologics and integration with existing HPLC infrastructure represents a significant advancement in development workflows.