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Rapid assessment of target-binding fractions in molecular imaging agents using size-exclusion HPLC
Ashtyn McAdoo1,2,3, Kamal Jouad3, Eben L Rosenthal2,3
1Chemical and Physical Biology, Vanderbilt University, Nashville, TN, USA.
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.
Insights
A new size-exclusion high-performance liquid chromatography (SE-HPLC) method rapidly determines target-binding fractions for labeled biologics. This breakthrough accelerates quality control, reducing timelines from weeks to minutes for precision oncology agents.
Area of Science:
- Oncology
- Analytical Chemistry
- Biotechnology
Background:
- Precision oncology relies on molecularly targeted therapeutics and imaging agents, driving a global theranostics market projected to exceed $25 billion by 2030.
- Development of these agents is hindered by analytical bottlenecks in quality control, specifically target-binding fraction assessment, which is a regulatory requirement.
- Current methods like ELISA and bead-based assays are time-consuming, resource-intensive, and not easily integrated into clinical manufacturing, delaying drug development and patient access.
Purpose of the Study:
- To develop and validate a rapid, efficient, and versatile method for determining the target-binding fraction of labeled biologics.
- To overcome the limitations of existing analytical techniques in quality control for theranostic agents.
- To streamline the development and manufacturing workflow for precision oncology therapeutics.
Main Methods:
- Development and validation of a size-exclusion high-performance liquid chromatography (SE-HPLC) method.
- Separation of unbound biologics from larger antigen-bound complexes for quantification.
- Validation using fluorescently labeled antibodies (panitumumab-IRDye800CW, nivolumab-IRDye800CW) and radiolabeled biologics ([18F]GEH200521, [18F]NOTA-ABY-030).
Main Results:
- 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 assay demonstrated high specificity and a total analysis time of less than 35 minutes, significantly faster than traditional methods.
Conclusions:
- SE-HPLC offers a rapid, specific, and cost-effective alternative for assessing binding fractions, reducing quality control timelines from weeks/hours to minutes.
- The method's compatibility with both fluorescent and radiolabeled biologics simplifies workflows.
- Integration with existing HPLC infrastructure represents a significant advancement for theranostic agent development.
