Researchers have developed and validated a rapid size-exclusion high-performance liquid chromatography technique capable of assessing the target-binding fractions of molecular imaging agents and targeted therapeutics in less than thirty-five minutes. This analytical approach, detailed in EJNMMI Radiopharmacy and Chemistry, addresses a major operational bottleneck in the clinical translation and manufacturing quality control of labeled biologics. The method relies on size-exclusion chromatography to separate unbound biologics from larger antigen-bound molecular complexes, enabling accurate and rapid quantification of binding potency. In the expanding field of precision oncology, where the global theranostics market is projected to surpass twenty-five billion dollars by 2030, regulatory authorities such as the United States Food and Drug Administration and the European Medicines Agency mandate rigorous proof of functional binding integrity. Determining that a conjugated biologic retains its capacity to bind its intended target represents a mandatory product release criterion, directly safeguarding therapeutic efficacy and patient safety across clinical manufacturing pipelines.
Current release testing protocols for targeted therapeutics and diagnostic companions frequently rely on conventional bioassays that impose severe logistical constraints on manufacturing facilities. Standard reactivity assays such as the enzyme-linked immunosorbent assay involve multiple labor-intensive incubation, blocking, and washing stages, often requiring either specialized laboratory resources or costly outsourcing to contract research organizations. For radiolabeled compounds, the cell-based Lindmo assay has served as a primary analytical benchmark, yet it depends on dedicated cell culture infrastructure, highly trained personnel, and extensive preparation time. Newer bead-based assays, although offering modest throughput improvements, still require complex multi-step procedures and specialized instrumentation that restrict their practical adoption in regulated manufacturing cleanrooms. Delays resulting from these prolonged analytical timelines can significantly postpone clinical trial initiation, drive up drug development expenditures, and slow down patient access to critically needed therapeutic and diagnostic agents entering the clinic.
To resolve these analytical obstacles, the team evaluated two size-exclusion columns to separate unbound panitumumab-IRDye800CW from its epidermal growth factor receptor complex. Initial assessment using a silica Biozen 1.8-micrometer dSEC-2 column (200 angstroms) primed with potassium phosphate and potassium chloride mobile phase achieved retention times of 7.2 plus or minus 0.1 minutes for unbound antibody and 5.7 plus or minus 0.1 minutes for the complex at 0.35 milliliters per minute. Although peak symmetry was high with tailing factors below 1.2, the resolution of 1.8 offered limited margin for method robustness. In contrast, a Superdex 200 Increase 10/300 GL column using a phosphate-buffered saline and sodium azide mobile phase demonstrated superior performance. On this column, unbound antibody eluted at 14.1 plus or minus 0.1 minutes while the complex eluted at 10.4 plus or minus 0.1 minutes at 0.8 milliliters per minute, achieving an improved resolution of 3.2.
The linearity of the method was established by evaluating samples prepared across varying molar ratios of epidermal growth factor receptor to panitumumab-IRDye800CW. The antigen volume remained constant while increasing quantities of labeled antibody were added before injection. Linear regression analysis across ratios from 0.25-to-1 up to 1-to-1 demonstrated an excellent linear relationship, yielding a coefficient of determination of 0.999 with p less than 0.001. The regression slope reached 94.2 plus or minus 1.1 percent, closely matching the theoretical value of 100 percent, while the y-intercept of minus 1.8 plus or minus 0.7 percent was not significantly different from zero (p = 0.052), indicating an absence of systematic bias. Triplicate testing confirmed reproducibility across runs, and complete saturation occurred at an antigen-to-antibody ratio of 1.3-to-1, where the antibody was 100 percent bound.
The researchers further demonstrated the robustness of the chromatographic protocol by examining whether variations in biologic reactant concentration influenced the resulting binding measurements. Testing panitumumab-IRDye800CW across a broad concentration range between 0.7 and 11.1 milligrams per milliliter revealed no statistically significant differences in the measured binding fraction. This concentration independence provides a crucial advantage for quality control workflows, particularly when evaluating radiolabeled agents that are frequently formulated and analyzed at microgram or nanogram concentrations per milliliter. To substantiate the chromatographic findings against an established standard, samples were evaluated using a conventional enzyme-linked immunosorbent assay protocol through an external contract research organization. The fluorescently labeled panitumumab exhibited an EC50 binding potency of 5 plus or minus 2.5 nanograms per milliliter, which proved directly comparable to unconjugated panitumumab, confirming that the fluorophore conjugation process left the critical target-binding region structurally intact and validating the model biologic.
Assay specificity was verified through a series of negative control experiments designed to detect non-specific interactions between unrelated antibodies and antigens. When panitumumab-IRDye800CW was incubated in the presence of non-target antigens, specifically human epidermal growth factor receptor 2 and programmed death-ligand 1, chromatography detected no binding whatsoever, with 100 percent of the fluorescent signal remaining exclusively in the unbound fraction. Reciprocal control experiments yielded equally definitive outcomes; incubating the epidermal growth factor receptor antigen with non-target antibodies, including the anti-amyloid monoclonal antibody lecanemab-IRDye800CW and an isotype control IgG2-IRDye800CW antibody, showed no observable complex formation or chromatographic peak shifts. These negative controls confirmed that chromatographic retention changes reflect genuine, highly selective molecular recognition events rather than non-specific physical aggregation or non-selective column interactions during size-exclusion separation.
To confirm that the size-exclusion protocol applies across distinct therapeutic immunoglobulins, the authors extended their methodology to nivolumab-IRDye800CW, a clinical monoclonal antibody targeting programmed cell death protein 1. Upon incubation of the fluorescently labeled nivolumab with its target antigen, the resulting antibody-antigen complex eluted at 12.2 plus or minus 0.1 minutes, while the unbound antibody eluted at 14.3 plus or minus 0.1 minutes, mirroring the baseline separation pattern observed with the panitumumab system. Chromatographic quantification revealed that 100 percent of the nivolumab-IRDye800CW was bound to the programmed cell death protein 1 antigen. This complete binding demonstrated that the size-exclusion analytical framework functions reliably for different fluorescently tagged therapeutic antibodies without requiring custom development of unique mobile phase systems or extensive re-engineering of chromatographic parameters.
The versatility of the chromatographic method was further established using radiolabeled molecular imaging agents representing fundamentally different molecular scaffolds, including a nanobody and an affibody. The researchers evaluated the immunoreactivity of [18F]GEH200521, a fluorine-18 radiolabeled nanobody directed against CD8. Baseline purity testing of [18F]GEH200521 identified a primary retention peak at 8.28 plus or minus 0.17 minutes, representing 92.04 plus or minus 4.14 percent of the sample. Incubation with recombinant CD8 antigen shifted the retention time to 5.57 plus or minus 0.04 minutes for the complex, while the unbound tracer appeared at 8.63 plus or minus 0.13 minutes. When evaluating the bound and unbound fractions, the method determined an immunoreactive binding fraction of 97.55 plus or minus 0.83 percent, with 2.45 plus or minus 0.83 percent remaining unbound. In parallel testing, the method measured the binding fraction of [18F]NOTA-ABY-030, an affibody radiopharmaceutical targeting the epidermal growth factor receptor, yielding an 89.67 percent binding fraction with a standard deviation of 1.46 percent.
The operational setup leverages standard high-performance liquid chromatography equipment, specifically testing on an Agilent 1260 Infinity II LC System and a Lablogic LOGI-Chrom system equipped with multi-wavelength ultraviolet-visible and in-line gamma detectors. Wavelength selection plays an essential role in analytical accuracy. While the conventional 280-nanometer detection channel captures all protein species indiscriminately, including excess uncomplexed antigen, monitoring at 780 nanometers enables selective quantification of optically labeled biologics without background interference. Similarly, in-line gamma radiation detection exclusively measures the radiolabeled tracer. Sample preparation requires minimal manipulation: five microliters of fluorescent agent or one hundred microliters of radiolabeled compound are mixed with antigen in a microcentrifuge tube, vortexed for three to five seconds at ambient room temperature between 20 and 25 degrees Celsius, and transferred into an autosampler vial. With sample preparation completed within five minutes and chromatographic runs lasting twenty to thirty minutes, total analysis time finishes well under thirty-five minutes.
From an economic and operational perspective, the size-exclusion chromatography strategy introduces substantial efficiency gains for manufacturing centers. The estimated per-sample analytical cost for the chromatographic method is approximately $75, representing a sharp reduction compared to in-house enzyme-linked immunosorbent assays, which range between $400 and $600 per sample, and outsourced commercial assays that cost from $1,200 to $2,500. Because standard liquid chromatography infrastructure is already ubiquitous within certified current Good Manufacturing Practice facilities, adopting this approach requires negligible additional capital investment. Furthermore, the rapid thirty-five-minute turnaround time supports same-day product release strategies, which are particularly indispensable for short-lived radiopharmaceuticals where radioactive decay renders multi-hour or multi-day testing methodologies practically unviable. By removing specialized cell-culture requirements and shortening analytical timelines, the cell-free protocol substantially lowers translation barriers for academic laboratories and biotechnology developers.
The authors also outlined several methodological considerations and study limitations that define the current operational scope of the assay. Because all experimental evaluations were conducted within a single research laboratory, formal inter-laboratory validation studies will be essential to establish reproducibility and promote widespread regulatory adoption. Additionally, while the chromatographic method was benchmarked against standard enzyme-linked immunosorbent assay data, the researchers noted that a direct head-to-head comparison against the traditional cell-based Lindmo assay was not performed for the same radiolabeled compound. Furthermore, chromatographic flow through size-exclusion columns can potentially perturb binding equilibria, causing partial dissociation of lower-affinity ligand-target complexes during separation. While any complex dissociation on the column would lead to an underestimation rather than an overestimation of the binding fraction, optimal quantification depends on high-affinity interactions. Future studies will need to define the precise affinity boundaries of the method to ensure reliable implementation across diverse molecular probes.
Subject of Research: Rapid chromatographic assessment of target-binding fractions in molecular imaging agents and targeted biologics
Article Title: Rapid assessment of target-binding fractions in molecular imaging agents using size-exclusion HPLC
Article References: McAdoo, A., Jouad, K., Rosenthal, E. L., & Rosenberg, A. J. (2026). Rapid assessment of target-binding fractions in molecular imaging agents using size-exclusion HPLC. EJNMMI Radiopharmacy and Chemistry, 11(1), Article 71. https://doi.org/10.1186/s41181-026-00469-5
Image Credits: AI Generated
DOI: 10.1186/s41181-026-00469-5
Keywords: size-exclusion chromatography, molecular imaging, theranostics, quality control, target-binding fraction, radiolabeled biologics, panitumumab, high-performance liquid chromatography, Rapid, assessment, target-binding, fractions
News Source: Bethany Barker. (November 11, 2026). Size-Exclusion HPLC Accelerates Target-Binding Quality Control for Molecular Imaging Agents. Scienmag.
