[89Zr]ZrCl4 for direct radiolabeling of DOTA-based precursors.

INTRODUCTION: Zirconium-89 (89Zr) is a positron emitter with several advantages over other shorter-lived positron emission tomography (PET) compatible radiometals such as gallium-68 or copper-64. These include practically unlimited availability, extremely low cost, greatly facilitated distribution logistics, positron energy fit for medical PET imaging, and sufficiently long physical half-life to enable PET imaging at later time points for patient-specific dosimetry estimations. Despite these apparent benefits, the reception of 89Zr in the nuclear medicine community has been tepid. The driving factor for the absence of broader adaptation is mostly routed in its final formulation - [89Zr]zirconium oxalate. While serving as a suitable precursor solution for the gold standard chelator deferoxamine (DFO), [89Zr]Zr-oxalate is inaccessible for the most commonly used chelators, such as the macrocyclic DOTA, due to its pre-chelated state. Consequently, pioneering work has been conducted by multiple research groups to create oxalate-free forms of [89Zr]Zr4+, either via chemical conversion of oxalate into other counterion forms or via direct radiochemical isolation of [89Zr]ZrCl4, showing that [89Zr]Zr-DOTA complexes are possible and stable. However, this success was accompanied by challenges, including complex and labor-intensive radiochemical processing and radiolabeling procedures as well as the relatively minuscule conversion rates. Here, we report on the direct production of [89Zr]ZrCl4 avoiding oxalate and metal contaminants to enable efficient radiolabeling of DOTA constructs. METHODS: We based our direct production of [89Zr]ZrCl4 on previously reported methods and further optimized its quality by including an additional iron-removing step using the TK400 Resin. Here, we avoided using oxalic acid and effectively minimized the content of trace metal contaminants. Our two-step purification procedure was automated, and we confirmed excellent radionuclide purity, minimal trace metals content, great reactivity over time, and high specific molar activity. In addition, DOTA-based PSMA-617 and DOTAGA-based PSMA-I&T were radiolabeled to demonstrate the feasibility of direct radiolabeling and to estimate the maximum apparent specific activities. Lastly, the biodistribution of [89Zr]Zr-PSMA-617 was assessed in mice bearing PC3-PIP xenografts, and the results were compared to the previously published data. RESULTS: A total of 18 batches, ranging from 6.9 to 20 GBq (186 to 541 mCi), were produced. The specific molar activity for [89Zr]ZrCl4 exceeded 0.96 GBq (26 mCi) per nanomole of zirconium. The radionuclidic purity was >99 %, and the trace metals content was in the <1 ppm range. The [89Zr]ZrCl4 remained in its reactive chemical form for at least five days when stored in cyclic olefin polymer (COP) vials. Batches of 11.1 GBq (300 mCi) of [89Zr]Zr-PSMA-617 and 14.4 GBq (390 mCi) of [89Zr]Zr-PSMA-I&T, corresponding to specific activities of 11.1 MBq/µg (0.3 mCi/µg), and 14.4 MBq/µg (0.39 mCi/µg), respectively, were produced. [89Zr]Zr-PSMA-617 animal PET imaging results were in agreement with the previously published data. CONCLUSION: In this work, we report on a suitable application of TK400 Resin to remove iron during [89Zr]ZrCl4 radiochemical isolation. The breakthrough allows for direct radiolabeling of DOTA-based constructs with [89Zr]ZrCl4, leading to high apparent molar activities and excellent conversion rates.

A rapid bead-based radioligand binding assay for the determination of target-binding fraction and quality control of radiopharmaceuticals.

INTRODUCTION: Determination of the target-binding fraction (TBF) of radiopharmaceuticals using cell-based assays is prone to inconsistencies arising from several intrinsic and extrinsic factors. Here, we report a cell-free quantitative method of analysis to determine the TBF of radioligands. METHODS: Magnetic beads functionalized with Ni-NTA or streptavidin were incubated with 1 µg of histidine-tagged or biotinylated antigen of choice for 15 min, followed by incubating 1 ng of the radioligand for 30 min. The beads, supernatant and wash fractions were measured for radioactivity on a gamma counter. The TBF was determined by quantifying the percentage of activity associated with the magnetic beads. RESULTS: The described method works robustly with a variety of radioisotopes and class of molecules used as radioligands. The entire assay can be completed within 2 h. CONCLUSION: The described method yields results in a rapid and reliable manner whilst improving and extending the scope of previously described bead-based radioimmunoassays. ADVANCES IN KNOWLEDGE: Using a bead-based radioligand binding assay overcomes the limitations of traditional cell-based assays. The described method is applicable to antibody as well as non-antibody based radioligands and is independent of the effect of target antigen density on cells, the choice of radioisotope used for synthesis of the radioligand and the temperature at which the assay is performed. IMPLICATIONS FOR PATIENT CARE: The bead-based radioligand binding assay is significantly easier to perform and is ideally suited for adoption by the radiopharmacy as a quality control method of analysis to fulfill the criteria for release of radiopharmaceuticals in the clinic. The use of this assay is likely to ensure a more reliable validation of radiopharmaceutical quality and result in fewer failed doses, which could ultimately translate to an efficient release of radiopharmaceuticals for administration to patients in the clinic.