cGMP-compliant high-yield automated production of [¹⁸F]AlF-FAPI-74: optimization of quality control and evaluation of molar dose impact.

Abstract

Background Fibroblast activation protein inhibitor (FAPI)-based positron emission tomography (PET) radiopharmaceuticals have shown promise for imaging cancer-associated fibroblasts (CAFs), a key component of the tumor microenvironment. FAPI radiopharmaceuticals offer high tumor-to-background contrast and are not influenced by hyperglycemia. Among these, [¹⁸F]AlF-FAPI-74, labeled using the Al¹⁸F-method, offers logistical advantages over ⁶⁸Ga-labeled radiopharmaceuticals, including a longer physical half-life and suitability for large-scale, centralized production. This study reports a fully automated, efficient, and GMP-compliant synthesis of [¹⁸F]AlF-FAPI-74 using the Trasis AllInOne ® platform, alongside a refined isocratic radio-HPLC method that enhances fluorine-18 recovery and impurity resolution. Additionally, the impact of molar dose on [¹⁸F]AlF-FAPI-74 biodistribution is evaluated in a preclinical model and a clinical case-study highlighting the performance of [¹⁸F]AlF-FAPI-74 produced at high apparent molar activity is provided. Results The automated GMP-compliant production was validated in three independent runs, with an average decay-corrected yield and apparent molar activity of 50 ± 10% and 1124 ± 254 GBq/µmol at the end of synthesis, respectively. Total synthesis time was 30 min. Quality control used validated analytical methods, including an optimized radio-HPLC protocol, ensuring regulatory compliance and batch consistency. [¹⁸F]AlF-FAPI-74 was produced with a radiochemical purity ≥ 95% and demonstrated excellent radiochemical stability in its final formulation for at least 10 h post-synthesis, at a concentration of 2019 MBq/mL. The in vitro binding kinetics of [¹⁸F]AlF-FAPI-74 were evaluated in a HEK293 cell line stably expressing human FAP, demonstrating rapid, specific uptake and internalization, along with high target affinity. In vivo biodistribution studies revealed a dose-dependent "molar amount effect," with doses > 30 nmol/kg yielding improved tumor-to-background ratios. This phenomenon was not observed in clinical imaging, where high molar activity supported excellent image contrast. Conclusions A robust, fully automated, and GMP-compliant production process for [¹⁸F]AlF-FAPI-74 is reported with high yield, purity, and stability, suitable for centralized production and distribution. The improved radio-HPLC method enhances quality control precision by accurately quantifying radiochemical and chemical purity. In vivo experiments confirmed fast tumor uptake of [¹⁸F]AlF-FAPI-74. While a clear mass effect on tumor-to-background ratios was observed in preclinical studies, high apparent molar activity resulted in excellent contrast in a clinical setting.