Fully automated [68Ga]Ga-FAPI-46 radiosynthesis using GalliAd® generator and MiniAIO® synthesizer: development and validation for PET imaging of fibroblast activation protein

Fibroblast activation protein (FAP) is a membrane-bound serine protease that is selectively overexpressed on cancer-associated fibroblasts (CAFs) in more than 90% of epithelial tumors. [68Ga]Ga labeled FAP inhibitors (FAPI) have emerged as promising PET tracers for oncological imaging and theranostic applications. [68Ga]Ga-DOTA-FAPI-46 (simplified as [68Ga]Ga-FAPI-46) is a DOTA-chelated FAPI that demonstrates superior tumor retention compared to earlier FAPI compounds. We describe the development, optimisation, and pharmaceutical validation of a combination not previously reported for the production of this radiopharmaceutical: a fully automated, one-step radiosynthesis of [68Ga]Ga-FAPI-46 without pre-purification of the generator eluate, performed at the hospital radiopharmacy of the Groupement Hospitalier Sud, Hospices Civils de Lyon, in the context of a clinical trial (EudraCT 2025-524897-40-00). Radiolabeling was performed via a miniAIO automated synthesizer (Trasis) and a GalliAd [68Ge]Ge/[68Ga]Ga generator (IRE-Elit). The process generator elution, direct radiolabeling at 95 °C for 7 min in sodium acetate buffer with L-ascorbic acid, HLB solid-phase purification and 0.22 μm sterilizing filtration was performed in a single uninterrupted automated sequence. The quality control testing included release tests (visual appearance, final volume, radioactivity, pH, radiochemical purity by radio-TLC and radio-HPLC, radionuclidic identity, filter integrity, and bacterial endotoxin testing), post-release tests (radionuclidic purity and sterility), and validation batch-only tests (L-ascorbic acid and ethanol content). Analytical methods were validated as per ICH Q2(R2). All the validation batches met the predefined release and post-release specifications. The decay-corrected radiochemical yields ranged from 66.9% to 86.0%, with a total synthesis time of 20 min. Radiochemical purity exceeded the acceptance threshold of 95.0% by radio-TLC and radio-HPLC at release. It remained ≥ 95.0% after 3 h for both control methods. No radiolytic impurities were detected. Radionuclidic purity was confirmed with [68Ge]Ge breakthrough < 0.001%, and the measured half-life values (66.0-69.0 min) were consistent with those of [68Ga]Ga. Endotoxin levels were < 1.0 IU/ml and all batches were sterile. Ethanol (43.6 ± 2.4 mg/ml - 5.5 ± 0.3% v/v) and L-ascorbic acid (7.5 ± 1.4 g/l) contents complied with predefined specifications. All analytical methods fulfilled the ICH Q2(R2) criteria, demonstrating adequate specificity, sensitivity and reproducibility. A fully validated one-step automated radiosynthesis of [68Ga]Ga-FAPI-46 was established via the use of a miniAIO synthesizer with a [68Ge]Ge/[68Ga]Ga generator. The process consistently produced a high-quality radiopharmaceutical suitable for clinical implementation and IMPD submission in FAP-targeted PET imaging. Trial registration EudraCT No 20,255,248,974,000 Clinical trials registry NCT07313033 15/01/2026.