Automated and manual synthesis of [68Ga]Ga-DOTA-4AH29 intended for PET-imaging of FAP-expression

Fibroblast activation protein is a well-established target for tumour imaging, and single-domain antibodies (sdAb) offer favourable pharmacokinetic properties for PET applications. However, the translation of sdAb-based radiotracers into robust and clinically applicable manufacturing processes remains limited. Previously, preclinical protocol for the production of [ 68 Ga]Ga-DOTA-4AH29 has been reported. In this study, this process was adapted for clinical use. Both a manual, kit-based approach and automated synthesis on two different platforms were developed. Process robustness was assessed through a stress study evaluating key parameters, and compatibility with two 68 Ga generator systems was investigated. Efficient radiolabelling was achieved at 50 °C for 10 min, with radiochemical conversion exceeding 90% and no need for purification. Both automated and manual processes showed consistent performance, with radiochemical purities of 99.5 ± 0.5% ( n = 9) and 96.3 ± 1.5% ( n = 8) as determined by iTLC and SE-HPLC respectively, with associated non-decay-corrected radiochemical yields of 78.3 ± 3.3% ( n = 9) and 76.9 ± 2.1% ( n = 8). The main source of activity loss was the radioactive decay of 68 Ga.The process remained stable across a range of activities and buffer conditions. Comparable results were obtained with both 68 Ga generator systems, with no observable impact on product quality. The final product was stable for at least 3 h at RT, with preserved binding capacity. A robust and reproducible manufacturing process for [ 68 Ga]Ga-DOTA-4AH29 was established, supporting both automated and manual clinical production. This approach will facilitate broader access to sdAb-based PET imaging across clinical centres.