Automated cassette-based production, purification, and radiolabeling of [89Zr]Zr-DFO-trastuzumab

Introduction Human epidermal growth factor receptor 2 (HER2)-positive breast cancer is an aggressive subtype of breast cancer in which accurate, non-invasive assessment of HER2 expression is critical for the stratification of patients and monitoring of treatment. Immuno-positron emission tomography using monoclonal antibodies radiolabeled with long-lived positron emitters, such as [ 89 Zr]zirconium, enables whole-body visualization and quantification of HER2 expression. This study aimed to develop and validate a fully integrated, sequential, and automated cassette-based synthesis process for the production, purification, and radiolabeling of [ 89 Zr]Zr-desferrioxamine (DFO)-trastuzumab for potential clinical application. Materials and methods In this study, [ 89 Zr]zirconium was produced via cyclotron irradiation of sintered [ 89 Y]yttrium solid targets, followed by purification using a hydroxamate-based solid-phase extraction (SPE) system. The resulting [ 89 Zr]Zr(C 2 O 4 ) 2 was used to radiolabel trastuzumab preconjugated with the chelator DFO, and the final radiopharmaceutical was purified via PD-10 size-exclusion chromatography. The automated workflow integrated target dissolution, [ 89 Zr]zirconium capture and elution, buffer adjustment, antibody radiolabeling, PD-10 purification, and sterile filtration within a single disposable cassette installed on an AllinOne synthesis module. The principal process parameters investigated were target dissolution time, acid concentration and washing volumes, [ 89 Zr]zirconium elution conditions, reaction temperature, reaction time, antibody concentration, and starting activity. Preliminary manual radiolabeling experiments were performed at 37 °C to define the radiolabeling conditions, whereas the subsequent automated radiolabeling reaction was performed at 30 °C for 60 min. Radiochemical purity (RCP) and radiochemical yield (RCY) were evaluated via high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC). All steps were integrated into a single-use cassette installed on a commercial synthesis module to minimize manual handling. Results Target dissolution and SPE purification yielded [ 89 Zr]Zr(C 2 O 4 ) 2 with an average recovery of approximately 90% and high radionuclidic purity. In preliminary manual experiments, radiolabeling yields of approximately 90% were obtained after 60 min. In contrast, four subsequent automated production runs using starting activities ranging from 37 to 185 MBq showed isolated radiochemical yields ranging from 65% to 78%. The HPLC radiochemical purity was 100% in all automated production runs, while TLC analysis of the final product showed 99%–100% radiochemical purity. Following PD-10 purification, the final product showed 99%–100% radiochemical purity in TLC and a pH of approximately 7. The automated cassette-based workflow demonstrated reproducible radiochemical purity and reduced direct operator handling of the radioactive material, while the lower isolated automated RCY indicated that activity recovery and fluidic transfer efficiency remain areas for further optimization. Discussion and conclusion The developed cassette-based system demonstrated the feasibility of integrating [ 89 Zr]zirconium target dissolution and purification with subsequent radiolabeling and purification of preconjugated DFO-trastuzumab in a single automated sequence. Although the overall automated RCY was lower than the labeling yields obtained in preliminary manual experiments, the consistently high RCP indicates that the main opportunity for further optimization lies in overall activity recovery and fluidic transfer efficiency rather than in the chemical purity of the final product. This process reduces direct operator handling of radioactive material and provides a standardized platform that may be further developed into a Good Manufacturing Practice–compliant production method for [ 89 Zr]-labeled monoclonal antibodies.