*2.3. Automation Adopted for <sup>89</sup>Zr Separation and Purification*

A mini AlliOne (miniAiO) cassette-based automatic synthesis unit (ASU) from TRASIS (Ans, Belgium), with dimensions 21.5W × 41.2H × 40.8D cm, was used for the automated separation and purification of <sup>89</sup>Zr (Figure 1). The miniAiO consists of 12 rotary actuators and 2 linear actuators for operating syringes. All liquids were transferred through PEEK tubing. All tube connections were assembled using non-metallic connectors to reduce exposure to the metals which may affect radionuclidic purity and specific activity. Many of the liquid connection pieces were flangeless fittings designed for high pressure fluidic connection and ferrules are manufactured from tefzel™ (ETFE). The fluidic path was controlled using the built-in three-way valve actuators. All liquids

and hydroxamate resin cartridge were preloaded onto the cassette prior to starting the sequence on the miniAiO. hydroxamate resin cartridge were preloaded onto the cassette prior to starting the sequence on the miniAiO.

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**Figure 1.** TRASIS mini AlliOne (miniAiO) setup for purification of zirconium-89. **Figure 1.** TRASIS mini AlliOne (miniAiO) setup for purification of zirconium-89.

The miniAiO ASU was located within a lead hot cell and remotely controlled through a graphical user interface (PC). A sequence was created on the TRASIS software using a graphical user interface which distributes commands and controls to the automation unit. The operator can initiate a preprogrammed 89Zr target dissolution and purification process. Radioactive detectors are built-in at different locations within the ASU to monitor and record the location of the radioactivity. The radioactivity in the dissolution vial, hydroxamate cartridge and final vial was monitored. The miniAiO ASU was located within a lead hot cell and remotely controlled through a graphical user interface (PC). A sequence was created on the TRASIS software using a graphical user interface which distributes commands and controls to the automation unit. The operator can initiate a pre-programmed <sup>89</sup>Zr target dissolution and purification process. Radioactive detectors are built-in at different locations within the ASU to monitor and record the location of the radioactivity. The radioactivity in the dissolution vial, hydroxamate cartridge and final vial was monitored.

#### *2.4. Target dissolution and Purification of 89Zr 2.4. Target Dissolution and Purification of <sup>89</sup>Zr*

#### 2.4.1. TRASIS MiniAiO Preparation TRASIS MiniAiO Preparation

Automation of the process for separation and purification of 89Zr, based on chemistry previously reported in the literature, was performed using a miniAiO ASU in a hot cell [1,12,16]. The miniAiO is a commonly used radiochemistry module for clinical GMP-grade radiopharmaceutical production. The ASU is designed for use with disposable kits and allows processing with full audit trail functionality for GMP production runs. Liquid transport was achieved using a syringe pump and transfer of liquids was controlled by three-way stopcock valves. For each liquid transfer step, the production protocols were adequately adapted and optimized. In brief, the following general steps were implemented in the production of 89Zr. The hydroxamate resin (100 mg) was packed into an empty reversible SPE tube (0.5 mL). The hydroxamate resin was preconditioned with acetonitrile (MeCN) (8 mL, trace metal grade), water (15 mL, trace metal grade) and 2.0 M HCl (2 mL) and installed on the cassette (valve #10) of the miniAiO ASU (Figure 2). The following vials were installed on the cassette: 2 M HCl (20 mL) in a glass vial with septa installed at cassette valve #4, water (10 mL) in a glass vial with septa installed at cassette valve #5, 1 M oxalic acid (1.5 mL) in a glass vial with septa installed at cassette valve #6, a waste vial (40 mL) connected to cassette valve #7 and a [89Zr]zirconium oxalate product vial (5 mL) connected to cassette valve #11. Automation of the process for separation and purification of <sup>89</sup>Zr, based on chemistry previously reported in the literature, was performed using a miniAiO ASU in a hot cell [1,12,16]. The miniAiO is a commonly used radiochemistry module for clinical GMP-grade radiopharmaceutical production. The ASU is designed for use with disposable kits and allows processing with full audit trail functionality for GMP production runs. Liquid transport was achieved using a syringe pump and transfer of liquids was controlled by three-way stopcock valves. For each liquid transfer step, the production protocols were adequately adapted and optimized. In brief, the following general steps were implemented in the production of <sup>89</sup>Zr. The hydroxamate resin (100 mg) was packed into an empty reversible SPE tube (0.5 mL). The hydroxamate resin was preconditioned with acetonitrile (MeCN) (8 mL, trace metal grade), water (15 mL, trace metal grade) and 2.0 M HCl (2 mL) and installed on the cassette (valve #10) of the miniAiO ASU (Figure 2). The following vials were installed on the cassette: 2 M HCl (20 mL) in a glass vial with septa installed at cassette valve #4, water (10 mL) in a glass vial with septa installed at cassette valve #5, 1 M oxalic acid (1.5 mL) in a glass vial with septa installed at cassette valve #6, a waste vial (40 mL) connected to cassette valve #7 and a [89Zr]zirconium oxalate product vial (5 mL) connected to cassette valve #11.

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**Figure 2.** TRASIS miniAiO layout for automated purification and isolation of 89Zr from irradiated yttrium coin. Valve 1 to dissolution vial; valve 3, 10 mL syringe; valve 4 (**A**) 2 M HCl (20 mL); valve 5 (**B**) water (10 mL); valve 6 (**C**) 1 M oxalic acid (1.5 mL); valve 10 hydroxamate resin; valve 11 to product vial. **Figure 2.** TRASIS miniAiO layout for automated purification and isolation of <sup>89</sup>Zr from irradiated yttrium coin. Valve 1 to dissolution vial; valve 3, 10 mL syringe; valve 4 (**A**) 2 M HCl (20 mL); valve 5 (**B**) water (10 mL); valve 6 (**C**) 1 M oxalic acid (1.5 mL); valve 10 hydroxamate resin; valve 11 to product vial.

The irradiated target was transferred to the custom-made dissolution vial and the vial was placed inside the aluminium block heater on a hot plate that had been pre-heated to 80 °C. After placing the vented glass cover with attached PEEK tubing on the dissolution vial, the hot cell was closed, and the purification sequence was initiated using the program interface for the miniAiO. Hydrochloric acid (2.0 M, 4 mL) was drawn into the syringe and pushed into the dissolution vial to dissolve the target. The target solution was heated at 80 °C for 20 min, and after cooling for 20 min, was passed through the hydroxamate resin using the syringe actuator at 1 mL/min flowrate to maximize trapping of 89Zr on the resin. The dissolution vial was rinsed with 2 M HCl (2 mL) and passed through the hydroxamate resin again at 1 mL/min flowrate. The 89Zr remained trapped on the resin as indicated by the in-process monitoring of the radiation detector at the column. The resin was washed with 2 M HCl (14 mL) at 3 mL/min flowrate followed by water (10 mL) at 3 mL/min flowrate. Finally, the 89Zr was eluted with 1 M oxalic acid solution (1.5 mL) at 0.5 mL/min flow rate and The irradiated target was transferred to the custom-made dissolution vial and the vial was placed inside the aluminium block heater on a hot plate that had been pre-heated to 80 ◦C. After placing the vented glass cover with attached PEEK tubing on the dissolution vial, the hot cell was closed, and the purification sequence was initiated using the program interface for the miniAiO. Hydrochloric acid (2.0 M, 4 mL) was drawn into the syringe and pushed into the dissolution vial to dissolve the target. The target solution was heated at 80 ◦C for 20 min, and after cooling for 20 min, was passed through the hydroxamate resin using the syringe actuator at 1 mL/min flowrate to maximize trapping of <sup>89</sup>Zr on the resin. The dissolution vial was rinsed with 2 M HCl (2 mL) and passed through the hydroxamate resin again at 1 mL/min flowrate. The <sup>89</sup>Zr remained trapped on the resin as indicated by the in-process monitoring of the radiation detector at the column. The resin was washed with 2 M HCl (14 mL) at 3 mL/min flowrate followed by water (10 mL) at 3 mL/min flowrate. Finally, the <sup>89</sup>Zr was eluted with 1 M oxalic acid solution (1.5 mL) at 0.5 mL/min flow rate and collected in a sterile 5 mL product vial.
