This method can help researchers in the fields of biology, radiochemistry, and nuclear medicine, to automate the production of short-lived positron-emission tomography tracers for basic research and clinical applications. The main advantages of automating radiochemical synthesis are to standardize the synthesis process, to enable repeat production, to improve synthesis reliability, and to protect the chemist from radiation exposure. By using a single flexible radiosynthesizer, multiple different positron-emission tomography, or PET tracers, suitable for clinical use, can be produced within a single hot cell.
To ensure the success of the automated synthesis, it is critical to take great care during the setup procedure before the radionuclide is introduced into the radiosynthesizer. To create an automated synthesis program for the PET tracer fluorine-18 labeled clofarabine, or CFA, the precursor is first reacted with dried activated fluoride-18 to form the intermediate followed by removal of the protecting groups to form the final compound. Use a paper and pen to divide the manual synthesis into high-level steps, and the high-level steps into discrete basic required processes, then map each process into the individual unit operations provided by the synthesizer software.
Using the radiosynthesizer programming interface, create a blank program by clicking menu, sequences, and new sequence, to program each of the identified unit operations and their parameters in sequence. For fluoride evaporation in unit operation three, drag the evaporation operation to the filmstrip view and enter the reactor used, temperature, duration and desired pressure of nitrogen stream. For precursor addition in unit operation eight, drag the addition operation to the filmstrip view.
For fluorination reaction in unit operation nine, drag a react operation to the filmstrip view and adjust their parameters. To set up the automated synthesis, power on the radiosynthesizer, and taking care that the dip tube of each new disposable cassette is pointed straight down. Install cassettes into the reactor number one and number two positions.
After inserting reaction vessels with magnetic stir bars, install the reagent vials into the cassettes according to the diagram and install an empty oxygen-18 water recovery vial in the W1 position of cassette number one. Connect the QMA cartridges to cassette number one. And the silica cartridge between cassette number one and number two.
Then, connect the output of cassette number two to the high pressure liquid chromatography or HPLC system of the purification module. After verifying that the cassette tubing connections match the schematic, confirm that no cassette tubing hangs in the interior where it may interfere with robotic movements. Connect the fluoride-18 source line from the cyclotron to the fluoride-18 input line on cassette number one.
To equilibrate the purification formulation subsystem before starting the synthesis, select HPLC to enter the purification formulation module control page. By default, the purification tab will already be selected. Set the flow rate to five milliliters per minute at the defined solvent composition.
And set the purification column position. Turn on the HPLC pump in the isocratic mode for at least 10 minutes and rinse the product line and all of the fraction collection lines with the mobile phase for one minute each. Then use a syringe to manually rinse each HPLC sample loop and HPLC sample loop transfer tubing with 10 milliliters of the mobile phase.
To prime the formulation subsystem, open the formulation tab of the purification formulation control page. To prime the concentrated sodium chloride, open the elute tab. Click initialize to initialize the syringe pump and dispense five milliliters of concentrated sodium chloride.
To prime the 0.9%saline, select the reconstitute tab and dispense five milliliters of saline. Next, connect the product and final product lines from the front of the purification formulation subsystem in a T-connection. Connect the output of the T-connection to a preassembled, vented, sterile, empty vial with filters and place the vial into a shielded lead pig.
Next, rotate the cassette wing knobs to secure the cassettes in place. Remove the Dewar from the instrument. Empty the cold trap, and add alcohol into the Dewar, followed by the slow addition of dry ice.
Finally, install the cold trap and Dewar back into the synthesizer and close the hot cell door. To run the synthesis program, open the sequences tab, select the fluoride-18 CFA program and click, run. Carefully review each item on the pre-run checklist, checking off each item as it is completed.
Then click, continue in the software to confirm that the setup is complete and to begin the automated synthesis. Before clicking continue, carefully review the setup to make sure everything is properly connected. Once the radioactivity is delivered into the synthesizer, no further manual manipulations are possible because of the radiation field within the hot cell.
During the fluoride-18 trapping operation, a pop-up will appear when it is time to deliver activity from the cyclotron. When this occurs, deliver the fluoride-18 from the cyclotron and monitor the radiation sensor to confirm the fluoride has been trapped on the QMA cartridge. Click continue to continue the automated program and monitor the synthesis in real-time via visual feedback, sensor readings and countdown timers.
In unit operation three, the liquid in the reaction vessel is evaporated to dry and activate the F-18 fluoride. The temperature, remaining time and remaining liquid level can be monitored on the interface. In unit operation eight, the precursor solution vial is picked up and moved to the loading position of cassette number one.
Upon which, the contents are delivered to the reaction vessel. The remaining time and liquid level in the reaction vessel can be monitored on the interface. In unit operation nine, the reaction vessel is sealed and heated to perform the fluorination reaction.
The temperature and remaining time, as well as a live video stream of the reaction vessel contents can be monitored on the interface. In unit operation 10, the dip tube is inserted into the reaction vessel and the contents are transferred through the silica cartridge for purification of the intermediate. The remaining time, level of remaining liquid in the reaction vessel and the radiation detector adjacent to the cartridge can be monitored on the interface.
During the final purification unit operation step when the product peak has begun to appear on the radiation detector chromatogram, select, product. Once the radiation detector chromatogram peak has returned to the baseline, select, waste to divert the flow path of the HPLC subsystem to the waste container. To set up the formulation program, under the sequence tab, open the fluoride-18 CFA formulation program and run the program.
The system will dilute the collected purified product fraction in the final sterile product vial through a sterilizing filter and dilute with sodium chloride and saline to ensure the isotonicity of the formulation. To retrieve the final product, open the hot cell door, disconnect the needles from the product vial and remove the formulated product vial from the hot cell. Then, using aseptic procedures, remove a sample to perform the necessary quality control testing.
To shut down the synthesizer, click the power button. A pop-up window will indicate when the power to the system can be turned off. Then close the appropriate shutoff valves to turn off the compressed air and inert gas supplies, allowing time for the residual radioactivity in the hot cell to decay to safe levels before performing another synthesis.
The obtained fluorine-18 CFA formulations passed all of the quality control tests. In these representative validation runs, the synthesis, purification and formulation were achieved in 110 minutes on average and the non-decay corrected radiochemical yield was nearly 8%While using this procedure to produce tracers for clinical use, it is necessary to create written, standard operating procedures that must be carefully followed to ensure that no setup or preparation steps are missed. Following this general procedure, the synthesis of many other radiotracers can be easily automated, facilitating the transition to current, good manufacturing practice compliant production for human clinical trials or clinical care.
Don't forget that working with radioactivity can be hazardous. Facility measures such as exposure monitoring, adequate shielding and safety procedures must be in place. Take good care to always work closely together with your radiation safety officer.
