An Automated Radiosynthesis of [⁶⁸Ga]Ga-FAPI-46 for Routine Clinical Use

This method presents the automated production of the positron emission tomography or PET tracer gallium-68 FAPI-46 on the iPHASE medicine synthesizer. Gallium-68 FAPI-46 is a novel PET imaging tracer targeting fibroblast activation protein, or FAP. FAP is over-expressed by cancer-associated fibroblasts in the microenvironment of most solid tumors.

Gallium-68 FAPI-46 has been shown to be a very promising PET tracer with a diagnostic advantage over the gold standard fluorine-18 FDG in multiple cancers. Although the manual radio labeling of gallium-68 FAPI-46 has been reported previously, the use of an automated synthesizer is more compliant with GMP and poses a lower radiation risk to personnel. The automated synthesis of gallium-68 FAPI-46 has been described on different commercial synthesizers using generator produced and cyclotron produced gallium-68.

The product was synthesized in 89.8%decay curated yield. The final product met all recommended quality control specifications and was stable for three hours post-synthesis. We anticipate any site manufacturing gallium-68 PSMA-11, or gallium-68 on the MultiSyn synthesizer should be fully equipped to produce gallium-68 FAPI-46 using this protocol.

To prepare the MS synthesizer, turn on the hot cell compressed air, nitrogen gas supply, ventilation, light, and power to the laptop and programmable logic controller. Check the level of the module waste bottle and replace it with an empty bottle if it is three quarters full. After logging on to the synthesizer software, press download recipe.

Select the Excel sequence file for the production of gallium-68 FAPI-46, and press open. Click okay after the sequence has downloaded successfully. Press start, input the batch number, reagent kit lot number, and any comments in the popup window before pressing okay.

Following the step message on the user interface, remove the old hardware kit and press next. Next, per the user interface instruction, remove the generator elution syringe from the synthesizer by pulling the thumb rest out of the syringe driver slot and unscrewing the syringe from the generator inlet line. Press next on the user interface.

To begin, pour approximately two milliliters of water into the cap of a 50 milliliter tube. Draw one milliliter of water in a five milliliter syringe labeled syringe A and cap it with a needle. Pipette 400 microliters of water.

Add it to the seven milligram ascorbic acid vial labeled vial A, and gently shake to dissolve. Open the provided 0.25 molar sodium acetate buffer vial, and the 50 microgram FAPI-46 glass vial. Using a pipette, transfer 0.9 milliliters of the buffer to the FAPI-46 glass vial labeled vial B and mix gently.

Withdraw the contents of vial A and vial B into syringe A and mix gently. Label the five milliliter syringe provided in the ancillary set as syringe B.With syringe B, withdraw five milliliters of 0.1 molar hydrochloric acid from the 0.1 molar hydrochloric acid bag and cap it with the provided dispensing pin. Label the three milliliter syringe provided in the ancillary kit as syringe C.From the acidified five molar sodium chloride solution vial, pipette one milliliter of the solution into syringe C and cap it with the provided dispensing pin.

Withdraw one milliliter of 0.9%saline into a five milliliter syringe labeled syringe D, and add it to vial C.To assemble a dedicated sterile gallium-68 cassette, unwrap the cassette envelope and check for any damage. Tighten each lure connection and rotate and align each stopcock on the cassette to ensure they're not stuck and will fit on the module. Remove all the spike caps.

To condition the strong cationic exchange pre-purification SPE cartridge, remove the cartridge positioned on manifold three valve seven from the cassette. Attach the syringe containing two milliliters of three molar hydrochloric acid provided in the reagent kit onto the SPE cartridge and slowly elute it through the cartridge dropwise into a dedicated glass waste bottle. Remove the syringe, withdraw five milliliters of air and flush the SPE cartridge with it.

Attach the syringe containing five milliliters of water provided in the reagent kit to the SPE cartridge and slowly elute dropwise into the glass waste bottle. Flush the cartridge with air as demonstrated previously. Press next on the user interface.

As shown on the user interface, proceed to assemble and install the new cassette without any reagents following the appropriate connections. First, connect the 10 milliliter syringe from the ancillary set to manifold two valve six. Install the four manifolds and lock the cassette using the synthesizer's magnetic locks.

Place the reactor in the oven. Connect tubings to G1, W2, W1, R, and G2 ports. Connect the manifold one valve three right side tubing to the cation exchange cartridge on manifold three valve seven.

Install an SPE post purification cartridge on manifold two valve four. Finally, press next on the user interface. To begin, remove the caps of the ethanol, saline, and water vials from the reagent kit, and swab each septum with an alcohol wipe.

When prompted by the synthesizer, install the respective vials at positions M4 valve 10, M4 valve 11, and M4 valve 12 before clicking next on the user interface. Remove the needle from syringe A and draw the plunger to five milliliters. After disconnecting the line to the reactor middle port, transfer the content of syringe A into the reactor vial via the reactor middle port.

Reconnect the line to the reactor middle port and click next on the user interface. Install the generator outlet lines on the cassette at position M1 valve two and click next. After removing the dispensing needle from syringe C, install it at position M1 valve three and click next.

Remove the dispensing needle from syringe B.Connect syringe B Luer lock on the generator inlet line fitting and push syringe B so the thumb rest of the plunger slides into the syringe driver slot before clicking next. To prepare the final product vial, in a class two biological safety cabinet or equivalent, place a labeled 25 milliliter sterile vial in a tungsten or suitably shielded pot. After swabbing the septum of the vial with an alcohol wipe, place a lid on the pot and insert a filtered vent needle.

Connect the outlet of a low protein binding sterilizing 0.22 micron PVDF vented filter to a sterile 20 gauge hypodermic needle. Label the filter with the product batch number and insert the needle into the vented 25 milliliter final product vial. Withdraw the content of vial C into empty syringe D and add four milliliters of air.

Connect syringe D to the inlet of the 0.22 micron vented filter and push the syringe contents through the filter and needle into the final product vial. Flush the filter twice with five milliliters of air before removing the syringe from the vented filter. After transferring the pot containing the final product vial to the hot cell, connect the end tubing from position M2 valve five to the product vial filter.

Click next and wait for the synthesizer to perform the preliminary steps. When the prompt Ready to elute Ga-68 generator"appears, press next on the user interface. Once the automated synthesis starts, monitor the radio labeling in real time on the schematic tab by observing the activity of the three strategically placed radio activity detectors, pre-purification cartridge, reactor, and post-purification cartridge.

Alternatively, see the trends tab showing the activity profile of each detector during the synthesis. When the message Synthesis Complete"appears, remove the sterilizing vented filter and the filtered vent needle from the final product vial and retrieve the tungsten pot from the hot cell.