All solvents, chemicals and reagents were purchased from commercial suppliers, namely Sigma Aldrich, Merck, ABX or VWR, and were used as received without further purification. Sterile water and saline (0.9%) for injection were respectively purchased from B. Braun and Aguettant. Sep-Pak® cartridges were purchased from ABX for pre-conditioned Light QMA Cartridge with potassium carbonate or from Waters Corporation for Accell Plus QMA Plus Light Cartridges (pre-conditioned with 10 mL of 0.5 M K2CO3 solution, followed by 20 mL of water) and tC18 Plus Short Cartridges (treated with 1 mL Ethanol and rinsed with 10 ml of water). Sodium Ascorbate (Na-Asc) was purchased from Cooper.
The cold [19F]LBT999 reference compound and its chlorinated precursor 4 (Fig. 2) were synthesized and supplied by Orphachem (Clermont-Ferrand, France), according to a procedure described in the literature (Dolle et al., 2008; Dolle et al., 2006a). The precursor and reference have been qualified as starting material or reference compound for radiopharmaceutical production. [18F]fluoride was produced by Cyclopharma Laboratories via the 18O(p,n)18F nuclear reaction with a GE cyclotron (PETtrace 800, 16.5 MeV). 18O-enriched water was purchased from CIL or Rotem. The bombardment of 18O-enriched water with protons at 80 µA during 30 min provided about 80 GBq of [18F]fluoride in 18O-enriched water (4 mL), then the activity was directly transferred under helium pressure to the radiosynthesis module.
Automated radiosynthesis of [18F]LBT999 was performed either on a TRACERlab FXFN module (GE) or on an AllinOne module (Trasis), including HPLC purification and formulation. Semi-preparative HPLC purification was carried out on an Alltima C18 column (250 X 10 mm, 5 µm, Grace, Alltech, France) for TRACER lab FXFN and for the AIO on an Zorbax EclipseXDB-C18 (250 X 9.4 mm, 5 µm, CA, Agilent, USA). In both cases, ammonium acetate 0.1M/acetonitrile: 40/60 was used as mobile phase at a 4 mL/min flow rate. Sodium ascorbate (0.5 %) was added for purification after AIO process optimization.
For the formulation, the collected fraction was diluted with water, trapped on a SPE cartridge and rinsed with water. [18F]LBT999 was eluted from the cartridge by injectable ethanol and the formulation complete by adding saline (0.9%). The formulation was done to not exceed 10% of ethanol (v/v). Sodium ascorbate (0.5 %) was added to the aqueous solution for the AIO process optimization. The final product was aseptically filtered (0.22 µm PES vented) into sterile vials for human injection. The filters were procured from RoweMed (A-6606) and sterile vials (12 mL) from Hospira.
For quality control, analytical HPLC analyses were performed by using two HPLC conditions with a gamma detection and UV signal detection at 220 nm. HPLC a: Equipment: ICS 3000 (Thermo Fisher Scientific); Column: µBondapak™ (C18, 3.9 X 300 mm, 10 µm); mobile phase: acetonitrile/water/TFA: 50/50/0.1 (v/v/v); Flow rate: 1 mL/min; HPLC b : Equipment: Ultimate 3000 (Thermo Fisher Scientific); Column: Brownlee CHOICE C18 (250 X 4.6 mm, 5 µm); mobile phase: ammonium acetate 0.1M/acetonitrile: 35/65 (v/v); Flow rate: 1 mL/min.
Thin layer chromatography (TLC) analyses were carried out on silica gel 60 F254 (Merck) by using a radioTLC reader (BioscanB-MS-1000) and ethyl acetate as mobile phase.
Na-Asc concentration was determined by using a reflectometer (Reflectoquant®, RQflex® 10, EMD Millipore).
Automated radiosynthesis of [18F]LBT999
For both modules, automated in-house programs were developed to synthesize the [18F]LBT999, including exactly the same sequence, fully described in Table 1, with four main steps: 1) Azeotropic drying of [18F]Fluoride, 2) [18F]radiofluorination of the precursor 4, 3) HPLC purification and 4) Formulation.
TRACERlab FXFN module
The module was used in its basic configuration without any modifications. After the end of bombardment (EOB), the [18F]fluoride produced by the cyclotron was delivered to the already conditioned automate. Then, [18F]Fluoride was trapped on a Sep-Pak Accell Plus QMA Plus Light Cartridge to remove [18O]H2O. [18F]Fluoride was eluted into the reaction vessel using 0.9 mL of an aqueous solution of Kryptofix (K2.2.2., 7.2 mg) in 715 µL of acetonitrile and potassium carbonate (K2CO3, 3.8 mg) in 285 µL of water. The [18F]fluoride was dried by azeotropic distillation under vacuum and helium flow by heating at 70 °C to 100°C. Following the drying step of [18F]fluoride, the chlorinated precursor 4 (3 mg) preliminary dissolved in 1 mL of anhydrous DMSO was added into the reaction vessel and heated at 165°C for 10 minutes. After this time, the reactor was cooled to 50°C and the crude reaction mixture was diluted with water (9 mL), passed through a Sep-Pak tC18 Plus Short Cartridge to remove unreacted [18F]fluoride and most polar impurities. The crude [18F]LBT999 was eluted from the cartridge by using acetonitrile (1 mL) prior to the semi-preparative HPLC purification. The [18F]LBT999 fraction was collected between 11-12 min, transferred into a flask preloaded with water (30 mL) then the resulting solution was passed through a Sep-Pak Alumina N Plus Light Cartridge and a Sep-Pak tC18 Plus Short Cartridge. The cartridges were rinsed with sterile water (5 mL) and [18F]LBT999 was eluted with injectable ethanol (1.5 mL) into the final product vial, containing 13.5 mL of saline (0.9%).
Automated radiosynthesis of [18F]LBT999 on AIO
For the radiosynthesis of [18F]LBT999 on AIO, a single-use cassette was designed with an appropriate home-made program, based on the work previously developed on TRACERlab FXFN with some modifications. As usual with the AIO, before receiving the activity from the cyclotron, the cassette was placed on the platform and all reagents loaded on it.
At EOB, the [18F]fluoride was delivered and trapped on a preconditioned QMA carbonate cartridge, and eluted into the reaction vessel with 0.9 mL of a solution of Kryptofix (K2.2.2., 7.2 mg) in 715 µL of acetonitrile and K2CO3 (3.8 mg) in 285 µL of water. Then, the [18F]fluoride was dried by azeotropic distillation under vacuum and nitrogen flow by heating with successive addition of acetonitrile. The precursor 4 (6 mg) in DMSO (0.8 mL) was added to the dried residue (18F]fluoride/K2.2.2. complex) and after heating at 155°C for 7.5 minutes, the reaction mixture was diluted in Na-Asc (0.5% in water, 9 mL). The resulting mixture was subsequently passed through a Sep-Pak tC18 cartridge, rinsed with water and the product was eluted with 1 mL of acetonitrile and mobile phase for injection into the HPLC loop for purification by semi-preparative HPLC. The [18F]LBT999 peak was detected between 11 and 12 minutes, collected and then diluted into 40 ml of Na-Asc (0.5% in water) and the resulting solution passed through a Sep-Pak tC18 cartridge and rinsed with water. The [18F]LBT999 was eluted with injectable EtOH (2.5 mL) and the formulation was completed with saline (0.9%) containing 0.5% of Na-Asc, affording 25 mL of mother solution.
Quality Control for human use
Visual inspection, testing for pH, filter integrity via the bubble test, radionuclide identity and purity tests via half-life and energy spectrum, radiochemical purity and identity by HPLC, chemical purity by HPLC, K2.2.2. spot test, sodium ascorbate concentration (reflectometer), bacterial endotoxin, the residual solvents analysis by GC, and sterility were performed following standard QC rules for fluorinated PET tracers.
Stability of the formulation
The stability of [18F]LBT999 was verified by analytical HPLC and radioTLC at room temperature (20°C) up to 8 h after the end of synthesis and on the day after production (data not reported).