General
For the production of [18F]fluoride, CYPRIS HM-18 manufactured by Sumitomo Heavy Industries was used, and [18O]H2O as the target water was purchased from Rotem (> 98 atom%, Mishor Yamin, Israel). The Cupid System (Sumitomo Heavy Industries, Tokyo, Japan) was also used to control the CFN-MPS200 in the automated synthesis of [18F]AlF-FAPI-74 and N2 gas (> 99.9999%) supplied to synthesizer was purchased from Taiyo Nippon Sanso (Tokyo, Japan).
The reagents used for the synthesis of [18F]AlF-FAPI-74 were purchased from the following suppliers; ultrapure water (ultrapure grade) and ethanol (trace analysis grade) from Kanto Chemical (Tokyo, Japan), dimethyl sulfoxide (DMSO, Molecular Biology grade) from FUJIFILM Wako Pure Chemical (Osaka, Japan), aluminum chloride hexahydrate (AlCl3 6H2O, ReagentPlus®) from Sigma-Aldrich (St. Louis, MI, USA), ascorbic acid (Biotechnology grade) from Nacalai Tesque (Kyoto, Japan), sodium ascorbate (USP grade) from Spectrum (New Brunswick, NJ, USA) and phosphate-buffered saline (pH 7.4) from ABX (Radeberg, Germany). These reagents were used without purification. Water for injection (WFI) and isotonic sodium chloride solution (saline) were all Japanese Pharmacopeia grade and were purchased from Otsuka Pharmaceutical (Tokyo, Japan). FAPI-74 precursors and a reference standard for [18F]AlF-FAPI-74 were provided by SOFIE (Dulles, VA, USA) (Fig. 1). Sep-Pak® Accell™ Plus QMA Plus Light cartridge (130 mg sorbent per cartridge), Sep-Pak® Accell™ QMA carbonate Plus Light Cartridge (46 mg sorbent per cartridge), and Oasis® Light HLB cartridges were purchased from Waters (Milford, MA, USA). For the quality control, high-performance liquid chromatography (HPLC) column and thin layer chromatography (TLC) plate were made by Merck (Kenilworth, NJ, USA).
Recovery of [
18
F]fluoride from QMA cartridges (manual operation)
Two types of QMA cartridges were preconditioned with 10 mL of 0.5 M sodium acetate buffer (pH 3.95 ± 0.05) and 20 mL of WFI. [18F]Fluoride was obtained by irradiating the proton beam to [18O]H2O using an 18 MeV energy cyclotron. After diluting part or all of the target water, containing [18F] fluoride, with 1–6 mL of WFI, 1–2 mL (ca. 20–1600 MBq) was introduced from the male or female side of the QMA cartridge. Then, 0.3 mL of 0.5 M sodium acetate buffer (pH 3.95 ± 0.05) was passed through the male or female side of the cartridge to recover [18F]fluoride. The recovery rate was calculated from the introduced radioactivity from male or female side (sum of the radioactivity that was retained in the QMA cartridge and the radioactivity that passed through it) and the radioactivity recovered from the male side or female side of the QMA cartridge.
Radio-synthesis of [
18
F]AlF-FAPI-74 with the automated-synthesizer
For the synthesis of [18F]AlF-FAPI-74 on CFN-MPS200, previously known conditions have been adapted (Giesel et al. 2021). An automated synthesizer for the radiosynthesis of [18F]AlF-FAPI-74 was used as a single-use cassette-type CFN-MPS200 controlled by the Cupid System. Silicon was used as the material for the cassette, and a new systematic diagram was created specifically for [18F]AlF-FAPI-74 synthesis (Fig. 2). The process is described as follows.
[18F]Fluoride was recovered from the cyclotron to an 18O-water recovery vial and then loaded onto a QMA cartridge preconditioned with 10 mL of 0.5 M sodium acetate buffer (pH 3.95 ± 0.05) and 20 mL of ultrapure water from the cartridge male side via VP3 and VP4 under vacuum and 50 mL/min N2 gas and discarded to 18O-water waste liquid vial via VP5 and VP7. The [18F]fluoride on QMA cartridge was eluted with 300 µL of 0.5 M sodium acetate buffer (pH 3.95 ± 0.05) from the female cartridge side via VP6 and VP5 using a vacuum and collected into a glass reactor (Nichiden-Rika Glass, Hyogo, Japan) via VP4 to VP37. Further, the precursor mixture for one-step fluorination was added into the reactor via VP36 and VP37 using vacuum and then mixed with 50 mL/min N2 gas. The precursor mixture was composed of 300 µL of DMSO, 6 µL of 10 mM aluminum chloride aq. solution, 4 µL of 20% w/v ascorbic acid (aq.) solution, and 20 µL of 4 mM FAPI-74 precursor aq. solution. The radio-fluorination was carried out continuously for 5 min at room temperature, followed by 15 min at 95 °C with a closed system (Fig. 3). After cooling the reactor, the reaction mixture was transferred to a dilution vial where 9 mL of saline was added beforehand via VP37 to VP42 using 100 mL/min N2 gas flow. To collect the remaining radioactivity in the reactor and transfer line, 1 mL of saline was added to the reactor via VP9 to VP37 under vacuum and then transferred to the dilution vial via the same route. The dilution solution was passed through the HLB cartridge preconditioned with 5 mL ethanol and 20 mL of ultrapure water via VP42 and VP43 with 50 mL/min N2 gas flow, then discarded to waste liquid vial via VP44 and VP45. The HLB cartridge was washed with 3 mL of saline via VP10 to VP43 with 50 mL/min N2 and discarded to waste liquid vial via VP44 and VP45. Trapped [18F]AlF-FAPI-74 onto the HLB cartridge was collected into a formulation vial containing 14 mL of formulation solution using 1 mL of ethanol with 50 mL/min N2 gas flow via VP13 to VP44 and VP43 to VP12. This time, seven different formulation solutions containing saline were tested to find a composition that would prevent radiolysis and would be stable until 4 h after the synthesis: saline, saline containing 10 mg ascorbic acid and 90 mg sodium ascorbate (pH 5.0), saline containing 100 mg sodium ascorbate, commercially available phosphate-buffered saline (pH 7.4), phosphate-buffered saline (pH 7.4) containing 100 mg sodium ascorbate, 10 mM phosphate-buffered saline (pH 6.7), and 10 mM phosphate-buffered saline (pH 6.7) containing 100 mg sodium ascorbate.
Quality control for [
18
F]AlF-FAPI-74
All radioactivity, including [18F]AlF-FAPI-74, was measured using a dose calibrator. Measurement of the radiochemical purity, non-radioactive AlF-FAPI-74 (AlF-FAPI-74), and chemical impurities were carried out using an SPD-20A ultraviolet (UV) detector (λ = 264 nm) using a Shimadzu HPLC system and a Gabi-Star radioactivity detector (Elysia-Raytest, Straubenhardt, Germany). A Chromolith performance RP-18e (100 mm × 4.6 mm) as an analysis column was selected, and the mobile phase was acetonitrile (solvent A) and 0.1% trifluoroacetic acid (solvent B) in gradient mode; its condition was at 95% of solvent B from 0 to 3 min and then at 95% to 50% from 3 to 15 min. The method was calibrated using AlF-FAPI-74 as the reference standard (Linearity in the range from 0.05 to 20 µg/mL and the coefficient of determination of > 0.995 were confirmed). [18F]AlF-FAPI-74 was determined based on the retention time (RT) of the reference standard and the amount of carrier AlF-FAPI-74 calculated by calibration; the amount of all other chemical impurities was calculated based on the same calibration assuming similar extinction coefficients as AlF-FAPI-74. The set value of the column oven was 30 °C, and the flow rate was 2.0 mL/min.
Residual [18F]fluoride and [18F]AlF were analyzed using a radio-TLC analyzer, mini-Gita (Elysia-Raytest, Straubenhardt, Germany). The TLC plate used was HPTLC Silica gel 60 RP-18 and 1 v/v% phosphoric acid in saline/acetonitrile (50/50) as a developing solvent was filled in the tank for more than an h before the plate was deployed.
The residual DMSO and ethanol contents were measured using a flame ionization detector of a Shimadzu GC system. A DB-624 30 m × 0.32 mm, 1.8 µm (Agilent, Santa Clara, CA, USA) as an analysis column was selected and split injection mode (split ratio = 30:1). The carrier gas (helium) was 2 mL/min, and the column, injector, and detector temperatures were 40 °C for 5 min to 200 °C (20 °C/min) for 3 min, 200 °C, and 250 °C, respectively.
pH adjustment of buffers, etc., was performed using a pH meter with electrodes set for low volume (HORIBA, Kyoto, Japan), calibrated with pH standard buffer solution before use.