All water was obtained from a Milli-Q® Direct-Q 3 UV system (Merck Millipore). Ultrapur hydrochloric acid 30% (Merck) was used directly and diluted with water during target dissolution and gallium-68 purification. Hydroxamate-based resin (ZR Resin), di(2-ehtylhexyl)orthophosphoric acid-based resin (LN Resin) and trioctylphosphine-based resin (TK200 Resin) were used for [68Ga]GaCl3 purification (Triskem International, Rennes, France). Labelling was performed with PSMA-11 and DOTATATE (ABX, Dresden, Germany).
Gallium-68 was produced via the 68Zn(p,n)68Ga nuclear reaction using highly enriched metallic zinc-68 solid targets (ARTMS, Vancouver, Canada, zinc-68: >98.2 %, Ø10 mm, 230-315 mg on water cooled silver backing). The zinc-68 solid targets were transferred and irradiated using the ARTMS QUANTM Irradiation System® (“QIS”, ARTMS, Vancouver, Canada) on a GE PETtrace 880 Cyclotron (GEMS PET Systems AB, Uppsala, Sweden) using a helium-cooled aluminium foil energy degrader to achieve a proton incident energy of 13.0 MeV on target. The targets were irradiated using proton beam currents of up to 80 µA for up to 2 hours. Prior to applying the maximum beam current of 80 µA on target, a series of beam current ramping experiments were performed on natural zinc targets. The irradiations were performed with increasing beam currents in steps of 5-10 µA (with all other parameters kept unchanged) followed by visual inspection of the irradiated target in each step.
After irradiation, the target capsule (Fig. 1) was pneumatically transferred to a hot cell by the QIS. The zinc-68 targets were then dissolved using 2 mL of hot 30% HCl on a QIS Dissolution System. Evolved hydrogen was vented.
The dissolved target solution containing [68Ga]GaCl3 and [68Zn]ZnCl2 was automatically transferred to a GE FASTlab 2 Developer synthesis module for separating the gallium-68 from the target material by three solid phase extraction columns (Fig. 2). First, the target solution was pumped through a column containing 250 mg ZR resin. 15 mL of 30% HCl were used to elute remaining zinc-68 from the ZR column, while gallium-68 was eluted from the column with 8 mL of 1 N HCl. The solution containing gallium-68 was pushed through the second column containing LN resin in order to bind Fe contaminants, while the eluted [68Ga]GaCl3 was directly loaded onto a third column containing TK200 resin. The TK200 resin was dried by purging with nitrogen, then eluted to a product vial using 2.5 mL of 0.1 N HCl. The overall process of target dissolution and purification took approximately 35 minutes.
Quality control of gallium-68
Quality control of the produced [68Ga]GaCl3 was performed according to the draft Ph. Eur. monograph on accelerator produced [68Ga]GaCl3 (5).
Gamma-ray spectroscopy and radionuclidic purity assays were performed using a calibrated Canberra Broad Energy Ge detector (BE2020, Canberra) equipped with Genie 2000 software. Half-life was determined by repeated measurements in a Capintec dose calibrator. pH was determined by pH sticks (Merck MColorpHast pH 0-6.0). The silver nitrate precipitation assay was performed by adding 5 µL gallium-68 chloride solution to 0.1 mL silver nitrate (17 g/L) to yield a white precipitate. The solid phase extraction (SPE) assay was done by application of the Phenomenex Strata-X-C cation exchange cartridge (33 µm, 10 mg/mL) preconditioned with 0.5 mL ethanol, then 1 mL 1.03 g/l HCl. The gallium-68 solution was slowly loaded onto the cartridge, the liquids collected and the column dried with air. The SPE column was washed with 1 mL HCl (1.03 g/l) and the liquids were collected. The column was then eluted with 1 mL 2 % (v/v) HCl (1.03 g/l) in acetone and dried with air to collect the final eluate. The quantity of radioactivity in each of the different fractions and the column were measured in the dose calibrator. Radiochemical purity was assessed by thin-layer chromatography on an Agilent ITLC-SG plate: A sample of the gallium-68 solution was added to the ITLC plate, which was eluted in 1:1 ammonium acetate (77 g/l): methanol over at least 7 cm. The radioactivity distribution was evaluated on a MiniGita TLC scanner (Elysia-Raytest). Contents of iron and zinc were determined with semi-quantitative test strips (QuantofixTM, Machery-Nagel) by comparing the colour change of the reactive pad against multi-stage colour scales. Further, the metal contents of decayed samples of the gallium-68 solution were quantified as described in draft Ph. Eur. monograph (5) by ICP-OES in an argon plasma (Thermo iCAP 6000 ICP Spectrometer, Thermo Scientific, Waltham, MA, USA). The contents of bacterial endotoxins was determined on an Endosafe Nexgen-PTSTM by adjusting and diluting the pH of a sample to pH 6-8 with NaOH (10 mM, in endotoxin free water (Charles River)) for minimum inhibition of the endotoxin assay before adding the solutions to the single use cartridge.
Radiolabelling of DOTATATE with gallium-68
Zinc-68 targets containing 230-315 mg zinc-68 were irradiated with 50-70 µA for 0.5-1 hours and the produced gallium-68 was separated from target material as described above. Radiolabelling of DOTATATE with gallium-68 was performed manually. For labelling with low activity levels (< 1 GBq, n = 3), 100 µl of the purified gallium-68 solution was used, while approximately 900 µl was used for the single 3.2 GBq labelling. For the low activity labelling, the [68Ga]GaCl3 was microwave heated (CEM PETwave) to 90 °C for 2 min. with 50 µg DOTATATE (34.8 nmol) in 125 µl acetate buffer (pH 4.6, Fluka) and 20 µl ethanol added. For higher activity labelling (3.2 GBq), the [68Ga]GaCl3 solution was mixed with 500 µl acetate buffer and then preheated to 95 °C before adding 500 µg of DOTATATE (348 nmol) in 100 µl acetate buffer. The solution was kept at 95 °C for 10 min. In all cases the labelling yield and radiochemical purity was assessed by ITLC (Biodex 150-771, 0.1 M sodium citrate, pH 5) and radio-HPLC on a Phenomenex Jupiter 300A (150x4.6, 5 µm) C18 column in water+0.1% TFA (A) and acetonitrile (B); 0-2 min. 14% B, 2-10 min. 14 to 63% B at 1 mL/min. and UV detection at 220 nm.
Radiolabelling of PSMA-11 with gallium-68
Zinc-68 targets containing 230-315 mg zinc-68 were irradiated with 80 µA for 2 hours and the produced gallium-68 was separated from the target material as described above. Radiolabelling of PSMA-11 with [68Ga]GaCl3 was achieved with a single use kit on an automated Modular-Lab PharmTracer (Eckert & Ziegler) in direct connection to the FASTlab synthesizer. The standard cassette was modified to accommodate accelerator-produced gallium-68 radiolabelling (Fig. 3), i.e. the cation exchange column was removed and the module was programmed to withdraw the gallium-68 solution from the FASTlab product vial and deliver 1.0 mL (out of the 2.2-2.4 mL total produced gallium-68 chloride solution) to the reactor. The reactor was also loaded with 50 mg of sodium ascorbate, 100 nmol PSMA-11 (ABX, Germany) and 1.0 mL sodium acetate buffer, pH 4.6. During the labelling, the reactor was heated to 95 °C for 600 s after which the solution was diluted with 3 mL deionized (DI) water and loaded onto a preconditioned SepPak C18 light column. The reactor was further washed with 3 mL DI water and loaded onto the C18 light column, which was washed with 3 mL of DI water. The column was eluted with 2 mL 50% (v/v) ethanol followed by 4 mL DI water into a product vial containing 50 mg sodium ascorbate in 6 mL phosphate buffered saline to yield a final product volume of approximately 12 mL containing the [68Ga]Ga-PSMA-11 at the end of synthesis (EOS).
For quality control, an aliquot was analysed by HPLC on a Phenomenex Kinetex XB-C18 column (150x4.6, 5 µm, 1 mL/min) applying a gradient program (A:B, DI water+0.1% TFA: acetonitrile) similar to that specified in the Ph. Eur [68Ga]Ga-PSMA-11 monograph draft, i.e. 0-0.5 min. 5% B then 5-40% B from 0.5 to 10 min (4). Also, the amounts of unlabelled and colloid gallium-68 were determined by TLC on silica gel on glass backing plates eluted in either 0.1 M sodium citrate, pH 5 or ammonium acetate in DI water (77 g/l):methanol 1:1, respectively. pH was checked by a calibrated pH meter.