Introduction

The demand for 68Ga has increased considerably over the last few years due to its extensive use in positron emission tomography (PET) imaging germanium 68Ge/68Ga generators have been used in the field of nuclear medicine for over half a century. The production of 68Ga using first-generation germanium generators began in the 1960s [1], but the first commercial use of the 68Ge/68Ga generators did not begin until the early twenty-first century [1].

The commercial generators usually consist of 1.85 GBq (50 mCi) 68Ge. The activity elution of 68Ga is limited during the operation of the generator since the activity of the parent 68Ge decreases over time (half-life 271 days) and there is a potential threat of 68Ge breakthrough. In the US, approved 68Ge/68Ga generators are currently being used, including E&Z Galliapharm and IRE Galli-Eo [2, 3].

The preparation of cyclotron 68Ga can be achieved through the irradiation of liquid or solid targets. Pandey et al. and Alves et al. dealt with the preparation of these liquid targets [4, 5]. The proton irradiation of a 68Zn solution may lead to the formation of 68Ga [4]. The production of radioisotopes with half-lives longer than that of 68Ga (T1/2 = 67.7 min) i.e. 66Ga, (T1/2 = 9.5 h), and 67Ga (T1/2 = 3.3 days) requires attention. As a result, the radionuclide purity of the 68Ga solution will be reduced. The elimination (66Ga and 67Ga) requires lowering the incident beam energy and the 66Ga production is determined by the presence of a 66Zn target impurity. The 67Ga radioisotope has an effective cross-section of 800 mb at Ep = 20 MeV [6]:

$$^{{{68}}} {\text{Zn }}\left( {{\text{p}},{\text{2n}}} \right)^{{{67}}} {\text{Ga}}$$
(1)

while the 66Ga radioisotope is formed by a nuclear reaction:

$$^{{{66}}} {\text{Zn }}\left( {{\text{p}},{\text{n}}} \right)^{{{66}}} {\text{Ga}}$$
(2)

and has an effective cross-section of 700 mb at Ep = 13 MeV [7].

Compared with generators, liquid targets have the advantage of being able to prepare the same activity 68Ga for every production. While liquid targets are simpler in terms of material handling, they still require some kind of recycling 68Zn with particular care being taken to accumulate long-lived and stable impurities.The main advantage of using liquid targets is the low radiation load for operators during handling with a radioactive solution. The application of liquid targets using protons to irradiate 68Zn is covered by a European patent [8]. When using liquid targets, the 68Ga separated from the 68Zn solution acts as a direct substitute for the 68Ga produced by the 68Ge/68Ga generators. The preparation of 68Ga from liquid targets is associated with a risk of damaging the cyclotron if the target fails. The risk of target failure could be eliminated by using proper beam shape and conducting regular target maintenance. Cyclotrons are usually separated from the targets by a beamline equipped with fast valves which help protect the cyclotron vacuum.

Solid targets provide much higher 68Ga activities than liquid targets. Such target materials could consist of ZnO or elemental Zn [9,10,11,12].

Another method for solid target preparation is the electrolytic deposition of enriched 68Zn on a platinum disk [13]. The dissolution time as well as the separation of 68Ga must be minimised as much as possible. The advantage of automation is that it helps minimise the operator radiation exposure as well as the contamination of the target itself during its transport [9, 11].

Solid targets are advantageous since they can produce a high activity of 68Ga. Up to 74–148 GBq of 68Ga can be obtained in 1–2 h of irradiation time, which is more than what can be achieved with liquid targets or 68Ge/68Ga generators. [68Ga]Ga-PSMA-11 has been prepared with an activity of 43 GBq using a solid target with a high activity of about 100 GBq alongside the separation of 68Ga using a TK − 400 column from TRISKEM, synthesised in FASTLAB [2].

The European Pharmacopoeia has approved the use of 68GaCl3 prepared by cyclotron since 2021. Enriched 68Zn is commonly used for the preparation of 68Ga by proton irradiation. In the European Pharmacopoeia monograph for cyclotron-produced 68Ga, the limit for its radionuclide purity is 98% [14]. In 2019, the Food and Drug Administration (FDA) approved the use of [68Ga]Ga-DOTA-TOC for imaging gastroenteropancreatic tumours [15]. In 2020, the FDA approved the use of [68Ga]Ga-PSMA-11 for the diagnosis and imaging of prostate cancer. In Europe (Austria, Germany, and France), [68Ga]Ga-DOTA-TOC was approved as early as 2016 [16]. Furthermore, 68Ga can be used to label [68Ga]Ga-DOTA-NOC and [68Ga]Ga-DOTA-TATE, which are used to diagnose and image neuroendocrine tumours.

Zinc and mainly iron can negatively impact the radiochemical yield (RCY) of radiopharmaceuticals due to their complexation reactions with peptides in competition. In its purest form without metal contaminants, 68Ga requires a separation procedure. Fe(III) has similar chemistry to Ga(III). One scheme to improve the separation of iron from gallium involves a reduction process with ascorbic acid from Fe(III) to (II) while Ga(III) is not affected by ascorbic acid [17]. Ascorbic acid is routinely used as a protective agent in peptide radiolabelling [11]. The principles of reduction of Fe(III) by ascorbic acid have also been used in the analytical determination of Fe(III) by its sorption on annex resin and elution with 5% ascorbic acid in 0.5 mol dm−3 hydrochloric acid (HCl). This method allows the preconcentration of iron and its determination by spectrophotometry [18].

We here describe a target preparation procedure using the proton irradiation of enriched 68Zn, and the dissolution, separation, and labelling procedure for [68Ga]Ga-DOTATOC, [68Ga]Ga-DOTANOC, and [68Ga]Ga-PSMA-11 radiopharmaceuticals. A chemical reaction that was used for iron reduction with ascorbic acid to separate iron from 68Ga is also presented.

Experimental

All chemicals used in this work were of pharmaceutical or supra pure quality. The corresponding PSMA-11 peptide used met good manufacturing practice (GMP) and active pharmaceutical ingredient (API) for clinical trials and was purchased from Advanced Biochemical Compounds (ABX, Germany). TK-400 ion exchange columns were purchased from TRISKEM (France) and contained octanol impregnated on an inert support. All chemicals used for the syntheses such as NaCl, ethanol, sterilised water for injection, phosphate buffer and water/ethanol (1:1), HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane-1-sulfone), and cartridges were purchased from ABX. The HCl (37% suprapur®) was purchased from Millipore Sigma (USA). Sterile FG Millex filters and sterile FG Millex ventilation filters were also obtained by Millipore Sigma.

The dissolution of the target containing enriched 98.2% 68Zn metal rough powder Campro Scientific GmbH (Germany) on a niobium coin was performed at the 68Ga-DISTA dissolution station (BIONT a.s., Bratislava, Slovakia). The target was dissolved in 7 mol dm−3 HCl. The 68Ga separation itself took place at the 68Ga–SEPUR separation station (BIONT a.s., Bratislava, Slovakia). The 68Ga was separated from 68Zn on a TK-400 column by washing with 7 mol dm−3 HCl and the 68Ga was eluted with 0.05 mol dm−3 HCl. The peptide was labelled with 68Ga on the appropriate commercial production cassette in the TRACERlab MX synthesis module made by GE-healthcare.

The radiopharmaceuticals were analysed according to European Pharmacopoeia procedures. The activity and half-life of [68Ga]Ga-PSMA-11 PET radiopharmaceuticals were determined using a Curiementor PTW (Germany) dose calibrator. The radionuclide purity of the radiopharmaceutical was determined with a Canberra Packard (USA) germanium detector. The [68Ga]Ga-DOTATOC, [68Ga]Ga-DOTANOC, and [68Ga]Ga-PSMA-11 were analysed for radiochemical and chemical purity by high-performance liquid chromatography (HPLC) on a 1260 Infinity instrument (Agilent Technologies, USA) using an Elysia s. POMO radiometric detector (Belgium) and an Agilent Technologies (USA) UV/Vis detector. An Elysia-Raytest (Belgium) MiniGita scanner was used for both thin layer chromatography (TLC) and instant TLC (iTLC) measurements. The solvent residues were measured on an Agilent 7890 B gas chromatograph (Agilent Technologies), with an Agilent 7697 A headspace sampler, on a gas chromatography (GC) column (Resteck Corporation, USA).

The metals in the radiopharmaceuticals (zinc and iron) were determined by voltammetric differential pulse polarography on a hanging mercury drop electrode (HMDE; Metrohm Ltd., Switzerland).

The voltammetric determination of iron with concentrations < 200 μg dm−3 was performed on a HMDE. The detection limit for this method was β (Fe) = 2 μg dm−3. The limit of quantification was β (Fe) = 6 μg dm−3. The sensitivity of the method could not be improved by deposition. Iron was determined using the HMDE method in an electrolyte consisting of solutions of triethanolamine at a concentration of 0.05 mol dm−3, potassium bromate at a concentration of 0.1 mol dm−3, and NaOH at a concentration of 0.3 mol dm−3. Ultrapure water (Merck suprapur® Millipore Sigma) with a resistance of > 18 MΩ cm (25 °C), type I (ASTM D1193) was used. The method was suitable for samples with iron concentrations of up to 200 μg dm−3. The required pH of the measured solution should be 12 and max. 12.4 [19].

Zinc was determined in a solution prepared from sodium acetate and potassium chloride on a HMDE using anodic stripping voltammetry. The reagents used were required to be of the purest quality and used 30% sodium hydroxide, 100% acetic acid, potassium chloride (TraceSelect® Sigma-Aldrich), a commercially available Zn standard solution with concentration 1 g dm−3 in ultrapure water. A standard solution of β (Zn(II)) = 10 mg dm−3 was prepared using c (HNO3) = 0.014 mol. If necessary, the pH of the solution was adjusted to 4.6 ± 0.2 [20].

Target preparation and irradiation

The target was prepared by pressing zinc (68Zn) powder with an enrichment of 98.2% and weighing about 25–35 mg onto a niobium coin with a purity of 99.99%. A space for zinc powder was excavated on the niobium coin, which was pressed with a hand press at a pressure of 195 MPa. The prepared target was placed in a 68Ga-solid target irradiation station and irradiated with protons at 10.9 MeV. The 18 MeV cyclotron beam was degraded to 10.9 MeV with a beam intensity of 35 µA hitting the target. The target coin was water-cooled from behind by triscus cooling. Following its irradiation, the target fell into the dissolution vessel using an automated control system 68Ga-DISTA.

Dissolution and separation

The dissolution of the irradiated target was monitored with the 68Ga − DISTA control system. Dissolution was performed using 10 cm3 of 7 mol dm−3 HCl. Subsequently, the dissolved 68Ga was transferred to the separation module through the transport capillary.

The separation of 68Ga from 68Zn took place in a 68Ga–SEPUR module. Initially, the TRISKEM sorbent TK-400 column was conditioned with 7 mol dm−3 HCl and the 68Ga solution from the dissolved target was thereafter loaded to the separation column. Seven mol dm−3 HCl was used to wash off the 68Zn out of the column. In the last step, 68Ga was eluted with 0.05 mol dm−3 HCl. The 68Ga was separated with 8 cm3 0.05 M HCl and diluted with another 7 cm3 of 0.05 HCl to achieve a total volume of 15 cm3. No further pH adjustments were made. More experimental details are included in the experimental section.

68 Ga labelling of peptides

All gallium-labelled radiopharmaceuticals such as [68Ga]Ga-DOTANOC (50 µg precursor), [68Ga]Ga − DOTATOC (50 µg precursor), and [68Ga]Ga-PSMA-11 (10 µg precursor) were prepared by the methods proposed by ABX for the TRACERlab MX module. DOTANOC (50 µg) and 68Ga (4.1 GBq), DOTATOC (50 µg) and 68Ga (2.76 GBq), and PSMA-11 (10 µg) and 68Ga (6.37 GBq) for all prepared radiopharmaceuticals the ratio of precursor to 68Ga was more than 250 times.

68Ga was transferred from the 68Ga–SEPUR separation unit to the TRACERlab MX module where it was a trapped on a PS-H+ cation exchange column. The acidity of the radiopharmaceutical 68Ga had to be adequate as the PS-H+ column loses sorption efficiency for gallium with increasing acid concentrations. 68Ga was eluted from the column with a 5 mol dm−3 NaCl solution and into a reactor containing the precursor dissolved in a 1.5 mol dm−3 HEPES buffer solution. The reaction was performed at 125 °C for 6 min. The product was separated from the impurities on a C-18 column and eluted with the aqueous-ethanol solution. Osmolality was adjusted with a phosphate buffer.

Quality control

The resulting radiopharmaceutical was subjected to several tests under the European Pharmacopoeia for quality control. A HPLC instrument equipped with a Zorbax Eclipse C-18 column (250 × 4.6 mm I.D. 5 µm) was used to determine its chemical and radiochemical purity. The composition of the mobile phase was: A) 5% ACN (V/V) + 10 mmol dm−3 TFA; B) CAN + 10 mmol dm−3 TFA. The temperature of the analytical column was set to 25 °C. The flow-rate of the mobile phase was 1 cm3/min. For the detection of the separated complexes and free peptides, the UV/DAD was used at wavelengths of 225 nm, 240 nm, and 280 nm. The gradient profile was from 10% B in 0–2 min, linearly increasing to 75% B for 10 min, 75% B for 2 min, and equilibration for 1 min to 10% B.

The radiopharmaceutical was measured by TLC using iTLC-SG chromatographic paper with a mobile phase containing 1 mol dm−3 of ammonium acetate with MeOH in a ratio of 1:1. The analysis was developed on 8 cm chromatographic paper. Subsequently, the TLC plate was scanned on a MiniGita scanner and evaluated with the GINA-Star TLC software.

Results and discussion

The 68Ga isotope was produced using a Cyclone IBA 18/9 cyclotron. Different types of degraders were examined and the optimal energy of 10.9 MeV was chosen to obtain the highest radionuclide purity of 68Ga. Proton irradiation of a 68Zn-in-niobium press powder target was performed with a 68Ga solid target station. The 68Ga-DISTA dissolving station was used to dissolve the target in 10 cm3 of HCl for six minutes inside a cyclotron vault at room temperature. A dissolution time of six minutes was sufficient to dissolve the target material completely. An ethylene tetrafluoroethylene (ETFE) transfer capillary was used to deliver the solution with dissolved zinc powder and 68Ga to a separation module in a "class-C" production room. The 68Ga-SEPUR module was used for the separation of 68Zn and 68Ga. The scheme of the separation 68Ga-SEPUR module is shown in Fig. 1.

Fig. 1
figure 1

Scheme of the 68Ga-SEPUR separation module

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The separation procedure consisted of conditioning the TK-400 column (the volume of the TK-400 resin was 2 cm3 and the particle size was 50–100 µm) with 5 cm3 of 7 mol dm−3 HCl. The TK-400 column was loaded with 10 cm3 of the 68Ga solution obtained from the dissolved target. The column was washed with 35 cm3 of 7 mol dm−3 HCl to eluate the zinc contents. The 68Ga was then eluted from the column after using 15 cm3 of 0.05 mol dm−3 HCl.

As previously mentioned, the protocol described here was used for the preparation of radiopharmaceuticals labelled with 68Ga, such as [68Ga]Ga-DOTANOC (50 µg DOTANOC), [68Ga]Ga -DOTATOC (50 µg DOTATOC), and [68Ga]Ga -PSMA-11 (10 µg PSMA-11). The time from the end of the beam (EOB) to the end of the analysis was 88 min, and the decay corrected radiochemical yield of the 68Ga-radiopharmaceuticals was between 50 and 60%.

The 68Ga-labelled radiopharmaceuticals underwent a quality control using the procedures described in the experimental part. Their radiochemical purity was determined by TLC and HPLC (Fig. 2 and Table 1). Around 93.0% of the radiochemical purity of 68Ga-PSMA11 (Fig. 2) referred only to the main peak on the chromatogram, but PSMA-11 formed two isomers with Ga, and the sum of the secondary and main peaks was included in the total radiochemical purity. In the end, a value of 99.9% was obtained.

Fig. 2
figure 2

Thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) analyses of [68Ga]Ga-PSMA-11, [68Ga]Ga-DOTANOC, and [68Ga]Ga-DOTANOC

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Table 1 Production and analysis of [68Ga]Ga-DOTATOC, [68Ga]Ga-PSMA-11, and [68Ga]Ga-DOTANOC
Full size table

With the exception of the sterility and LAL tests that were determined several days after the synthesis, the quality control tests were performed immediately after the synthesis was completed. The data in Table 1 fulfill the criteria determined by the European Pharmacopoeia.

According to the European Pharmacopeia, the purity of a radionuclide is expected to be 98% for 68Ga produced by cyclotrons. The main radionuclides causing a decrease of 68Ga purity are 66Ga and 67Ga. These radionuclides are produced through the irradiation of 66Zn and 68Zn nuclear reaction 66Zn(p,n)66Ga and 68Zn(p,2n)67Ga. The quantity of 66Ga depends on the purity of the enriched starting material while the activity of 67Ga depends on the energy of the protons used for irradiation. According to our results, the purity stayed above 98% until seven hours after the EOB in the proton energy of 10.9 MeV (Fig. 3).

Fig. 3
figure 3

Radionuclide purity at 10.9 MeV of proton irradiation (68Ga ≥ 98%; 7:00 h), EOB = 8:27

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Separation scheme for iron and gallium separation

The reduction potential of Fe(III) to Fe(II) at a low pH was around + 0.8 V and that of Ga(III) to Ga(II) was around − 0.6 V [14, 21]. Ascorbic acid can selectively reduce Fe(III) because of the large difference in their potentials. The fact that TK-400 resins do not strongly bind Fe(II) means that Fe(II) could be eluted with no loss of 68Ga. The 68Ga was eluted from the TK-400 column with 0.05 mol dm−3 HCl. In this study, 50 mg of iron solutions in 0.5 dm3 of 7 mol dm−3 HCl were used for the experiments with iron. The TK-400 resins sorbed very well iron from 7 mol dm−3 HCl concentration (step 1, Table 2), and only a small amount of iron(III) was desorbed using 1% ascorbic acid in a strongly acidic medium (steps 2–5, Table 2).

Table 2 Sorption and elution of iron from the TK-400 using 1% ascorbic acid in 7 mol dm−3 HCl
Full size table

By decreasing the acidity of the HCl, we were able to increase the efficacy of washing 68Ga from the TK-400 column. Mixing a 1:1 solution of 4 mol dm−3 HCl and 1% ascorbic acid with 5 mol dm−3 NaCl provided a solution containing sufficient chloride for complexing 68Ga, allowing the development of negative chloride complexes and sufficient protons for protonating octanol to support a positive surface on the TK-400 resin. The sorption of gallium was due to the ion exchange mechanism of the negative gallium complex with a positive surface of the TK-400. The TK-400 resin was eluted with a solution consisting of 2.5 mol dm−3 NaCl, 2 mol dm−3 HCl, and 0.5% ascorbic acid, resulting in a very effective separation of iron from gallium. The 68Ga was washed out from the column with 0.05 mol dm−3 HCl. The acidity of the solution left in the dead volume of the resin was less acidic. The 68Ga eluted solution did not require dilution to achieve an acidity compatible with the 68Ga separation step of the TRACERlab MX synthesiser module. The determined of H+ proton concentration was 0.49 mol dm−3 and that of Cl− was 0.98 mol dm−3 based on titration (step 5).

Step 1: Loading iron and 68Ga in 7 mol dm−3 HCl into the TK-400 column; step 2: washing with acid in 7 mol dm−3 HCl; step 3: washing with 1% ascorbic acid in 7 mol dm−3 HCl; step 4: washing with a solution consisting of 2.5 mol dm−3 NaCl, 2 mol dm−3 HCl, and 0.5% ascorbic acid; step 5: elution with 0.05 mol dm−3 HCl. The volume 5 cm3 in the all steps.

The efficiency of the separation process was 95.3% decay corrected. The 68Ga was obtained from the Galli-Eo 68Ge/68Ga generator. The Curiementor isotope calibrator was used to measure the 68Ga activity of loading solutions No. 1 and 5, while a NaI scintillation detector was used to measure the activity of washing solutions Nos. 2–4 because their activity was too low to be determined with Curiementor. The elution profile of 68Ga from Fe influenced by the mobile phase is presented in Fig. 4. The initial iron activity was 500 µg/cm3. After applying the sample (step 1), 1.94% of iron flowed into the eluate. After using the reducing solution (step 4), 98% of the iron was leached. A 95.3% activity was achieved when 68Ga was eluted with 0.05 M HCl.

Fig. 4
figure 4

Elution profiles of 68Ga and Fe depending on the mobile phase composition

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Conclusions

The separation of 68Ga prepared by irradiating powder pressed 68Zn targets and dissolving the targets in the 68Ga-DISTA module was rapid and fully automated. A 68Ga-SEPUR separation station was developed. The separation of gallium from transition metal interferents showed a reliable radiochemical yield. The TRACERlab MX, a disposable sterile cassette, and chemicals from ABX were used to label the 68Ga peptides according to good manufacturing practices. The labelled product exhibited a five-fold higher activity than a 68Ge/68Ga generator with a starting activity of 1.85 GBq. The resulting product would allow twice the number of patients to be examined by cyclotron production 68Ga compared to the generator produced 68Ga. The product was manufactured in accordance with the regulations specified in the European Pharmaceutical Pharmacopoeia. The final product met the quality requirements stated in the pharmacopoeia 10.3. A separation procedure for the effective separation of iron was developed. The radiochemical yield of 68Ga from such a separation process exceeded 95%. The final 68Ga product was prepared up to 6 GBq at the end of synthesis.