*3.1. Irradiation and Purification of [61Cu]CuCl<sup>2</sup>*

Irradiation of zinc liquid targets, both natural and enriched, and further copper-61 purification was conducted following the previously published and described methodology [48,49]. Briefly, copper-61 was obtained from the irradiation of both highly pure zinc nitrate hexahydrate and enriched zinc-64 solutions using an IBA Cyclone 18/9 (IBA, Louvain-la-Neuve, Belgium) [20]. Zinc nitrate was directly dissolved in 10 mM nitric acid, yielding a concentration of 0.2 g/mL, whereas zinc-64 (metal form) had to be initially dissolved with highly concentrated nitric acid, left overnight, evaporated to dryness, and finally re-dissolved in 10 mM nitric acid, yielding likewise a concentration of 0.2 g/mL. Zinc-64 solutions were recycled and re-irradiated up to 4 times. Since only water and HNO<sup>3</sup> were added to the original zinc-64 solution during the purification process, the recycling process was made possible simply by evaporating the excess of water. These solutions were irradiated at 65–75 µA for 180 min. Copper-61 automatic purification was conducted using a Synthera® Extension module (IBA, Louvain-la-Neuve, Belgium) without any manual intervention, and it was completed in less than 40 min from the EOB [45].

#### *3.2. EtOH as Radiolytic Scavenger*

The considerably high percentage of radiolysis in the final product vial (FPV), caused by the presence of free radicals in solution (e.g., superoxide or hydroxyl radicals) [50], impaired the establishment of the most favourable labelling conditions. Although not explicitly measured, for higher activity concentrations, the radiolysis percentage was anticipated to increase. Several compounds are known to act as radiolytic stabilizers and protect against radiolysis. Antioxidant compounds, such as ascorbic acid (AA) and gentisic acid (GA), are commonly known to protect against radiolysis and are mainly described in the literature for radiolabelling biomolecules with β - -emitting radionuclides (e.g., yttrium-90 and luthetium-177) [51,52]. Notwithstanding, these compounds might have a negative impact on copper-based radiopharmaceuticals, given the redox properties of copper. More recently, EtOH also gained relevance in <sup>68</sup>Ga-based radiopharmaceuticals and proved to be of great value, as confirmed by Eppard et al. [53–55]. To evaluate the applicability of EtOH as a radiolytic scavenger for <sup>61</sup>Cu-based radiopharmaceuticals, a single test with and without EtOH was performed before establishing optimal labelling conditions. Figure 4 shows the results attained when using EtOH up to 300 µL (maximum 5 vol% ethanol). Compared with the labelling of [61Cu]Cu-DOTA-NOC without EtOH (Figure 4A), it is evident that there is a significant decrease in the rate of radiolysis using the ethanol-based method (Figure 4B–D). This method showed that 5 vol% EtOH leads to a decrease in radiolysis from more than 15% in [61Cu]Cu-DOTA-NOC to less than 5% for [61Cu]Cu-DOTA-NOC and less than 1% for both [61Cu]Cu-DOTA-TOC and [61Cu]Cu-DOTA-TATE. We found that [61Cu]Cu-DOTA-NOC is the most sensitive peptide to radiolysis, even in the presence of EtOH. Based on these findings, the use of EtOH was implemented in all further labelling formulations to act as a radiolytic stabilizing agent during the labelling reaction.

during the labelling reaction.

a decrease in radiolysis from more than 15% in [61Cu]Cu-DOTA-NOC to less than 5% for [61Cu]Cu-DOTA-NOC and less than 1% for both [61Cu]Cu-DOTA-TOC and [61Cu]Cu-DOTA-TATE. We found that [61Cu]Cu-DOTA-NOC is the most sensitive peptide to radiolysis, even in the presence of EtOH. Based on these findings, the use of EtOH was implemented in all further labelling formulations to act as a radiolytic stabilizing agent

**Figure 4.** Representative chromatograms of [61Cu]Cu-DOTA-NOC, [61Cu]Cu-DOTA-TOC and [61Cu]Cu-DOTA-TATE without (**A**) and with (**B**–**D**) using EtOH (maximum 5 vol% EtOH) during the labelling reaction. Two different HPLC methods were used, as described in Table 4. For a more practical comparative analysis between chromatograms, raw data was normalised as percentage of total radioactivity. Percentage of radiolysis is the ratio of radiolysis counts to total counts. **Figure 4.** Representative chromatograms of [61Cu]Cu-DOTA-NOC, [61Cu]Cu-DOTA-TOC and [ <sup>61</sup>Cu]Cu-DOTA-TATE without (**A**) and with (**B**–**D**) using EtOH (maximum 5 vol% EtOH) during the labelling reaction. Two different HPLC methods were used, as described in Table 4. For a more practical comparative analysis between chromatograms, raw data was normalised as percentage of total radioactivity. Percentage of radiolysis is the ratio of radiolysis counts to total counts. *Pharmaceuticals* **2022**, *15*, x FOR PEER REVIEW 10 of 14

#### *3.3. Synthesis of [61Cu]Cu-Conjugated Peptides on the IBA Synthera® Extension Module*  Fully automated post-processing synthesis was performed using a Synthera® *3.3. Synthesis of [61Cu]Cu-Conjugated Peptides on the IBA Synthera® Extension Module* 5. Reaction mixture is cooled down with water (12 mL) (A) and passed through a C18

Extension module (Figure 5) and completed within a maximum of 25 min from the EOP, without any manual intervention. After the purification process, [61Cu]CuCl2 was automatically transferred to the reaction vial (B). Then, the peptide (DOTA-NOC acetate, DOTA-TOC acetate or DOTA-TATE acetate), dissolved in 2.5 M sodium acetate buffer, was transferred to the same reaction vial, where the reaction occurred. After the labelling reaction, the mixture was cooled down with water, and the product was then purified using a C18 cartridge (Sep-Pak Plus Short C18, Waters, Milford, Massachusetts, USA). After a rinse step, the 61Cu-conjugated peptide was eluted from the C18 cartridge with a mixture of (50/50%) water/ethanol. The general automated synthesis/radiolabelling steps are as follows: 1. The C18 cartridge (C) is preconditioned with ethanol (10 mL) followed by water (10 Fully automated post-processing synthesis was performed using a Synthera® Extension module (Figure 5) and completed within a maximum of 25 min from the EOP, without any manual intervention. After the purification process, [61Cu]CuCl<sup>2</sup> was automatically transferred to the reaction vial (B). Then, the peptide (DOTA-NOC acetate, DOTA-TOC acetate or DOTA-TATE acetate), dissolved in 2.5 M sodium acetate buffer, was transferred to the same reaction vial, where the reaction occurred. After the labelling reaction, the mixture was cooled down with water, and the product was then purified using a C18 cartridge (Sep-Pak Plus Short C18, Waters, Milford, Massachusetts, USA). After a rinse step, the <sup>61</sup>Cu-conjugated peptide was eluted from the C18 cartridge with a mixture of (50/50%) water/ethanol. cartridge at 3 mL/min flow to the waste container (Waste 2); 6. C18 cartridge is then rinsed with water (10 mL) (A), which rinses the column at a 3 mL/min flow; 7. [61Cu]Cu-labelled peptide is finally eluted from the C18 column with a solution of water/EtOH (50/50%) (E) to the final product vial (F) with a 3 mL/min flow. After labelling and purification, the FPV was transferred to the Quality Control (QC) laboratory, and all the components were measured, after which the radiochemical yield was determined.

**Figure 5.** Schematic flow and Synthera® Extension module device and disposable cassette: (A) Water, (B) Reaction vial and oven, (C) SPE C18 cartridge, (D) Buffer and peptide, (E) Ethanol/Water (50%/50%) solution and (F) product vial. **Figure 5.** Schematic flow and Synthera® Extension module device and disposable cassette: (A) Water, (B) Reaction vial and oven, (C) SPE C18 cartridge, (D) Buffer and peptide, (E) Ethanol/Water (50%/50%) solution and (F) product vial.

The HPGe was calibrated with 154Eu and 133Ba radioactive sources and placed in a lowbackground shielding. γ-spectra were acquired using point-source-like samples with a dead-time below 4%. GammaVision (ORTEC Inc., Easley, South Carolina, USA) software

RCP was measured by HPLC (Agilent 1200 series HPLC system, Agilent Technologies, Santa Clara, CA, USA) equipped with a GABIStar NaI(Tl) radiometric detector (Raytest Isotopenmessgeraete GmbH, Straubenhardt, Germany) (20 μL sample volume). Two different methods (Table 4) were used, one for [61Cu]Cu-DOTA-NOC (Method A) and a second to evaluate both [61Cu]Cu-DOTA-TATE and [61Cu]Cu-DOTA-TOC (Method B). An ACE 3 C18 150 × 3 mm HPLC column (ACE, Reading, UK) was used

The stability of 61Cu-conjugated peptides was evaluated under various conditions: in the final formulation (10% EtOH/0.9% NaCl), in the presence of PBS and in mouse serum. All stability measurements were quantified by HPLC, as incubation solutions could affect the accuracy of Thin Layer Chromatography (TLC). The HPLC methods used to evaluate stability were previously described (in Table 4), with exception of [61Cu]Cu-DOTA-NOC. In this case, stability was evaluated using a faster method, with the following gradient: 0–

*3.4. Quality Control*  The general automated synthesis/radiolabelling steps are as follows:

in both methods, and the flow was fixed at 0.6 mL/min.

3.4.1. Radionuclidic Purity (HPGe)

was used to determine photopeak areas.

3.4.2. Radiochemical Purity (Radio-HPLC)

3.4.3. Stability Experiments

5 min Mobile Phase A (100% to 0%).


After labelling and purification, the FPV was transferred to the Quality Control (QC) laboratory, and all the components were measured, after which the radiochemical yield was determined.

#### *3.4. Quality Control*

#### 3.4.1. Radionuclidic Purity (HPGe)

The RNP of copper-61 at EOB was determined through γ-spectroscopy of the final solution using a High Purity Germanium detector (HPGe), several hours after the EOP. The HPGe was calibrated with <sup>154</sup>Eu and <sup>133</sup>Ba radioactive sources and placed in a lowbackground shielding. γ-spectra were acquired using point-source-like samples with a dead-time below 4%. GammaVision (ORTEC Inc., Easley, SC, USA) software was used to determine photopeak areas.

#### 3.4.2. Radiochemical Purity (Radio-HPLC)

RCP was measured by HPLC (Agilent 1200 series HPLC system, Agilent Technologies, Santa Clara, CA, USA) equipped with a GABIStar NaI(Tl) radiometric detector (Raytest Isotopenmessgeraete GmbH, Straubenhardt, Germany) (20 µL sample volume). Two different methods (Table 4) were used, one for [61Cu]Cu-DOTA-NOC (Method A) and a second to evaluate both [61Cu]Cu-DOTA-TATE and [61Cu]Cu-DOTA-TOC (Method B). An ACE 3 C18 150 × 3 mm HPLC column (ACE, Reading, UK) was used in both methods, and the flow was fixed at 0.6 mL/min.

#### 3.4.3. Stability Experiments

The stability of <sup>61</sup>Cu-conjugated peptides was evaluated under various conditions: in the final formulation (10% EtOH/0.9% NaCl), in the presence of PBS and in mouse serum. All stability measurements were quantified by HPLC, as incubation solutions could affect the accuracy of Thin Layer Chromatography (TLC). The HPLC methods used to evaluate stability were previously described (in Table 4), with exception of [61Cu]Cu-DOTA-NOC. In this case, stability was evaluated using a faster method, with the following gradient: 0–5 min Mobile Phase A (100% to 0%).

#### 3.4.4. Stability in Aqueous Solvents

The published protocol was followed with minor changes [56]. Briefly, 50 µL of the final purified solution (Water/EtOH: 50%/50%) containing the radiolabelled <sup>61</sup>Cuconjugated compound under study was added to 450 µL of each medium (0.9% NaCl or PBS), and the mixtures were incubated at 37 ◦C (T0). At different time points (T0, T0 + 1 h, T0 + 2 h, T0 + 4 h, T0 + 6 h and T0 + 12 h), aliquots were taken and measured using the HPLC methods formerly characterised (Table 4).

3.4.5. Stability in Mice Serum

For stability in mice serum, 500 µL of serum was incubated with 50 µL of <sup>61</sup>Cuconjugated peptides dissolved in the final formulation, at 37 ◦C. At different time points (T0, T0 + 1 h, T0 + 2 h, T0 + 4 h, T0 + 6 h and T0 + 12 h), 50 µL aliquots were taken, and 150 µL of ethanol was added to precipitate the plasma proteins. The mixture was centrifuged at 3000 rpm for 10 min and the supernatant was collected and diluted in NaCl 0.9% for HPLC analysis.

#### **4. Conclusions**

In this study, we demonstrated that clinical amounts of <sup>61</sup>Cu-based radiopharmaceuticals can be produced, under GMP, in a medical cyclotron, using liquid targets. Production yields are higher using enriched target in comparison to irradiating natural zinc. The high radionuclidic and radiochemical purity of the produced <sup>61</sup>Cu-labelled radiopharmaceuticals ([61Cu]Cu-DOTA-NOC, [61Cu]Cu-DOTA-TOC and [61Cu]Cu-DOTA-TATE), opens the possibility for them to be used as an alternative to the current clinically used versions with gallium-68. This work serves as background for future preclinical in vitro and in vivo studies aiming at bringing copper-61 radiopharmaceuticals to the clinical setting in the near future.

**Supplementary Materials:** The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/ph15060723/s1, Table S1: Comparison of copper-61 activity produced and purified, corrected at EOB and EOP, respectively, when using non-recycled, once recycled, twice recycled, and three times recycled zinc-64 solution (mean ± SD, N = 8). Isotopic enrichment of the irradiated zinc-64 recycled solution determined by ICP-MS analysis.

**Author Contributions:** Conceptualization, A.I.F., V.H.A. and F.A.; methodology, A.I.F., V.H.A. and S.J.C.d.C.; resources, M.S., I.H. and S.J.C.d.C.; investigation, A.I.F.; writing—original draft preparation, A.I.F.; writing—review and editing, A.J.A., F.A. and V.H.A.; supervision—A.J.A., F.A. and A.F.; project administration, A.J.A. and F.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was founded by the Portuguese Foundation for Science and Technology (FCT) through PhD grants (grant number PD/BDE/150681/2020 and PD/BDE/150331/2019) and by ICNAS-P.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article and supplementary material.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

