*2.1. [61Cu]CuCl<sup>2</sup> Production*

Copper-61 was produced using the target system previously described in [20] and then in [43]. Several cyclotron irradiations were performed with both natural and enriched zinc. Table 1 summarises the number of runs, the irradiation conditions and the activity produced for each target. The same solution of zinc-64, with an initial concentration of 200 mg/mL, was irradiated a maximum of four times.


**Table 1.** Irradiation conditions applied to each target and total activity produced (GBq) at EOB.

Initial concentration before recycling. EOB: End Of Bombardment.

A direct comparison between the 180 min long irradiation of natural zinc and first-time irradiated enriched zinc-64 showed that, under the same irradiation conditions, i.e., time, concentration, current and pressure, the use of zinc-64 allowed the production of twice the activity of copper-61 than natural zinc: 3.65 ± 0.18 GBq (N = 8) and 1.84 ± 0.24 GBq (N = 20), respectively. These correspond to low yields when compared to solid targets, as stated in [44]; however, the latter also come with high cost and tremendous operational complexity. These studies confirmed the expected higher activities of copper-61 from the enriched target, considering the 49.2% abundance of the zinc-64 isotope in the natural zinc. Although higher activities of copper-61 are produced using enriched target material, zinc-64 is approximately 200 times more expensive than the natural target (550–669 €/g zinc-64 vs. 2.92 €/g natural zinc). Given this tremendous difference, a cost–benefit analysis is required.

## *2.2. Recovery and Recycling of <sup>64</sup>Zn*

One of the advantages of using liquid targets is that the recycling of enriched material is simplified. This is especially important when considering the high cost of zinc-64.

Notwithstanding, few authors have actually described this. In this study, the zinc-64 target was recovered from the CU resin waste container several days after been irradiated. It was then evaporated and re-dissolved into the initial form of 10 mM HNO3. Moreover, the recycling process was simple, since no solvent other than HNO<sup>3</sup> was introduced during the purification process, and the zinc-64 solution could be re-used directly after filtration. The percentage of zinc-64 recovered and re-irradiated was collectively determined to be higher than 90% each time it was recycled. We found a slight variation in the activity produced (corrected at EOB), depending on how many times the zinc-64 solution was recycled and subsequently irradiated (Table S1). The second irradiation of the same batch of zinc-64 did not show a significant decrease in the amounts of produced nor purified copper-61. On the other hand, with more than two irradiations, there was a statistically significant decrease in the amount of copper-61 produced and, consequently, in purified copper (Table S1). This decrease in the activity of copper-61 is explained by the loss of zinc-64 during the several steps of the process: recycling, purification, evaporation and final filtration of the solution. Regarding isotopic enrichment of the recycled solutions (Table S1), the recovery process did not lead to a significant decrease in zinc-64 enrichment.

The purification process was performed as described earlier [45] without further modifications.

## *2.3. [61Cu]Cu-DOTA-NOC, [61Cu]Cu-DOTA-TATE and [61Cu]Cu-DOTA-TOC Production Activity Distribution*

[ <sup>61</sup>Cu]Cu-DOTA-NOC, [61Cu]Cu-DOTA-TOC and [61Cu]Cu-DOTA-TATE were produced using the Synthera® Extension automated module (IBA, Louvain-la-Neuve, Belgium). This fully automated process complies with GMPs to produce radiopharmaceuticals (EudraLex, Volume 4, Annex 3) (i.e., the use of disposable cassettes and tubing systems, ensuring high quality and reproducibility of the final radiopharmaceutical product and the narrowing of the risk of radioactive cross-contamination). A total of 50 µg of DOTA-NOC (N = 10), DOTA-TATE (N = 3) and DOTA-TOC (N = 3) were labelled with purified [ <sup>61</sup>Cu]CuCl<sup>2</sup> at a 85–100 ◦C reaction temperature and 10 min reaction time. Specifications are summarised in Table 2.

**Table 2.** Summary of activities and Yields (i.e., Labelling Yield and RCY) achieved in the radiopharmaceutical synthesis of [61Cu]Cu-DOTA-NOC, [61Cu]Cu-DOTA-TATE and [61Cu]Cu-DOTA-TOC produced from either natural or enriched zinc.


RCY: radiochemical yield. EOS: End Of Synthesis.

As expected, depending on the production route of [61Cu]CuCl<sup>2</sup> used, the greatest difference found was in the amount of [61Cu]Cu-DOTA-NOC activity at the End Of Synthesis (EOS): 0.99 ± 0.16 GBq or 1.95 ± 0.21 GBq from natural or enriched targets, respectively. To evaluate the efficacy and reproducibility of this synthesis method, we determined the radiochemical and labelling yields of the process. Radiochemical Yield (RCY) refers to the final activity in the product of [61Cu]Cu-DOTA-NOC/TOC/TATE, expressed as the percentage (%) of starting activity of [61Cu]CuCl<sup>2</sup> obtained after purification [46]. The quantity of both was decay-corrected to the same time point. All radioactivity lost during transfer, labelling reaction, solid phase purification (SPE) and dispensing were accounted for in the RCY. Whereas the labelling yield indicated the direct yield of the labelling reaction.

Nonetheless, labelling yield referred only to the extent of the labelling reaction, comparing the amount of [61Cu]CuCl<sup>2</sup> that reacted into [61Cu]Cu-DOTA-NOC/TOC/TATE, and did not consider any other process losses. The data showed that neither labelling yields nor RCY were affected by the amount of activity, which confirms that activities of copper-61 up to 2.7 GBq (at the End Of Purification (EOP)) do not have negative effects on the synthesis process of these radiopharmaceuticals. [61Cu]Cu-DOTA-NOC N = 5 Natural Zinc 32 ± 4 0.99 ± 0.16 98.48 ± 0.89 94.73 ± 3.03 [61Cu]Cu-DOTA-NOC N = 5 Zinc-64 38 ± 2 1.95 ± 0.21 97.72 ± 2.01 94.03 ± 1.84 [61Cu]Cu-DOTA-TATE N = 3 Zinc-64 37 ± 6 2.06 ± 0.08 98.61 ± 0.84 95.91 ± 1.50

*Pharmaceuticals* **2022**, *15*, x FOR PEER REVIEW 5 of 14

efficacy of the automated synthesis process.

**(min)** 

**Radiopharmaceutical Target Process Duration** 

DOTA-TOC produced from either natural or enriched zinc.

results for all peptides. It is important to note the low residual activity in the different components and the small SD of its values, which reflects both high reproducibility and

**Table 2.** Summary of activities and Yields (i.e., Labelling Yield and RCY) achieved in the radiopharmaceutical synthesis of [61Cu]Cu-DOTA-NOC, [61Cu]Cu-DOTA-TATE and [61Cu]Cu-

**Labelling Yield** 

**(%) RCY (%)** 

**Activity @EOS (GBq)** 

We also compared the distribution patterns regarding the different cassette components for all radiopharmaceuticals (Figure 1). Activity distribution revealed similar results for all peptides. It is important to note the low residual activity in the different components and the small SD of its values, which reflects both high reproducibility and efficacy of the automated synthesis process. [61Cu]Cu-DOTA-TOC N = 3 Zinc-64 38 ± 4 1.77 ± 0.12 97.87 ± 1.10 94.67 ± 1.19 RCY: radiochemical yield. EOS: End Of Synthesis.

**Figure 1.** Activity distribution of the different cassette components after synthesis on Synthera® Extension module: Final Product Vial, C18 SPE cartridge, Waste and Reaction vial. Data comprises the different radiopharmaceuticals produced (mean ± SD, N ≥ 3). **Figure 1.** Activity distribution of the different cassette components after synthesis on Synthera® Extension module: Final Product Vial, C18 SPE cartridge, Waste and Reaction vial. Data comprises the different radiopharmaceuticals produced (mean ± SD, N ≥ 3).

#### *2.4. Quality Control*

*2.4. Quality Control*  Table 3 outlines the final product specifications obtained, including radiochemical and radionuclidic purity, radionuclidic identity and pH. Radiochemical purity was evaluated by radio-HPLC, using the methods described in the next section (Table 4 in Materials and Methods). Radionuclidic purity was evaluated using a High Purity Germanium (HPGe) detector, several hours after EOS. A single value of radionuclidic purity is presented for [61Cu]Cu-DOTA-NOC, [61Cu]Cu-DOTA-TOC and [61Cu]Cu-DOTA-TATE produced from the enriched target, as radionuclidic purity is only Table 3 outlines the final product specifications obtained, including radiochemical and radionuclidic purity, radionuclidic identity and pH. Radiochemical purity was evaluated by radio-HPLC, using the methods described in the next section (Table 4 in Materials and Methods). Radionuclidic purity was evaluated using a High Purity Germanium (HPGe) detector, several hours after EOS. A single value of radionuclidic purity is presented for [ <sup>61</sup>Cu]Cu-DOTA-NOC, [61Cu]Cu-DOTA-TOC and [61Cu]Cu-DOTA-TATE produced from the enriched target, as radionuclidic purity is only dependent on the method of copper-61 production, regardless of the subsequent synthesis process.

dependent on the method of copper-61 production, regardless of the subsequent synthesis process. As expected, the choice of target material has an impact on radionuclidic purity. When copper-61 is produced from natural zinc, 1.5% of copper-64 (at EOB) is produced simultaneously with copper-61 [20], whereas when an enriched target is used, almost no copper impurities are expected to be produced; however, small amounts of copper-64 are present as a side product, resulting from the (p,α) nuclear reactions on residuals zinc-67 and zinc-68 present in the enriched target material. This amounts to about 0.03% of copper-64 co-produced when irradiating zinc-64. Figure 2 indicates the impact of this percentage of copper-64 on the shelf life of the product when each target is used.


**Table 3.** Final product specifications for [61Cu]Cu-DOTA-NOC, [61Cu]Cu-DOTA-TATE and [61Cu]Cu-DOTA-TOC (mean ± SD, N ≥ 3).

MA: Molar activity. RCP: Radiochemical Purity. RNP: Radionuclidic Purity. *Pharmaceuticals* **2022**, *15*, x FOR PEER REVIEW 6 of 14

**Table 4.** HPLC methods for RCP determination.


**Figure 2.** Radionuclidic purity (%) of copper-61 produced from liquid targets before purification (blue lines obtain from calculations [20]) and after purification (black lines obtain from HPGe measurements) either from natural Zinc (dashed lines) or enriched Zinc-64 (solid lines) experimental values determined by HPGe. **Figure 2.** Radionuclidic purity (%) of copper-61 produced from liquid targets before purification (blue lines obtain from calculations [20]) and after purification (black lines obtain from HPGe measurements) either from natural Zinc (dashed lines) or enriched Zinc-64 (solid lines) experimental values determined by HPGe.

**Table 3.** Final product specifications for [61Cu]Cu-DOTA-NOC, [61Cu]Cu-DOTA-TATE and

**Production Route natZn(p,α)61Cu 64Zn(p,α)61Cu**  124

RCP (%) 99.48 ± 0.51 98.71 ± 0.57 99.90 ± 0.03 99.77 ± 0.16

pH 3–5 3–5 3–5 3–5 Visual Inspection Clear, Colourless Clear, Colourless Clear, Colourless Clear, Colourless Volume (mL) 5–10 5–10 5–10 5–10

MA: Molar activity. RCP: Radiochemical Purity. RNP: Radionuclidic Purity.

MA (MBq/nmol) 28.93 ± 4.58 56.82 ± 6.25 52.31 ± 9.83 50.27 ± 3.40 Activity at EOS (GBq) 0.99 ± 0.16 1.95 ± 0.21 2.06 ± 0.08 1.77 ± 0.12

Radionuclidic identity (h) 3.33 ± 0.04 3.33 ± 0.04 3.33 ± 0.04 3.33 ± 0.04

**TEST [61Cu]Cu-DOTA-NOC [61Cu]Cu-DOTA-NOC [61Cu]Cu-DOTA-TATE [61Cu]Cu-DOTA-TOC** 

[61Cu]Cu-DOTA-TOC (mean ± SD, N ≥ 3).

RNP (%) 98.49 ± 0.07 99.97 ± 0.03

Currently, no European Pharmacopeia (Ph. Eur.) monograph exists for copper-61 [47]. Taking into consideration the limits set for radionuclidic impurities in the Gallium (68Ga) Chloride (accelerator produced) monograph of 2% (mon. 3109), the production of copper-61 from natural Zinc would require it to be used immediately after purification. On the other hand, when produced from zinc-64, the radionuclidic purity of copper-61 is higher than 99% and remains at this level for many hours after production (Figure 2). [47]. Taking into consideration the limits set for radionuclidic impurities in the Gallium (68Ga) Chloride (accelerator produced) monograph of 2% (mon. 3109), the production of copper-61 from natural Zinc would require it to be used immediately after purification. On the other hand, when produced from zinc-64, the radionuclidic purity of copper-61 is higher than 99% and remains at this level for many hours after production (Figure 2). *2.5. In Vitro Stability* 

Currently, no European Pharmacopeia (Ph. Eur.) monograph exists for copper-61

**Time (min) Mobile Phase A** 

Solvents Water/0.1% TFA ACN/0.1% TFA

**(Per Cent** *v***/***v***)** 

0–11 74 → 60 26 → 40 11–12 60 → 40 40 → 60 12–14 40 60

0–8 78 22 8–9 78 → 40 22 → 60 9–14 40 60

**Mobile Phase B (Per Cent** *v***/***v)*

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**Table 4.** HPLC methods for RCP determination.

#### *2.5. In Vitro Stability* The stability of [61Cu]Cu-DOTA-NOC, [61Cu]Cu-DOTA-TOC and [61Cu]Cu-DOTA-

Method A

Method B

The stability of [61Cu]Cu-DOTA-NOC, [61Cu]Cu-DOTA-TOC and [61Cu]Cu-DOTA-TATE in aqueous solution (NaCl 0.9% or PBS) and in mouse serum was evaluated up to 12 h after the EOS. Figure 3 shows that all radiopharmaceuticals were stable under the conditions tested. Radiochemical purity results indicated that these compounds are highly stable (over 95%) at 37 ◦C up to 12 h after the EOS, in the final formulation (NaCl 0.9%), PBS and serum. TATE in aqueous solution (NaCl 0.9% or PBS) and in mouse serum was evaluated up to 12 h after the EOS. Figure 3 shows that all radiopharmaceuticals were stable under the conditions tested. Radiochemical purity results indicated that these compounds are highly stable (over 95%) at 37 °C up to 12 h after the EOS, in the final formulation (NaCl 0.9%), PBS and serum.

**Figure 3.** Stability of [61Cu]Cu-DOTA-NOC (**A**), [61Cu]Cu-DOTA-TATE (**B**) and [61Cu]Cu-DOTA-TOC (**C**) in NaCl 0.9%, PBS and mouse serum. Radiochemical purity results were obtained by radioHPLC at: T0, T0 + 1 h, T0 + 2 h, T0 + 4 h, T0 + 6 h and T0 + 12 h, where T0 represents the EOS. **Figure 3.** Stability of [61Cu]Cu-DOTA-NOC (**A**), [61Cu]Cu-DOTA-TATE (**B**) and [61Cu]Cu-DOTA-TOC (**C**) in NaCl 0.9%, PBS and mouse serum. Radiochemical purity results were obtained by radioHPLC at: T0, T0 + 1 h, T0 + 2 h, T0 + 4 h, T0 + 6 h and T0 + 12 h, where T0 represents the EOS.

#### **3. Materials and Methods**

All chemicals and solvents used for purification of [61Cu]CuCl<sup>2</sup> and synthesis of <sup>61</sup>Cuconjugated peptides were trace metal grade, and HPLC solvents were HPLC grade. The remaining solvents and reagents (i.e., hydrochloric acid > 30% and nitric acid > 69% (Honeywell Fluka, Charlotte, NC, USA), bi-distilled water (BBraun, Melsungen, Germany), ethanol (Rotem, Israel), sodium acetate anhydrous (Honeyweell Fluka, Germany), *L*-ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA) and DTPA (Alfa Aesar, Kandel, Germany)) were also trace metal basis, to prevent metal cross-contamination.

Zinc (99.998%) was acquired from Alfa Aesar, whereas the enriched zinc metal form ( <sup>64</sup>Zn—99.89%) was obtained from CMR (Moscow, Russia). Purification and labelling disposable kits were purchased from Fluidomica (Cantanhede, Portugal) and purification resins (i.e., CU-B25-A resin and SAX 1 × 8 200–400 mesh, Cl<sup>−</sup> form resin) from Triskem (Bruz, Belgium). Peptides DOTA-NOC acetate, DOTA-TATE acetate and DOTA-TOC acetate, fractioned and kept at −20 ◦C in an aqueous solution, were manufactured by ABX (Radeberg, Germany). The usage of polyethylene and polypropylene materials was favoured over that of glass materials.
