*3.3. Specific Activity and Radionuclidic Purity*

The specific activity in the <sup>89</sup>Zr-immuno-PET imaging is vitally important because the amount of antibody injected in the preclinical or clinical subjects can alter the quality immuno-PET images and quantification. Particularly, tumour-specific antibodies or peptides for diagnostic and therapeutic applications where only small amounts of antibodies are injected to ensure site-specific uptake. Metallic impurities can compete with the radioisotope for binding sites or ligands during the radiolabelling process with a resulting decrease in radiolabelling efficiency. In order to achieve higher radiolabelling efficiency, radionuclides need to be free from metallic impurities and this can be achieved by selecting high purity target material, energy window for irradiation and trace metal grade solutions for processing. The conventional method to determine effective specific activity (ESA) of <sup>89</sup>Zr is titration with commonly used chelator deferoxamine (DFO). In this method, the known quantities of *p-SCN-Bz*-DFO is titrated with <sup>89</sup>Zr. The specific activity of three production batches was calculated and was found to be in the range of 1351–2323 MBq/µmol. Our effective specific activities by titration method were low compared to reported data by Queern et al. [20], but similar to those of Wooten et al. [14]. Our ICP-MS analysis showed significantly lower concentration of metallic impurities compared with those of Queern et al. [20].

The radionuclidic purity of <sup>89</sup>Zr mainly depends on the purity of the target material and irradiation conditions. The [89Zr]zirconium oxalate solution was tested for radionuclidic identity and purity using a high purity germanium (HPGe) detector (Figure 3A). The analysis showed the presence of 909 and 511 keV peaks. No additional radioactive contamination was detected. In these studies, the radionuclidic purity of the isolated <sup>89</sup>Zr fractions was found to be >99.99%. Similar values for radionuclidic purity have been reported by other authors [1,14,20]. presence of 909 and 511 keV peaks. No additional radioactive contamination was detected. In these studies, the radionuclidic purity of the isolated 89Zr fractions was found to be >99.99%. Similar values for radionuclidic purity have been reported by other authors [1,14,20]. Gamma spectrometry of waste was also performed to determine radioactive impurities produced during irradiation. These impurities were successfully removed using hydroxamate resin as these impurities were not adhered on hydroxamate resin cartridge. The radioactive impurities produced during production were 52Mn, 54Mn, 56Co, 65Zn, 67Ga, 96Tc, 48V (Figure 3B).

**Figure 3. A**) Gamma spectrum of purified sample of 89Zr taken 8h after the end-of-bombardment (EOB). **B**) Gamma spectrum of impurities found in waste vial following 89Zr production. **Figure 3.** (**A**) Gamma spectrum of purified sample of <sup>89</sup>Zr taken 8h after the end-of-bombardment (EOB). (**B**) Gamma spectrum of impurities found in waste vial following <sup>89</sup>Zr production.

*3.4. Radiolabeling and Characterization of 89Zr-DFO-Trastuzumab* Radiolabeling experiments were performed as described in the literature [3,19]. To obtain the quantitative radiolabeling yield and to determine specific activity, several reactions with different concentrations of DFO-trastuzumab were performed and analysed on iTLC (Figure 4 A and B). Gamma spectrometry of waste was also performed to determine radioactive impurities produced during irradiation. These impurities were successfully removed using hydroxamate resin as these impurities were not adhered on hydroxamate resin cartridge. The radioactive impurities produced during production were <sup>52</sup>Mn, <sup>54</sup>Mn, <sup>56</sup>Co, <sup>65</sup>Zn, <sup>67</sup>Ga, <sup>96</sup>Tc, <sup>48</sup>V (Figure 3B).

Reported specific activities of 89Zr-DFO-trastuzumab typically range from 0.067 to 0.296 MBq/µg

#### [20,22,23]. *3.4. Radiolabeling and Characterization of <sup>89</sup>Zr-DFO-Trastuzumab*

was added to the DFO-trastuzumab solution for labeling.

The labeling efficiency of 89Zr-DFO-trastuzumab was 100% ± 5% when 25 µg of DFOtrastuzumab and 3.3 MBq 89Zr was used. The specific activity was 0.308 MBq/µg. which is comparable to Queern et al*.* [20] (specific activity 0.296 MBq/µg with DFO-trastuzumab). The results of radiolabelling efficiencies are summarized in Table 1. **Table 1.** The labelling efficiency of 89Zr with DFO conjugated trastuzumab were evaluated at 37 °C Radiolabeling experiments were performed as described in the literature [3,19]. To obtain the quantitative radiolabeling yield and to determine specific activity, several reactions with different concentrations of DFO-trastuzumab were performed and analysed on iTLC (Figure 4A,B). Reported specific activities of <sup>89</sup>Zr-DFO-trastuzumab typically range from 0.067 to 0.296 MBq/µg [20,22,23].

with varying concentrations of conjugate in HEPES buffer, pH 7. A constant ~3.3 MBq of 89Zr-oxalate

**Mass of DFO-trastuzumab (µg) Labeling Efficiency (%)** 200 100 50 100 25 100 3.125 74.24 0.39 73.41 0.048 62.43

**Figure 4. A**). Sigmoidal dose–response curve of *p*-isothiocyanatobenzyl-desferoxamine (*p-SCN-Bz*-DFO)– <sup>89</sup>Zr titration, showing effective specific activity of 2323 MBq/µmol. **B**) Sigmoidal dose– response curve of <sup>89</sup>Zr-DFO-trastuzumab titration showing effective specific activity of 0.308 MBq/µg. *3.5. Elemental Analysis* **Figure 4.** (**A**). Sigmoidal dose–response curve of *p*-isothiocyanatobenzyl-desferoxamine (*p-SCN-Bz*-DFO)–89Zr titration, showing effective specific activity of 2323 MBq/µmol. (**B**) Sigmoidal dose–response curve of <sup>89</sup>Zr-DFO-trastuzumab titration showing effective specific activity of 0.308 MBq/µg.

The ICP-MS analyses of three batches of the final 89Zr solution (1.0 mL) were performed after decay of 89Zr. The concentrations of impurities such as yttrium, zirconium, zinc, aluminium, copper, nickel, iron, chromium, niobium and magnesium were quantified by ICP-MS (Table 2). All impurity levels were significantly lower than previously reported values [15,20] and always below <1.0 ppm The labeling efficiency of <sup>89</sup>Zr-DFO-trastuzumab was 100% <sup>±</sup> 5% when 25 <sup>µ</sup>g of DFO-trastuzumab and 3.3 MBq <sup>89</sup>Zr was used. The specific activity was 0.308 MBq/µg. which is comparable to Queern et al. [20] (specific activity 0.296 MBq/µg with DFO-trastuzumab). The results of radiolabelling efficiencies are summarized in Table 1.

**Table 2.** ICP-MS analysis of three consecutive <sup>89</sup>Zr production runs. **Sample # Zr (ppm) Al (ppm) Y (ppm) Fe (ppm) Cu (ppm) Cr (ppm) Ni (ppm) Zn (ppm) Mg (ppm) Nb (ppm)** PV1 **Table 1.** The labelling efficiency of <sup>89</sup>Zr with DFO conjugated trastuzumab were evaluated at 37 ◦C with varying concentrations of conjugate in HEPES buffer, pH 7. A constant ~3.3 MBq of <sup>89</sup>Zr-oxalate was added to the DFO-trastuzumab solution for labeling.


#### with high radionuclidic purity using the commercially available TRASIS miniAiO automation module. This method showed reproducible results for the production of [89Zr]zirconium oxalate. *3.5. Elemental Analysis*

Additionally, the automated process with disposable cassettes provides sterile [89Zr]zirconium oxalate and documentation of the manufacturing process that can be used to fulfil cGMP requirements. This automated process can also be adopted for [89Zr]Cl<sup>2</sup> production. With adequate shielding, the module ensures safe user operation and environmental radiation protection. This study The ICP-MS analyses of three batches of the final <sup>89</sup>Zr solution (1.0 mL) were performed after decay of <sup>89</sup>Zr. The concentrations of impurities such as yttrium, zirconium, zinc, aluminium, copper, nickel, iron, chromium, niobium and magnesium were quantified by ICP-MS (Table 2). All impurity levels were significantly lower than previously reported values [15,20] and always below <1.0 ppm

**Table 2.** ICP-MS analysis of three consecutive <sup>89</sup>Zr production runs.

