2.2. Radiolabelling and In Vitro Stability

For labelling with ⁶⁶Ga, the DFO-ZEGFR:2377 was reconstituted in water to obtain a concentration of 2 mg/mL. The DFO-ZEGFR:2377 solution (30 µg, 15 µL) was mixed with 80 μL of 1.25 M of NaOAc, pH 3.6. The radionuclide solution (50–100 μL, 15 MBq) was added; the mixture was thoroughly vortexed and incubated for 10 min at 85 °C. Radiochemical yield and purity of the labelled conjugate were analyzed using ITLC-SG (Agilent Technologies) developed with PBS (affibody: Rf = 0.0; other forms of ⁶⁶Ga: Rf = 1.0). To further cross-validate radio-ITLC data, reverse phase-HPLC conducted on an Elite LaChrom system (Hitachi, VWR, Darmstadt, Germany) consisting of an L-2130 pump, a UV detector (L-2400), and a radiation flow detector (Bioscan, Washington, DC, USA) coupled in series was used. Purity analysis of labelled compounds was performed using an analytical column (Phenomenex, Aschaffenburg, Germany; Luna® 5 µm C18, 100 Å; 150 × 4.6 mm column). HPLC conditions were as follows: A = 10 mM TFA/H₂O; B = 10 mM TFA/acetonitrile; UV-detection at 220 nm; gradient elution: 0–15 min at 5 to 70% B, 15–18 min at 70 to 95% B, 19–20 min at 5% B; and flow rate of 1.0 mL/min. The retention time of [⁶⁶Ga]Ga-DFO-ZEGFR:2377 is 11.2 min.

Labelling of DFO-ZEGFR:2377 with ⁶⁸Ga was performed using the method described earlier [28]. A generator eluate (120 μL, 100 MBq) was used. DFO-ZEGFR:2377 was labelled with ⁸⁹Zr at 85^°C according to the protocol described earlier [32].

In Vitro stability of [⁶⁶Ga]Ga-DFO-ZEGFR:2377 was evaluated in PBS and also in the presence of 1000-fold molar excess of ethylenediaminetetraacetic acid (EDTA). Namely, after purification, samples of freshly labelled conjugate (1.4 µg, 50 µL) were mixed with EDTA (60 µg, 2 mg/mL in PBS) to obtain a 1000-fold molar excess of EDTA and incubated at room temperature for 1 h. Control samples were mixed with an equal volume of PBS. The experiment was performed in triplicate.