Itlc paper

DFO^Z(35BPA)-huA33 and DFO-huA33 (0.2 mg) were diluted in Chelex-treated PBS (pH 7.4). [⁸⁹Zr]Zr⁴⁺ [74.0 – 185.0 MBq (2.0 – 5.0 mCi)] in 1.0 M oxalic acid was diluted with Chelex-treated PBS, and the pH of the solution was adjusted to 7.4 with 1.0 M Na₂CO₃. The mAb solutions were combined with pH-adjusted zirconium-89, mixed thoroughly, and incubated on a ThermoMixer for 1 h at 37 °C and 500 rpm. The reaction was monitored using glass fiber silica-impregnated instant thin-layer chromatography (iTLC) paper (Pall Corp.; East Hills, NY, USA) using an EDTA solution as the eluent (50 mM, pH 5.5). The iTLC plates were analyzed on an AR-2000 radioi-TLC plate reader with WinScan Software (Bioscan, Inc.; Washington, DC, USA). Following the reaction, the radioimmunoconjugates were purified using size exclusion chromatography (PD-10 Column). Radiochemical purities were assayed using radio-iTLC with EDTA as the eluent (50 mM, pH 5.5).

DFO-pertuzumab, ^SSKDFO-pertuzumab, and ^malDFO-pertuzumab (0.5 mg) were diluted in Chelex PBS (pH 7.4) to a final concentration of 1 mg/mL. [⁸⁹Zr]Zr⁴⁺ [92.5 MBq – 185.0 MBq (2.5 mCi – 5 mCi)] in 1.0 M oxalic acid was diluted with Chelex PBS and the solution pH was adjusted to 7.0-7.5 with 1.0 M Na₂CO₃ (final volume: 100 μL). After CO₂ bubbling ceased, the ⁸⁹Zr solution was added to the antibody solution, mixed thoroughly, and reacted on a thermomixer (500 rpm, 37 °C, 15 min). The reaction progress was assayed using glass-fiber, silica-impregnated instant thin-layer chromatography (iTLC) paper (Pall Corp.; East Hills, NY), eluted with 50 mM EDTA (pH 5.0), and analyzed on an AR-2000 radio-iTLC plate reader using Winscan Radio-TLC software (Bioscan, Inc.; Washington, DC). Following the completion of the reaction, any free [⁸⁹Zr]Zr⁴⁺ was removed from the radioimmunoconjugate using size exclusion chromatography (PD-10 column). The radiochemical purity of the final radiolabeled construct was assayed using radio-iTLC with EDTA as the eluent (50 mM, pH 5.0). All radiolabeling studies were performed in triplicate.