Amicon ultra 4 centrifugal filtration units

For each antibody construct, 500 μg of immunoconjugate solution was diluted to 400μL with PBS, pH 7.4. [⁸⁹Zr]Zr-oxalate (1500 μCi) in 150 μL of 1.0 M oxalic acid was adjusted to pH 7.0–7.5 with 1.0 M Na₂CO₃. After the bubbling of CO₂ stopped, the ⁸⁹Zr solution was added to the antibody solution, and the resulting mixture was incubated at room temperature for 1h. The reaction progress was then assayed using iTLC and an eluent of 50mM EDTA (pH 5). Subsequently, the reaction was quenched with 13 μL of 50mM of EDTA (pH=5), and the antibody construct was purified using size exclusion chromatography (Sephadex G-25 M, PD-10 column, GE Healthcare; dead volume = 2.5 mL, eluted with 500 μL fractions of PBS, pH 7.4) and if necessary concentrated via centrifugal filtration units with a 50,000 molecular weight cut off (Amicon™ Ultra 4 Centrifugal Filtration Units, Millipore Corp. Billerica, MA). The radiochemical purity of the final radiolabeled bioconjugate was assayed by radio-TLC again using 50mM EDTA (pH 5) as an eluent. In the iTLC experiments, free ⁸⁹Zr⁴⁺ cations and [⁸⁹Zr]-EDTA elute with solvent front, while radiolabeled antibody construct remains at the baseline.

To a suspension of 200 μg of antibody in PBS pH 7.4 (1 mg/mL) was added 1.33 μL of a fresh TCEP solution (10 mM in water, 10 eq.) and the appropriate volume of a solution of PODS-FL (1 mM in DMSO). The reaction mixture was stirred on a thermomixer (25°C or 37°C) for 30 min, 2 h, or 24 h. The conjugate was then purified on a size exclusion column (Sephadex G-25 M, PD-10 column, GE Healthcare; dead volume = 2.5 mL, eluted with 2 mL of PBS, pH 7.4) and concentrated using centrifugal filtration units with a 50,000 Da molecular weight cut off (Amicon™ Ultra 4 Centrifugal Filtration Units, Millipore Corp. Billerica, MA).

To a suspension of 1 mg of antibody in PBS pH 7.4 (1 mg/mL) was added 6.7 μL of a fresh TCEP solution (10 mM in water, 10 eq.) and 33 μL of a solution of PODS-DFO-Fe (2 mM in DMSO). The reaction mixture was stirred on a thermomixer at 2 5°C for 2 hours. To this yellow solution was then added 100 μL of EDTA (tetrasodic salt, 25 g/L in water), and the pH was adjusted to 4.5 with 0.25 M H₂SO₄. The mixture was stirred at 25 °C for 30 min to yield a colorless solution, indicating the transchelation of the Fe(III) from the DFO. The conjugate was then purified on a size exclusion column (Sephadex G-25 M, PD-10 column, GE Healthcare; dead volume = 2.5 mL, eluted with 2 mL of PBS, pH 7.4) and concentrated using centrifugal filtration units with a 50,000 Da molecular weight cut off (Amicon™ Ultra 4 Centrifugal Filtration Units, Millipore Corp. Billerica, MA).