FIGURE 1. Schematic representation of premodification and postlabeling of mAbs with ⁸⁹Zr. Step 1 is synthesis of N-sucDf, as described in Materials and Methods. Step 2 is complexation of N-sucDf with Fe(III). N-sucDf (9 mg, 13.6 µmol) is dissolved in 3 mL of 0.9% NaCl, containing 60 µL of 0.1 mol/L Na₂CO₃ (final pH 6.5–7.0). To this solution, 300 µL (14.8 µmol) of FeCl₃ solution (8 mg/mL in 0.1 mol/L HCl) is added. Step 3 is esterification of N-sucDf-Fe. After 10 min, to N-sucDf-Fe solution are added 300 µL (0.36 mmol) of TFP solution (200 mg/mL in MeCN) and 120 mg (0.63 mmol) of solid EDC (final pH 5.8–6.0). After 45 min of incubation, reaction mixture is loaded onto conditioned Sep-Pak C₁₈ cartridge (Waters), followed by washing with 60 mL of sterile water for injection. TFP-N-sucDf-Fe is eluted from Sep-Pak cartridge with 1.5 mL of MeCN. Step 4 is conjugation of TFP-N-sucDf-Fe to mAb. To 1 mL (33 nmol) of mAb solution (5 mg/mL), pH 9.5–9.8 (adjusted with 0.1 mol/L Na₂CO₃), 20 µL (63 nmol) of TFP ester solution (2.5 mg/mL in MeCN) are added to obtain final chelate:mAb ratio of 1:1 (based on 54% reaction efficiency). After 30 min, 2 times 25 µL of gentisic acid solution (100 mg/mL in 0.32 mol/L Na₂CO₃) are added to reaction mixture and pH is adjusted to 4.3–4.5 with 4 times 6 µL of 0.25 mol/L H₂SO₄. Step 5 is removal of Fe(III) from mAb-N-sucDf-Fe. To reaction mixture, 50 µL (3.3 µmol) of an EDTA solution (25 mg/mL) is added and solution is incubated for 30 min at 35°C (final pH 4.3–4.5). After 30 min, EDTA, TFP, iron (as [Fe(III)EDTA]^-), and unreacted hydrolyzed ester (N-sucDf) are removed by gel filtration using PD-10 column (eluent: 0.9% NaCl/gentisic acid [5 mg/mL], pH 5): First 2.6 mL (containing reaction volume and first 1.5 mL) are discarded, and modified mAb is collected in next 2 mL. Step 6 is labeling of mAb-N-sucDf with ⁸⁹Zr. To 600 µL of ⁸⁹Zr oxalic acid solution (1 mol/L oxalic acid), 130 µL of 0.9% NaCl, 270 µL of 2 mol/L Na₂CO₃, and 3 mL of 0.5 mol/L HEPES (pH 7.2–7.4) are added, followed by 2 mL (33 nmol) of modified mAb solution (2.5 mg/mL in 0.9% NaCl/gentisic acid [5 mg/mL], pH 5), final pH 7.2–7.4. Reaction volume can be varied provided amounts of oxalic acid, Na₂CO₃, and HEPES buffer are adjusted accordingly. After 30 min, reaction mixture (6 mL) is divided over 3 PD-10 columns (eluent: 0.9% NaCl/gentisic acid [5 mg/mL], pH 5): First 2.5 mL (2-mL sample volume and first 0.5 mL) are discarded, and radiolabeled mAb is collected in next 3 mL. Bold arches represent -(CH₂)₂CONH(CH₂)₅-. Desferal (Df) (Novartis) is term used instead of desferrioxamine B, and Df-Fe is term used to represent corresponding iron(III) complex (ferrioxamine).

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