Lachrom system

The HPLC apparatus was comprised of a HITACHI ELITE LaChrom system (L2130) equipped with a Hitachi L-3000 detector and a D-2500 chromato-integrator. Peptides were purified by RP-HPLC using a Cosmosil 5C₁₈-AR-II column (4.6 × 150 mm, Nacalai tesque Inc., Kyoto, Japan). The peptides were separated by a linear gradient of CH₃CN in 0.05% TFA increasing at a rate of 1%/min from solvent A (0.05% TFA/H₂O) to solvent B (0.05% TFA/CH₃CN) at a flow rate of 1 mL/min [8 (link),21 (link)].

All reagents were of analytical grade and used without further purification unless otherwise stated. Chelex-100 resin was purchased from Bio-Rad Laboratories (Richmond, CA) and used with aqueous buffers for radiolabeling experiments to ensure metal-free conditions. Trastuzumab and pertuzumab were obtained from Genentech (San Francisco, CA). Control IgG1 antibodies, 88R20 and mAb-69 were kindly provided by Dr. Kendra Carmon and Dr. Barret R. Harvey, the University of Texas Health Science Center at Houston. IRDye800CW-NHS was purchased from LI-COR Biosciences (Lincoln, NE). Desferrioxamine-p-benzyl-isothiocyanate (DFO-Bz-NCS) was purchased from Macrocyclics (Plano, TX). Zirconium-89 (⁸⁹Zr)-oxalate was produced by Washington University School of Medicine (St. Louis, MO). Size-exclusion high-performance liquid chromatography (SEC-HPLC) was performed on an analytical Hitachi LaChrom system using a TSKgel G3000SW (5 μm) column and mobile phases of A = 0.1 M sodium phosphate buffer (pH 7.3) and B = CH₃CN (isocratic: 90% A and 10% B) and a flow rate of 1 mL/min. Radiochemical yield and purity were measured with a radio-thin-layer chromatography (TLC)/HPLC detector system (LabLogic) using previously described methods [15 (link)].