*2.2. <sup>165</sup>Er Radiochemical Isolation*

After irradiation, holmium cyclotron targets were dissolved in 11 M HCl (2 mL, Trace Select Ultra, Fluka Analytical, Buchs, Switzerland), followed by evaporation to dryness at 120 ◦C under Ar flow. The resulting yellow/pink salts were dissolved in 70 mM αHIB (2 mL, pH = 4.7). The αHIB solution was freshly prepared by dissolving α-hydroxyisobutyric acid (99%, Sigma Aldrich, St. Louis, MO, USA or 98%, Acros Organics, Geel, Belgium) with 18 MΩ·cm ultrapure water (Milli-Q) and pH adjusted with 25% ammonia solution (Fisher Scientific, Waltham, MA, USA) using a pH probe (Checker® pH tester, Hanna Instruments, Woonsocket, RI, USA). A 30–60 µL aliquot of the dissolved, reconstituted target was assayed for <sup>165</sup>Er radioactivity by ionization chamber dose calibrator (CRC-15R, setting #260, Capintec Inc., Florham Park, NJ, USA). The dose calibrator setting #260 was experimentally determined by cross-calibration with HPGe gamma spectrometry. The <sup>165</sup>Er activity in highly concentrated (50–90 mg/mL) holmium solutions was corrected for self-attenuation of the 46–55 keV X-rays.

Preparation of radiopharmaceutical quality <sup>165</sup>Er from bulk holmium was accomplished through a three-step radiochemical isolation process. First, a 1 cm diameter, 25 cm-long CX column (AG50W-X8, 230–400 mesh, 63–150 µm, 1.7 meq/mL, NH<sup>4</sup> + form, Bio-Rad, Hercules, CA, USA) was equilibrated with water (~100 mL), then 70 mM αHIB (pH = 4.7, ~100 mL), followed by injection of the <sup>165</sup>Er/Ho/αHIB solution and elution with 5 mL/min 70 mM αHIB (pH = 4.7, 440–820 mL). Under these conditions, <sup>165</sup>Er elutes before holmium and the first ~90% of eluted <sup>165</sup>Er was collected (150–350 mL) for secondary purification, as determined by monitoring the column effluent using a shielded inline radiation detection system consisting of a CsI(Tl) scintillator (1 × 1 cm, Hilger Crystals Ltd., Kent, UK) coupled to a photomultiplier tube (E849-35, Hamamatsu Photonics, Hamamatsu City, Japan) powered and processed with bench-top electronics (925-SCINT, Ortec, Oak Ridge, TN, USA) and logged using a digital counter (USB-6008, National Instruments Corp., Austin, TX, USA). Then, the mobile phase was changed to 0.5 M αHIB (pH = 4.7, 250 mL) for stripping the remaining bulk holmium from the cation exchange column followed by water (200–500 mL) for column storage. The <sup>165</sup>Er radioactivity eluting in each collected CX fraction was quantified by a dose calibrator immediately after elution.

The second step utilized EXC with a commercially available HEHEHP-impregnated resin (LN2, 20–50 µm, Triskem Int., Bruz, France) [37], based on previous literature work on its use for Ho/Er separations [41]. Fritted polypropylene columns (5.5 mm diameter, 1 mL, Supelco Inc., Bellefonte, PA, USA) were dry-packed with resin (500 mg) and preconditioned with 1 M HNO<sup>3</sup> (5 mL) followed by 0.1 M HNO<sup>3</sup> (25 mL). All nitric acid solutions were prepared using 16 M HNO<sup>3</sup> (TraceSelect, Fluka Analytical, Buchs, Switzerland) and 18 MΩ·cm ultrapure water (Milli-Q). The <sup>165</sup>Er/Ho/αHIB eluate from separation step 1 was acidified to 0.1 M HNO<sup>3</sup> using 16 M HNO<sup>3</sup> and passed through the EXC column at 5.8 ± 0.9 mL/min using a peristaltic pump (*n* = 10, WPM1-P1CA-WP Welco Co. Ltd., Fuchu, Japan). With a lower flow rate of 1.2 ¯ ± 0.1 mL/min (*n* = 10, WPM1-P1BB-BP, Welco Co. Ltd., Fuchu, Japan), holmium was eluted with 0.4 M HNO ¯ <sup>3</sup> (40–60 mL), followed by <sup>165</sup>Er eluted with 1 M HNO<sup>3</sup> (4–6 mL).

In the third separation step, the <sup>165</sup>Er-rich fractions from separation step 2 were acidified from 1 M to 5 M HNO3, loaded onto a N,N,N0 ,N0 -tetra-2-ethylhexyldiglycolamide (100 mg, bDGA, 50–100 µm, Triskem Int., Bruz, France) EXC column. The column was then rinsed with 3 M HNO<sup>3</sup> (15 mL) and 0.5 M HNO<sup>3</sup> (2 mL). The <sup>165</sup>Er was subsequently eluted with 0.01 M HCl (1–1.5 mL).
