Step 1: CX/αHIB

The first step of the <sup>165</sup>Er isolation procedure accomplishes a bulk holmium/erbium separation while accommodating 180 mg holmium loading masses through CX/αHIB column chromatography with commercially available CX resin in a standard stainless steel semipreparative high pressure chromatography column housing. This 19.6 mL column has a theoretical capacity of 1.8 g of trivalent Ho3+, ten times larger than the intended holmium loading masses. Based on the Dy/Ho separation process of Mocko et al. [33], 70 mM αHIB (pH = 4.7) was used as mobile phase. When mobile phase was freshly prepared and carefully pH adjusted to within 0.05 pH units, consistent retention times were observed with 90% of the total <sup>165</sup>Er radioactivity eluting in a Gaussian-shaped peak 40–90 min after injection, as shown in the representative radiochromatogram (Figure 2). Holmium elutes after <sup>165</sup>Er with its leading edge beginning at ~350 mL as seen through the diminishing Ho/Er SF with increasing <sup>165</sup>Er fraction collection volume. Use of mobile phase that was not freshly prepared or incorrectly adjusted to too low of a pH resulted in significantly longer retention times and diminished separation of <sup>165</sup>Er and Ho. With 5 mL/min flow, the column pressure was routinely 17–19 MPa. Following <sup>165</sup>Er elution, the column was stripped with freshly prepared 0.5 M αHIB (100 mL, pH = 4.7), followed by water (250 mL). The column was re-used until the flow pressure significantly increased (>22 MPa), upon which it was disassembled and repacked with fresh resin slurry, every ~10 uses.

value, resulting in ~90% 165Er recovery.

**Figure 2.** Representative <sup>165</sup>Er radioactivity elution profile from a cation exchange column loaded with 178 mg holmium and eluted with 5 mL/min 0.07 M αHIB (pH = 4.7). Dotted lines and corresponding text highlight three possible <sup>165</sup>Er-rich fraction collection volumes demonstrating the balance between <sup>165</sup>Er recovery and Ho/Er SF. The 0.5 M αHIB (pH = 4.7) rapidly elutes remaining <sup>165</sup>Er along with bulk holmium. **Figure 2.** Representative <sup>165</sup>Er radioactivity elution profile from a cation exchange column loaded with 178 mg holmium and eluted with 5 mL/min 0.07 M αHIB (pH = 4.7). Dotted lines and corresponding text highlight three possible <sup>165</sup>Er-rich fraction collection volumes demonstrating the balance between <sup>165</sup>Er recovery and Ho/Er SF. The 0.5 M αHIB (pH = 4.7) rapidly elutes remaining <sup>165</sup>Er along with bulk holmium.

Step 2: LN2 EXC The second step of the 165Er isolation procedure accomplishes a high Ho/Er SF while accommodating milligram quantity holmium masses through EXC using commercially available LN2 resin in a polypropylene column. When filled to maximum capacity (500 As determined by dose calibrator measurements of <sup>165</sup>Er and MP-AES measurements of Ho in the eluted CX fractions, the CX/αHIB column accommodated 180 mg Ho loading mass and effectively removed bulk Ho with acceptable <sup>165</sup>Er yield. Loading 111 <sup>±</sup> 17 mg Ho and recovering 94.7 <sup>±</sup> 2.5 % of <sup>165</sup>Er resulted in a SF of 320 <sup>±</sup> 210 (*<sup>n</sup>* = 6). Loading <sup>174</sup> <sup>±</sup> 8 mg Ho and recovering 95.3 <sup>±</sup> 1.8 % of <sup>165</sup>Er resulted in a SF of 130 <sup>±</sup> 60 (*<sup>n</sup>* = 5). For the larger loading masses, decreasing <sup>165</sup>Er recovery to 90.5 <sup>±</sup> 1.4 % resulted in an SF of 250 <sup>±</sup> 150 (*<sup>n</sup>* = 5) and further decreasing <sup>165</sup>Er recovery to 80% resulted in a SF of 1000 ± 400 (*n* = 1). These results indicate the sensitivity of the CX/αHIB separation to Ho loading mass and demonstrate the challenging balance between <sup>165</sup>Er recovery and Ho/Er SF. To ensure a reproducible, optimal balance between yield and SF for this step, an inline radiation detector was used to determine when to stop collecting the <sup>165</sup>Er fraction. Following loading <sup>≤</sup>120 mg Ho, <sup>165</sup>Er fraction collection was ended when the radioactivity signal was ~1/10th maximum value, resulting in ~95% <sup>165</sup>Er recovery. Following loading ~180 mg Ho, <sup>165</sup>Er fraction collection was ended when the radioactivity signal was ~1/4th maximum value, resulting in ~90% <sup>165</sup>Er recovery.

mL). The column was re-used until the flow pressure significantly increased (>22 MPa), upon which it was disassembled and repacked with fresh resin slurry, every ~10 uses.

As determined by dose calibrator measurements of 165Er and MP-AES measurements of Ho in the eluted CX fractions, the CX/αHIB column accommodated 180 mg Ho loading mass and effectively removed bulk Ho with acceptable 165Er yield. Loading 111 ± 17 mg Ho and recovering 94.7 ± 2.5 % of 165Er resulted in a SF of 320 ± 210 (n = 6). Loading 174 ± 8 mg Ho and recovering 95.3 ± 1.8 % of 165Er resulted in a SF of 130 ± 60 (n = 5). For the larger loading masses, decreasing 165Er recovery to 90.5 ± 1.4 % resulted in an SF of 250 ± 150 (n = 5) and further decreasing 165Er recovery to 80% resulted in a SF of 1000 ± 400 (n = 1). These results indicate the sensitivity of the CX/αHIB separation to Ho loading mass and demonstrate the challenging balance between 165Er recovery and Ho/Er SF. To ensure a reproducible, optimal balance between yield and SF for this step, an inline radiation detector was used to determine when to stop collecting the 165Er fraction. Following loading ≤120 mg Ho, 165Er fraction collection was ended when the radioactivity signal was ~1/10th maximum value, resulting in ~95% 165Er recovery. Following loading ~180 mg Ho, <sup>165</sup>Er fraction collection was ended when the radioactivity signal was ~1/4th maximum
