In the last decade, holmium microspheres containing ¹⁶⁶Ho (t_1/2=26.8 h) have been successfully employed for the treatment of several malignances, particularly liver tumors [1]. Thanks to its high-energy β^- particles (E_β-,ave=667 keV), ¹⁶⁶Ho is also a potential candidate for targeted radioligand therapy. Furthermore, the versatile coordination chemistry of lanthanides enables the development of theranostic approaches based on chemically similar radiometals. In this context, our work focused on the development of an indirect production route through the neutron irradiation of enriched ¹⁶⁴Dy₂O₃ as target material. No-carrier-added (n.c.a.) ¹⁶⁶Ho is obtained from the decay of ¹⁶⁶Dy (t_1/2=81.3 h, β^- 100%), its parent radionuclide.  This approach avoids the co-production of the long-lived impurity ^166mHo (t_1/2=1132 y) and enables repeated separations in a pseudo-generator configuration.

Several quartz ampoules containing 8 μg of ¹⁶⁴Dy₂O₃ (enr. 96.8%) were irradiated at the Institut Laue-Langevin’s (ILL) high-flux reactor. The production yields of the activation products were thoroughly assessed using γ-spectrometry measurements. The end-of-irradiation production yields of ¹⁶⁶Dy was 143 (9) GBq/mg using a neutron flux of 1.1×10¹⁵ cm^-2s^-1.

Extraction chromatographic (TK211/2/3) and cation exchange (Sykrogel H S 18 X-16) resins were evaluated for Dy/Ho separation. ICP-OES analysis of the collected fractions was used to assess the influence of eluent concentration, column dimensions, solution pH, and flow rate on separation performance and processing time.

The overall separation setup consisted of three columns and an additional one to enable the pseudo-generator.  The TK211 extraction resin ensured rapid Dy recovery (>80% within 1 hour from loading), while removing stable holmium (¹⁶⁵Ho) and ^166mHo. ¹⁶⁶Ho was isolated using Sykrogel cation exchange resin in combination with α-hydroxyisobutyric acid (α-HIBA). The TK213 extraction resin was used to remove metal contaminants, convert the product into chloride form, and elute ¹⁶⁶Ho in 1 mL volume. ¹⁶⁶Dy was recovered from the Sykrogel column using concentrated α-HIBA and loaded onto a TK212 resin to convert it into 0.5 HNO₃. This enabled recovery yields of up to 98% of ¹⁶⁶Dy, allowing ¹⁶⁶Ho ingrowth and supporting repeated generator-like processing using the Sykrogel column.

Gigabecquerel-scale production of ¹⁶⁶Ho was successfully demonstrated, yielding up to 3 GBq of n.c.a. and radionuclidically pure ¹⁶⁶Ho in approximately 1 mL of 0.1 M HCl. Ongoing optimization of the separation procedure is expected to improve activity concentration and process robustness. In parallel, the successful implementation of TK212 resin enabled the generator-based approach. Upon optimization of process reproducibility, routine production of high specific activity of ¹⁶⁶Ho could support preclinical investigations and facilitate the development of ¹⁶⁶Ho-based targeted radioligand therapy.