**1. Introduction**

The recent phase III clinical trial of Lutathera® ([177Lu]DOTATATE) for neuroendocrine tumors [1] and phase II clinical trial of [177Lu]PSMA-617 for prostate cancer [2] show receptor targeted, medium energy electron-emitting radiopharmaceuticals are effective in treating these solid tumors. However, for the treatment of micrometastatic or disseminated cancers, radiopharmaceuticals emitting shorter range, higher linear energy

**Citation:** Da Silva, I.; Johnson, T.R.; Mixdorf, J.C.; Aluicio-Sarduy, E.; Barnhart, T.E.; Nickles, R.J.; Engle, J.W.; Ellison, P.A. A High Separation Factor for <sup>165</sup>Er from Ho for Targeted Radionuclide Therapy. *Molecules* **2021**, *26*, 7513. https://doi.org/10.3390/ molecules26247513

Academic Editors: Alessandra Boschi and Petra Martini

Received: 8 November 2021 Accepted: 7 December 2021 Published: 11 December 2021

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transfer (LET) radiations, such as Auger electrons (AEs, also known as Auger–Meitner or Meitner–Auger electrons [3,4]), show potential in preclinical studies [5–7]. Following decay, AE-emitting radionuclides release a cascade of 0.02–50 keV electrons that stop with high LET over subcellular, nano- to micrometer ranges. These properties give AE-based radiopharmaceuticals reduced crossfire irradiation of healthy tissues and off-target dose burden compared with β −-emitting radionuclide therapeutics. Furthermore, when AE-emitting radiopharmaceuticals are specifically localized to radiation-sensitive subcellular structures such as DNA [8], this may result in higher maximum tolerated doses and expanded therapeutic windows. The biological impact of these AEs is also evident in comparison studies of <sup>177</sup>Lu- and <sup>161</sup>Tb-based radiopharmaceuticals [9–11], which are biologically, chemically, and physically matched, with the exception that <sup>161</sup>Tb has ten times larger AE emission yields (Table 1) [12]. Fundamental radiation biology studies of the effects of AEs require a pure AE-emitting radionuclide, with minimal concomitant medium/high energy electron or photon emissions. Erbium-165 decays by electron capture with only low energy X-ray and AE emissions (Table 1) [12]. As a heavy lanthanide, <sup>165</sup>Er can radiolabel the same biological targeting vectors used to deliver <sup>177</sup>Lu and <sup>161</sup>Tb [13], allowing for comparative studies of pure AE-emitting (165Er), β <sup>−</sup>-emitting (177Lu), and mixed AE- and β −-emitting ( <sup>161</sup>Tb) radiopharmaceuticals. Thus, <sup>165</sup>Er is a useful tool for fundamental studies on the biological effects of AEs and, if incorporated into an appropriate biological targeting vector, for the targeted radionuclide therapy of metastatic and disseminated disease.

**Table 1.** Comparison of AEs and β <sup>−</sup>-particles emitted by <sup>165</sup>Er, <sup>161</sup>Tb, and <sup>177</sup>Lu.


Proton, deuteron, or alpha particle irradiation of erbium or holmium targets produces no-carrier-added <sup>165</sup>Er through a variety of nuclear reaction routes [14]. Proton or deuteron irradiation of erbium produces <sup>165</sup>Tm (*t*1/2 = 30.06 h) via natEr(*p,xn*) <sup>165</sup>Tm [15,16] or natEr(*d,xn*) <sup>165</sup>Tm [17], respectively, which can be chemically isolated prior to its β − decay to <sup>165</sup>Er. While these routes offer high <sup>165</sup>Er yields, they require a medium energy, multiparticle research cyclotron and expensive, isotopically enriched targets for highest yields. Irradiation of naturally monoisotopic holmium targets produces <sup>165</sup>Er using 35–70 MeV alpha particles via <sup>165</sup>Ho(*α,4n*) <sup>165</sup>Tm(β – decay)165Er [18,19], 10–20 MeV deuterons via <sup>165</sup>Ho(*d,2n*) <sup>165</sup>Er [20,21], or 6–16 MeV protons via <sup>165</sup>Ho(*p,n*) <sup>165</sup>Er [22–24]. The existence of more than 500 biomedical cyclotrons capable of accelerating low energy protons makes the latter route particularly accessible to the research community worldwide [25].

Receptor-targeted therapeutic radiopharmaceuticals for human use are radiolabeled using 10–40 GBq <sup>177</sup>LuCl<sup>3</sup> with ~90% radiolabeling yield at ~60 MBq/nmol molar activity [26–28]. To accomplish these molar activities for biomedical cyclotron-produced <sup>165</sup>Er, the radionuclide must be chemically isolated from the holmium target material with a high (>10<sup>5</sup> ) separation factor (SF) due to residual target material competition with <sup>165</sup>Er during radiolabeling chemistry. Radiochemical separation of <sup>165</sup>Er from a Ho cyclotron target is challenging as adjacent lanthanides have identical oxidation states, similar coordination chemistry, and unfavorable <sup>165</sup>Er (~10 ng) to Ho (~100 mg) mass ratio. Radiochemical separations of adjacent lanthanides have traditionally been performed using cation exchange (CX) chromatography with the complexing agent α-hydroxy isobutyrate (αHIB) [29,30], with recent reports effectively separating Gd/Tb [31,32], Dy/Ho [33], Ho/Er [22,24], and Er/Tm [34,35]. Additionally, extraction chromatography (EXC) resins [36,37] impregnated with acidic organophosphorous extractants including bis(2-ethylhexyl) phosphoric acid (HDEHP, LN resin) [38,39], 2-ethylhexyl phosphonic acid mono-2-ethylhexyl ester

(HEHEHP, LN2 resin) [40–43], and bis(2,4,4-trimethyl-1-pentyl)phosphinic acid (H[TMPeP], LN3 resin) [24,43] have been used to separate adjacent heavy lanthanides.

A recent publication of the biomedical cyclotron production and radiochemical isolation of <sup>165</sup>Er from holmium reported the production of 1.6 GBq <sup>165</sup>Er in a 10 h, 10 µA proton irradiation [24]. The <sup>165</sup>Er was isolated in a 10 h process through a CX/αHIB column using a proprietary resin with 12–22 µm particle size followed by an EXC column using LN3 resin to concentrate the product. While the isolation of <sup>165</sup>Er from macroscopic Ho was achieved, neither the residual mass of Ho in the <sup>165</sup>Er product, the Ho/Er SF, nor radiopharmaceutical labeling results were reported [24]. The present work aims to improve the irradiation intensity tolerance of holmium targets to allow for the production of GBq-scale <sup>165</sup>Er in shorter irradiations, develop a radiochemical isolation process that utilizes commercially available resins to achieve a high Ho/Er SF in shorter times, and demonstrate the radiopharmaceutical quality of the produced <sup>165</sup>Er through chelator-based titration apparent molar activity (AMA) measurements and labeling a clinically relevant DOTA-based radiopharmaceutical (PSMA-617).
