**4. Discussion**

For application in receptor-targeted therapeutic radiopharmaceuticals, a high molar activity is necessary to achieve good radiolabeling yield of small pharmaceutical masses with large amounts of radioactivity. This high molar activity ensures that the biologically administered mass of radiopharmaceutical is sufficiently small to not saturate the targeted receptor on diseased cells. When prepared for human use, [177Lu]PSMA-617 and [177Lu]DOTATATE are radiolabeled with high yield at a molar activity of 60 MBq/nmol [26,27]. For cyclotronproduced <sup>165</sup>Er, achieving a high molar activity must begin with careful consideration of the holmium target material. Many commercial sources of holmium have significant erbium impurity, which, in turn, will limit the maximum attainable molar activity of <sup>165</sup>Er produced in the proton irradiation of an impure holmium target. Additionally, as shown in Table 2, the holmium target and irradiation parameters significantly affect the overall quantity of <sup>165</sup>Er that can be produced in a bombardment, with larger targets having greater overall yields compared to their smaller counterparts. However, larger holmium mass targets come with the added challenges of a more difficult <sup>165</sup>Er/Ho separation and a higher cold erbium and holmium burden in the purified <sup>165</sup>Er fraction.

In addition to sourcing Ho target material with low Er impurity content, an effective Ho/Er radiochemical isolation procedure is necessary. Because of the chemical similarity between the adjacent lanthanide elements, any residual holmium in the <sup>165</sup>Er final formulation will affect the molar activity of <sup>165</sup>Er. A high Ho/Er SF is accomplished in this work by a multi-step separation process utilizing cation exchange and extraction chromatography. Based on MP-AES analysis of the final <sup>165</sup>Er product, this <sup>165</sup>Er radiochemical isolation process gives a Ho/Er SF of (2.8 <sup>±</sup> 1.1) · <sup>10</sup><sup>5</sup> . The <sup>165</sup>Er radiochemical yield was 64 <sup>±</sup> 2% calculated by the ratio of the decay-corrected <sup>165</sup>Er activity in the final product and the <sup>165</sup>Er activity produced in the irradiated target.

Two main sources of erbium/holmium—cold erbium target impurity and residual holmium due to incomplete separation—are the two dominating terms in the denominator of supplementary Equation (S1), which calculates a maximum achievable EoB MA for a given <sup>165</sup>Er preparation. With the experimental <sup>165</sup>Er production and radiochemical separation results presented above, supplementary Equation (S1) yields a calculated EoB <sup>165</sup>Er MA of 9.9 ± 0.5 MBq/nmol for a 1 h, 12.5 MeV, 130 mg, 9.5 mm Ø, low purity (100 ppm Er) holmium target PETtrace irradiation. This calculated MAEoB is in good agreement with the measured DOTA/DTPA <sup>165</sup>Er AMAs (Table 4). In this case, the calculated EoB MA is nearly entirely driven by the cold erbium impurity present in the holmium target material (second term in supplementary Equation (S1) denominator). This underscores the fact

that under the investigated irradiation conditions, utilization of holmium targets with high erbium impurity content will limit the molar activity of the resulting <sup>165</sup>Er to values lower than typically acceptable for therapeutic radiopharmaceutical research applications. Producing high MA <sup>165</sup>Er requires holmium with extremely low erbium content, such as target material that has been prepurified from erbium, or the DOE Ames Laboratory MPC metal used in this work.

For an identical irradiation of a high-purity (0.5 ppm Er) holmium target, supplementary Equation (S1) yields an <sup>165</sup>Er EoB MA of 240 <sup>±</sup> 60 MBq/nmol, with this molar activity being driven by cold holmium remaining in the <sup>165</sup>Er preparation after their separation by a factor of (2.8 <sup>±</sup> 1.1) · <sup>10</sup><sup>5</sup> (third term in supplementary Equation (S1) denominator). However, the EoB <sup>165</sup>Er AMAs (Table 4) with DOTA (1.2–47 GBq/µmolDOTA, *n* = 3) and DTPA (4.9–250 GBq/µmolDTPA, *n* = 5) and radiolabeled [165Er]PSMA-617 MAs (37–130 GBq/µmolPSMA-617, *n* = 5, Table 5) do not reflect this high value of MA. This disagreement may be a result of nanomole, sub-ppm trace metal (Zn, Fe, Cu) impurities in the radiolabeling reactions, which are not considered in supplementary Equation (S1). This hypothesis is supported by the fact that, for <sup>165</sup>Er isolated from high-purity holmium targets, the titration-based AMA measured using DTPA were 5–22 times higher than for DOTA (*n* = 3). This difference in DTPA/DOTA AMA values, which has also been observed for cyclotron-produced <sup>86</sup>Y [49], is likely due to the non-selective nature of DOTA's metal binding properties. Compared with DTPA, DOTA binds 10,000 times stronger to Fe2+ (LogKFeDOTA = 20.22 ± 0.07 [50], LogKFeDTPA = 16.0 ± 0.1 [51]), 100 times stronger to Zn2+ (LogKZnDOTA = 20.8 <sup>±</sup> 0.2, LogKZnDTPA = 18.6 <sup>±</sup> 0.1 [51]), and 10 times stronger to Cu2+ (LogKCuDOTA = 22.3 <sup>±</sup> 0.1, LogKCuDTPA = 21.5 <sup>±</sup> 0.1 [51]), causing these three common trace metal impurities to be significantly more problematic in DOTA versus DTPA radiochemical labelings. Thus, a systematically higher DTPA-based AMA compared with DOTA-based AMA is indicative that Fe/Zn/Cu-based trace metal impurities in the radiolabeling solutions are significantly impacting the <sup>165</sup>Er AMA values. The impact of trace Zn, Fe, and Cu on the [165Er]PSMA-617 radiolabeling experiments is supported by the presence of UV-absorbing impurities with analytical HPLC retention times equivalent to natZn-PSMA-617, natFe-PSMA-617, and natCu-PSMA-617 in the final radiopharmaceutical preparation (Supplementary Figure S7).

This work represents the first published radiosynthesis of [165Er]PSMA-617, a radiopharmaceutical that could serve as a useful in vitro and in vivo tool that can be used to assess the role of AEs in the efficacy of PSMA-targeted radionuclide therapy of prostate cancer using [161Tb]PSMA-617 [11]. The radiopharmaceutical is stable in serum for at least 12 h and has an octanol–water partition coefficient of LogD = −3.3 ± 0.3, less polar than [161Tb]PSMA-617 (−3.9 <sup>±</sup> 0.1) [11], [44Sc]PSMA-617 (−4.21 <sup>±</sup> 0.04), [177Lu]PSMA-617 (−4.18 <sup>±</sup> 0.06), and [68Ga]PSMA-617 (−4.3 <sup>±</sup> 0.1) [52].
