High-yield reactor production of terbium-161 using thin-layer gadolinium-160 targets, simplified chloride based separation and radiolabeling of DOTA based ligands

Abstract

Background

Terbium-161 (¹⁶¹ Tb) is an emerging therapeutic radionuclide combining β⁻ emission with a high yield of Auger electrons and conversion electrons, enabling enhanced localized dose deposition. However, its widespread clinical adoption is limited by inefficient target utilization and multistep post-irradiation processing. This study aimed to develop a simplified high-yield production and direct chloride-based purification workflow for clinical-grade terbium-161 using thin-layer enriched gadolinium-160 targets under limited-resource reactor conditions. Terbium-161 was produced by neutron irradiation of enriched Gadolinium-160 targets fabricated as conventional oxide powder and novel thin-layer chloride films at a thermal neutron flux of 1.5 × 10¹⁴ n cm⁻² s⁻¹ for 24 h in Pakistan Atomic Research Reactor-1 (PARR-I). Post-irradiation separation of terbium was carried out using LN resin employing HCl and HNO₃ media, and compared with α-hydroxyisobutyric acid (α-HIBA) cation-exchange chromatography. Radionuclidic purity was assessed by HPGe γ spectrometry, while radiochemical purity and labeling efficiency with DOTA conjugated Peptides were evaluated using ITLC and HPLC.

Results

Thin-layer targets significantly enhanced neutron utilization, compared to oxide targets due to reduced self-shielding of target. The specific activities achieved across three production batches (34.25–79.01 MBq/mg) demonstrated an approximately 2.3-fold higher specific activity with ¹⁶⁰GdCl₃ layered targets than bulk Gd₂O₃ targets. LN–HCl separation provided superior recovery (91.4%) and enabled direct formulation of [¹⁶¹ Tb]TbCl₃, eliminating post-processing steps required in nitrate systems and cation exchange with α -HIBA. Radionuclidic purity exceeded 99.99%, with minimal Terbium-160 impurity and Radiolabeling yields with DOTA-conjugated peptides exceeded 95.0%.

Conclusion

Target geometry plays a critical role in reactor-based radionuclide production. The integrated thin-layer target and chloride-based separation workflow provides a simplified, high-yield approach for production of terbium-161, enables efficient radiolabeling with DOTA-based ligands. These findings provide a foundation for further analytical qualification required for preclinical and clinical translations.

Graphical abstract

Introduction

Terbium (Tb), often referred to as the “Swiss army knife” of nuclear medicine, comprises four clinically significant radionuclides: terbium-149 for targeted alpha therapy (TAT), terbium-152 for positron emission tomography (PET), terbium-155 for single photon emission computed tomography (SPECT), and terbium-161 for targeted radionuclide therapy. Among them, terbium-161 (T_1/2 = 6.89 d) is particularly promising for β¯/auger electron therapy due to clustered emission of β¯particles (E_β⁻_max = 0.593 MeV), gamma radiation (Eγ = 75 keV, I = 10.2%), (Borgna et al. 2021) low-energy conversion (1.8), and Auger electrons (10.9) (Bolcaen et al. 2023) per decay as detailed in Fig. 1 (Desorgher et al. 2021) and structured in (Table 1“Nuclear National Data Center (Upton N.Y.) NuDat 2.6”. Accessed 2026). Auger electrons have a higher linear energy transfer (~ 4–26 keV/μm) and shorter range (0.5–30 μm) (“Nuclear National Data Center (Upton N.Y.) NuDat 2.6”. Accessed 2026; De Nardo et al. 2024) compared to β¯particles (0.04–1.8 mm) (De Nardo et al. 2024) resulting in dense local energy deposition, enhanced tumor cell killing, and reduced renal toxicity (Alcocer-Ávila et al. 2020).

Fig. 1.

Simplified decay scheme of terbium-161 (Bolcaen et al. 2023; Desorgher et al. 2021)

Table 1.

Clustered radiation emitting energy layers of terbium-161 (“Nuclear National Data Center (Upton N.Y.) NuDat 2.6”. Accessed 2026)

Radiation type	Energy (keV)	Intensity (%)	Energy potential
Beta emissions (β⁻ average)	154	100	Bulk tumor irradiation
Gamma emissions (γ)	25.7	23	SPECT imaging
	48.9	17	Imaging/dosimetry
	74.6	10.2	Quantification
Auger/CE emissions	3.4	17.5	Nanoscale damage
	5.5	87.9
	9–20	41.56	DNA-level irradiation
	20–50	77.66	Local energy deposition
	50–100	2.57	Minor contribution

Additionally, its chemical similarity to lutetium-177 allows it to be stably coordinated with a macrocyclic chelators like 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and other targeting vectors currently used with lutetium-177 without altering established clinical protocols (Müller et al. 2018). It has been demonstrated that terbium-161 offers superior efficacy for single tumor cells and micro metastases, requiring 40% less absorbed dose, delivering over threefold higher cellular dose, and exhibiting a higher electron-to-photon dose ratio than lutetium-177 (Borgna et al. 2022; Schaefer-Schuler et al. 2024; Buteau et al. 2024; Tschan et al. 2023; Gracheva et al. 2019; Rizvi et al. 2025). Preclinical studies using [¹⁶¹ Tb][Tb-DOTATOC], [¹⁶¹ Tb][Tb-DOTA-LM3] ((Fricke et al. 2024; Borgna et al. 2022)), [161 Tb]Tb-PSMA-617(Schaefer-Schuler et al. 2024), [¹⁶¹ Tb][Tb-PSMA-I&T] (Buteau et al. 2024), [¹⁶¹ Tb][Tb-SibuDAB] (Tschan et al. 2023) showed excellent anti-tumor efficacy.

The reactor-based production through ¹⁶⁰Gd(n,γ) ¹⁶¹Gd → ¹⁶¹ Tb reaction (¹⁶⁰Gd σ = 1.5 b) is most efficient and scalable route for obtaining high-purity non-carrier-added (n.c.a.) terbium-161 (Gracheva et al. 2019). However, two major bottlenecks persist, namely neutron self-shielding in bulk targets that reduces effective activation, and complex post-separation processing. Specifically, traditional methods require an arduous, time-consuming evaporation phase to convert the isolated product from a nitrate form into a chloride matrix prior to radiolabeling, increasing processing time and radiation exposure to operators.

To address these hurdles, alternative separation systems continue to be developed. Recently, Furkina et al. reported extraction chromatographic separation of terbium-161 from neutron-irradiated gadolinium targets using LN resin systems, while Gizawy et al. demonstrated reactor-scale production and purification strategies using irradiated gadolinium targets, highlighting the continuing need for simplified separation approaches that facilitate downstream radiopharmaceutical applications (Furkina et al. 2026).

The present study focuses on the development of an integrated workflow for the production and radiopharmaceutical evaluation of terbium-161 under resource-limited conditions. A novel thin-layer gadolinium-160 target fabrication strategy was introduced to minimize neutron self-shielding and enhance effective burn-up compared to conventional oxide powder targets. The study further evaluates and compares two chromatographic separation systems using LN resin with nitric acid and hydrochloric acid eluents to optimize terbium-161 recovery, radionuclidic purity, and compatibility with subsequent radiolabeling using DOTA-based ligands.

Materials and methods

All the chemicals used in this work were analytical grade. Nitric acid (≥ 65% w/v), Hydrochloric acid (HCl), α-hydroxyisobutyric acid (α-HIBA Acros), Dowex 50W × 8 and HPLC grade acetonitrile (MeCN) were purchased from Sigma Aldrich. LN resin was procured from Eichrom Inc. (USA) and highly (98.04% Gd₂O₃) enriched gadolinium oxide target from Trace Sciences International (Canada). DOTA-Tyr³-Octreotate (DOTATTATE), and prostate-specific membrane antigen 617 (PSMA-617), were acquired from ABX Advanced Biochemical Compounds GmbH (Germany). All aqueous solutions were prepared using ultrapure water (derived from a Millipore Direct-Q 5 water purification system.

Radionuclidic purity, radiochemical purity, and radiolabeling yield were determined using a high-purity germanium (HPGe) co-axial gamma spectrometer (GCDX-60200, Baltic Scientific Instruments Latvia), a radio-thin-layer chromatography (radio-TLC)) scanning system (LabLogic Systems, Sheffield, UK) equipped with a 2.54 × 2.54 cm thallium-doped sodium iodide NaI(Tl) scintillation detector coupled to a digital multi-channel analyzer (MCA(, and an ultra-high performance liquid chromatography system (uHPLC Ultimate 3000, Thermo Fisher Scientific, USA).

Target fabrication and irradiation

Highly enriched gadolinium-160 oxide ([¹⁶⁰Gd]Gd₂O₃ ,98.04% isotopic enrichment) was used as irradiation target. The target matrix contained ¹⁵⁴Gd (0.04%,), ¹⁵⁵Gd (0.24%), ¹⁵⁶Gd (0.39%,), ¹⁵⁷Gd (0.27%) and ¹⁵⁸Gd trace. Two target configurations were engineered to investigate spatial flux attenuation:

A powdered target (Sample I) was prepared by directly sealing [¹⁶⁰Gd]Gd₂O₃ in a quartz capsule of 5mm internal diameter. For the preparation of thin-film targets, Gd₂O₃ was dissolved in 4 M HCl, vacuum dried and reconstituted in ultrapure double-distilled water to obtain a stock solution of [¹⁶⁰Gd]GdCl₃ (~ 0.1 mg µL⁻¹). Aliquots of this solution were transferred into quartz ampoules to form thin-film targets (~1.0 ± 0.03 mm thickness). The samples were irradiated at Pakistan Atomic Research Reactor –I (PARR-I) at thermal neutron flux of 1.5 × 10¹⁴ n cm⁻²sec⁻¹ for 24 hours at maximum reactor power of 10 MW. Gold and Zirconium flux monitors were also irradiated for accurate flux measurements during the irradiation period. To ensure complete process validation and data traceability across the production campaigns, independent experimental runs were performed and tracked under validation batch identifiers: Batch Tb161-2025-01A (Sample I), Batch Tb161-2025-02B (Sample II), and Batch Tb161-2025-03C (Sample III). All production yields and specific activities were decay corrected to end of bombardment (EOB).

Calculation of neutron self shielding

To evaluate spatial flux depression across different target geometries, the experimental neutron self-shielding factor (f_SS) was derived according to the relationship:

where (A_EoB) represents the experimentally measured activity determined via gamma spectrometry at EoB, and (A_th) is the theoretical unshielded activity yielded by the classic activation equation:

where N₀ is the initial atom inventory of target ¹⁶⁰Gd nuclei, σ is the thermal capture cross-Sect. 1.50 b,) φ is the unperturbed thermal neutron flux 1.5 × 10¹⁴ n cm⁻² s⁻¹, λ is the decay constant of gadolinium-161 ln(2) /T_1/2, and t_irr is the total irradiation time (24.0 h).

Chemical processing of irradiated target

Irradiated samples were processed for chemical separation following the decay cooling period of 24 h post irradataion to allow for the complete decay of short-lived intermediate activation products, the targets were transferred to a lead-shielded hot cell for chemical dissolution. The bulk oxide target (Sample I) was dissolved in 2.00 mL of 3.0 N HCl, followed by multiple cycles of drying and washing with double distilled water to neutralize the solution of [¹⁶¹ Tb]TbCl₃ for subsequent use. The thin film targets (Samples II and III) were dissolved directly in ultra-pure double-distilled water with gentle thermal heating (45.00 °C) to form chloride solution without requiring post-dissolution acid conversion steps.

Radiochemical separation of terbium-161/gadolinium

The isolation of non-carrier-added (n.c.a.) terbium-161 from the macro-gadolinium target matrix was investigated using extraction chromatography (LN-3 resin) and cation exchange chromatography (Dowex 50W × 8 resin). All chromatographic workflows were executed within 72.00 h post-EoB. Due to resource limitations restricting access to routine offline inductively coupled plasma mass spectrometry ICP-MS, co-activated gadolinium-159 (¹⁵⁹Gd, T1/2 = 18.48 h) was successfully leveraged as an internal radiotracer to dynamically monitor, profile, and quantify the separation kinetics of the macro-target mass from the target terbium-161 fractions.

Cation exchange chromatography

A glass chromatographic column (ϕ:10 mm, l:170 mm) was packed with 1 g Dowex cation exchange resin (Dowex 50W × 8, (100–200 mesh H⁺ form) that has swollen overnight in 0.10 M NH₄OH. The column was sequentially preconditioned with 5.00 mL of distilled water, 5.00 ml 0.50 M α-HIBA solution (pH = 4.5) and again with 10.00 mL of distilled water. A aliquot of crude [¹⁶¹ Tb]TbCl₃ stock solution treated with 0.05 M NH₄Cl solution (pH = 3) was gravity loaded onto the column at a flow rate of 0.1 mL/min.

Ammoniated α-HIBA solutions (titrated to pH = 4.5 using57.00 g L⁻¹ ammonia) were evaluated as mobile-phase eluents. Gradient steps were evaluated at concentrations of 0.11 M, 0.12 M, 0.13 M, 0.15 M α-HIBA solutions using a peristaltic pump at constant velocity of 0.2 mL.min⁻¹. Eluate radioactivity profiles were tracked in real-time. Upon full recovery of the terbium-161 fractions, the eluent concentration was increased to 0.50 M, α-HIBA to strip the retained gadolinium isotopes off the matrix. The activity level in the eluates were observed by the dose rate. As soon as, terbium—161 activity reached negligible level, the concentration of the ammoniated α-HIBA -solution was increased to 0.5 M to elute gadolinium isotopes.

Extraction chromatography

Two distinct extraction chromatography columns (ϕ:10 mm, l:170 mm) packed with 800.0 gm each of LN-3 resin (25–50 μm particle diameter) pre soaked overnight in 0.10 M HNO_3.. Column 1(C₁) was allocated for systematic elution studies utilizing nitric acid while column 2 (C₂) was designated for elution studies with hydrochloric acid. Columns C1 and C2 were preconditioned with 5.00 mL of water followed by 5.00 mL of 0.15 M HNO3 and 0.15 M HCl, respectively, and washed with 10.00 mL of water. Equal activity fractions of the crude target solutions were loaded onto both column structures at a rate of 0.10 mL min⁻¹. Stepwise gradient elutions were executed using progressive concentrations (0.05 M, 0.1 M, 0.2 M, 0.3 M, 0.4 M, upto 1 M) of either HNO₃ (C1) or HCl (C2) driven at 0.20 mL min⁻¹ using peristaltic pump. Fractions were isolated in 0.50 mL volumes and quantified on a dose calibrator and the coaxial HPGe spectrometer. The summary of the operational parameters is given in Table 2.

Table 2.

Operational parameters of evaluated radiochemical separation systems

Resin	Mesh size (μm)	Column dimensions (mm)	Preconditioning	Eluent
Dowex 50W × 8	100–200	ϕ:10, l:170	0.50 M α-HIBA (pH = 4.50)	α-HIBA (0.11 M – 0.50 M)
LN-3 (C-1)	25–50	ϕ:10, l:170	5.00 mL H2O, 0.15 M HNO3	Nitric acid (0.20 M – 1.00 M)
LN-3 (C-2)	25–50	ϕ:10, l:170	5.00 mL H2O, 0.15 M HNO3	Hydrochloric acid (0.20 M—0.75 M)

Radionuclidic purity of the [¹⁶¹ Tb]TbCl₃ after purification

The radionuclidic purity of the isolated [¹⁶¹ Tb]TbCl₃ product was verified via high-resolution gamma-ray spectrometry using a coaxial HPGe detector possessing a relative efficiency of 60\%. Energy calibrations were established using a certified multi-nuclide standard source covering a energy envelope from from 53.16 keV (Eγ of ¹³³Ba) to 1457 keV Eluate fractions were measured dynamically until counting statistics yielded a 3σ confidence interval below 5%.. Terbium-161 was identified and quantified via its primary photopeaks at 25.7, 48.9, 57.2, 74.6, 87.9, 103.2, 106.1, and 292.4 keV. Co-extracted macro-gadolinium concentrations were tracked via the 348.0 keV and 363.0 keV gamma -lines of gadolinium-159. Traces of the activation impurity terbium-160 were monitored using its characteristic high-energy photopeaks at 298.00, 879.00, 966.00, 1177.00, and 1271.00 keV.

Radiolabeling

Radiolabeling of the macrocyclic DOTA-conjugated targeting vectors DOTATATE and PSMA-617 was executed in accordance with modified standard validation procedures. Briefly, n.c.a. [¹⁶¹ Tb]TbCl₃ solution was added a in peptide solution containing either DOTATATE or PSMA-617 (1.00 mg mL⁻¹) prepared in 10% (v/v) ethanol in water to achieve a final peptide concentration of 1.00 mg mL⁻¹ to achieve a defined nuclide-to-peptide labeling ratio. Reactions were conducted inside 10.0 mL borosilicate glass vials sealed with chlorobutyl rubber stoppers that had been pre-sterilized via a 10% ethanol rinse. The reaction environment was buffered to a stable labeling pH of 5.50 utilizing a 0.2 M sodium ascorbate solution, which functioned as both a hydrogen ion buffer and an analytical radical scavenger to mitigate radiolytic peptide backbone degradation. The matrix was thoroughly homogenized via vortexing for 5.0 min and subsequently incubated at 95.0 °C for 20.0 min inside a calibrated digital dry heating block.(Rizvi et al. 2025).

Radiochemical yield of [.¹⁶¹ Tb]TbCl₃,[161 Tb]Tb-DOTATATE and [161 Tb]Tb-PSMA-617

The radiochemical yield of the final product was determined by applying 10.0 μL drop of free [¹⁶¹ Tb]TbCl₃ solution, [161 Tb]Tb-DOTATATE and [161 Tb]Tb-PSMA-617on separate iTLC-SG plates. The plates were run in 0.1 M sodium citrate solution (pH 5.5). After development, the plates were dried and analyzed using a radio TLC scanner (Lablogic) equipped with Laura software.

Radiochemical purity of [161 Tb]Tb-DOTATATE and [161 Tb]Tb-PSMA-617

Radiochemical purity of the labeled peptides was assessed by radio-HPLC using a Thermo Fisher Scientific uHPLC system (Ultimate 3000) equipped with UV detection at 220 nm equipped with Chromeleon 7.0 software. Radioactivity was monitored using a Bioscan (Japan) system fitted with a NaI(Tl) scintillation detector. A 20 µL sample was injected onto an RP-18 column (LiChrospher® 100 RP-18 end capped, 5 µm). The method employed a binary solvent system consisting of (A) 0.1% TFA in water and (B) 0.1% TFA in acetonitrile, with a gradient elution from 5 to 100% B at a flow rate of 1.0 mL min⁻¹ over 30 min.

Results

Production of terbium-161

Table 3 details the production yields of terbium-161 alongside a comparative evaluation of the irradiation metrics across the different target morphologies evaluated. The thin-layer chloride targets (Sample II and Sample III) demonstrated significantly higher experimental activities and relative yields than the traditional bulk powder matrix (Sample I).

Table 3.

Comparative figures for production of terbium-161 (m = 10 mg, Φ = 1.5 × 10¹⁴ n.m⁻² s⁻¹, T_irr = 24 h, σ = 1.5 b)

Sample ID	Target material	Thickness (mm)	Experimental activity (MBq)	Theoretical activity (MBq)	Relative yield (%)	Specific activity (MBq/mg Gd)	Measured burn-up (× 10^-7%)	Self-shielding factor
Sample I	Gd2O3	5.00 ± 0.04	297.35 ± 28.44	702.00	42.36 ± 4.05	34.30 ± 3.28	0.82 ± 0.08	0.42 ± 0.04
Sample II	GdCl3	1.00 ± 0.03	521.11 ± 29.81	702.00	74.23 ± 4.25	60.11 ± 3.44	1.44 ± 0.08	0.74 ± 0.04
Sample III	GdCl3	1.00 ± 0.03	685.83 ± 15.77	702.00	97.70 ± 2.25	79.10 ± 1.82	1.89 ± 0.04	0.98 ± 0.02

^* each sample measured three times

Considering the hygroscopic nature of GdCl₃, the target material was thoroughly dried before encapsulation and hermetically sealed in quartz ampoules under dry condition to minimize the moisture uptake before irradiation (Talip et al. 2020; International Atomic Energy Agency (IAEA)). It is pertinent to mention that under the specified irradiation conditions (24 h, thermal neutron flux 1.5 × 10¹⁴ n m⁻² s⁻¹), the quartz ampoules remained intact, without obvious cracks, deformation, discoloration, or other signs of irradiation-induced damage. Furthermore, no gaseous discharge was observed during the opening of ampoule after irradiation, indicating absence of gaseous chlorine. These observations suggest that any radiolytic gas generation was insufficient to adversely affect target integrity. However, future studies employing analytical gas measurements or pressure monitoring will provide a more accurate evaluation of the radiation stability of GdCl₃ targets during extended neutron exposure.

Radionuclide composition

High-resolution gamma spectrometry of the raw irradiated targets identified terbium-161 as the primary activation product. Minimal co-induced isotopic impurities were detected, consisting of trace levels of the long-lived activation byproduct terbium-160 and residual target matrix gadolinium-159, as displayed in the raw spectrum in Fig. 2.

Fig. 2.

a Representative HPGe gamma-ray spectrum of the irradiated enriched ¹⁶⁰Gd target following neutron irradiation and dissolution, showing the characteristic photo peaks of terbium-161 together with trace radionuclidic impurities. b Radionuclide composition of irradiated sample for impurity quantification studied over period of 50 days

Radiochemical separation of ¹⁶¹ Tb/Gd

The net chemical extraction efficiencies and separation mechanics achieved across the three evaluated chromatographic matrix styles (as outlined in Sects. “Cation Exchange Chromatography” and “Extraction Chromatography”) are summarized in Table 4

Table 4.

Percentage recovery of terbium-161 in different chromatographic systems

Chromatographic system	Terbium-161 loaded (MBq)	Terbium-161 recovered (MBq)	% Recovery	Separation factor (α)
LN-3, HCl eluent (n = 3)	37.0 ± 2.6	33.83 ± 0.48	91.4 ± 1.3	4.41
LN-3, HNO3 eluent (n = 3)	15.2 ± 1.98	13.37 ± 0.41	89.13 ± 2.7	3.35
Dowex resin (n = 3)	11.1 ± 0.96	8.65 ± 0.93	77.93 ± 8.3	2.71

^*All campaigns were processed in triplicate independent validation batches (n = 3)

Cation exchange chromatography

Separation of terbium-161 from gadolinium-160 target using Dowex cation Exchange Resin was performed with increasing molarities of ammoniated α-HIBA as a complexing agent. The elution profile (Fig. 3a) showed that terbium-161 was released in the 0.12 M-0.14 M concentration of α-HIBA in the form of [Tb(IBA)]₃⁺. Maximum activity of terbium-161 was released with 0.14 M concentration of eluent. Gadolinium-159 eluted later with 0.5 M α-HIBA (fractions 17–24). HPGe γ-spectrometry confirmed that terbium-161 fractions contained characteristic γ-lines at 25.7, 48.9 and 74.6 keV, whereas gadolinium-159 (226.0, 348.0, 363.0 keV was absent from the main terbium peak region. The total activity recovered was 8.65 MBq from 11.1 MBq loaded, corresponding to 77.93% recovery. Only trace terbium-160 impurity was observed.

Fig. 3.

High-resolution radiochemical elution profiles of terbium-161/gadolinium separations across the investigated column architectures: a Dowex 50W × 8 cation exchange matrix using ammoniated α-HIBA mobile phase; b LN-3 extraction chromatography matrix using nitric acid mobile phase; and c LN-3 extraction chromatography matrix using hydrochloric acid mobile phase

Extraction chromatography

1. Nitric acid mobile phase system

   Within the nitric acid eluent configuration on the LN-3 extraction column matrix, the target macro-gadolinium ions eluted prior to the target radiometal. Quantitative gamma spectrometry profiling showed that the gadolinium-159 fraction cleared the column rapidly within a low-acid concentration threshold of 0.20 M HNO₃ (encompassing fractions 1–5). Conversely, trivalent terbium-161 ions displayed intense retention on the resin bed at low acid strengths and were sharply recovered within an elevated gradient step of 0.75 M–1.00 M HNO₃defining fractions 10–17 of the total elution volume (Fig. 3b). This system isolated 13.37 MBq of terbium-161 from a column loading of 15.22 MBq, corresponding to an 89.13% radiochemical extraction yield with trace co-eluted terbium-160 impurities.

2. Hydrochloric acid mobile phase system

   For the parallel LN-3 extraction matrix column (C2), an elution sequence was performed utilizing a gradient hydrochloric acid mobile phase (0.2–0.75 M HCl. This fluidic layout exhibited reverse chemical selectivity compared to the nitric acid configuration. Here, terbium-161 was stripped during the initial steps at low acid strength, while the bulk gadolinium matrix remained retained on the column.

   Terbium-161 was isolated as a sharp, high-intensity peak within a mobile phase concentration of 0.50 M HCl (defining fractions 4–10; Fig. 3c). The retained gadolinium-159 tracer was only released when the acid strength was increased to 0.75 M HCl (fractions 19–23). This optimized setup achieved a high radiochemical recovery of 33.83 MBq from a column loading of 37.02 MBq, establishing a chemical extraction yield of 91.40 directly in a chloride form suitable for radiolabelling.

Post separation radionuclidic purity of [.¹⁶¹ Tb]TbCl₃

Following column purification, the final radionuclidic purity of the isolated [¹⁶¹ Tb]TbCl₃ formulation was verified by analyzing the characteristic gamma emissions 25.7, 48.90, 57.2, 74.60, 87.90, 103.20, 106.10, and 292.40 keV using the calibrated HPGe spectrometer. The absolute radionuclidic purity was quantified at 99.99 ± 0.003%, as shown in Fig. 4. Long-lived activation contaminants, specifically terbium-160, were measured after allowing for ten half-lives of intermediate cooling, revealing an exposure level below 0.008% relative to the primary terbium-161 activity profile at the end of chemical separation.

Fig. 4.

Comparative HPGe gamma ray spectra of the target matrix recorded before and after extraction chromatography, illustrating the removal of radionuclidic impurities’

To evaluate possible gadolinium target contamination, the purified terbium-161 fraction was examined by high-resolution HPGe gamma-ray spectrometry for the characteristic gamma emissions of gadolinium-159. No detectable gadolinium-159 photopeaks were observed within the detection limits of the measurement, indicating negligible radioactive gadolinium activity in the final product also indicated by spectrum of purified terbium-161 shown in Fig. 4. Although a quantitative decontamination factor was not determined because residual gadolinium-159 activity was not quantified, the absence of detectable gadolinium-159 gamma emissions supports the high selectivity of the chromatographic separation.

Radiochemical yield of [¹⁶¹ Tb]TbCl_3, [¹⁶¹ Tb]Tb-DOTATATE and [.¹⁶¹ Tb]Tb-PSMA-617

The radiochemical purity of the uncomplexed [¹⁶¹ Tb]TbCl₃ starting solution was monitored via instant thin-layer chromatography using iTLC-SG matrices paired with a 0.10 M sodium citrate mobile phase pH 5.5. Unbound free radiometal migrated rapidly alongside the solvent front Rf = 0.70–0.80 confirming a baseline radiochemical purity of 99.00 ± 0.10% (Fig. 5a).The radiopharmaceutical coordination complexation of both DOTATATE and PSMA-617 precursors with the direct-chloride terbium-161 product was conducted in a sodium ascorbate buffer 0.20 M, pH 5.5 at 95.00 °C for a duration of 20.0 min. This protocol yielded a radiochemical complexation efficiency consistently exceeding 95.0 ± 1.20, as confirmed via radio-TLC screening under identical conditions. The fully chelated radiopharmaceuticals, [¹⁶¹ Tb]Tb-DOTATATE and [¹⁶¹ Tb]Tb-PSMA-617remained strictly at the application origin Rf = 0.00–0.30, with no evidence of secondary migration peaks at the solvent front (Figs. 5b and c). This confirms the complete consumption of free radiometal and establishes high labeling efficiencies without requiring post-labeling cartridge purification steps.

Fig. 5.

Radio-iTLC scans recorded on iTLC-SG sheets developed using a 0.10 M sodium citrate mobile phase (pH 5.5) to evaluate radiochemical profiles: a uncomplexed n.c.a. [161 Tb]TbCl3 solution; b synthesized [161 Tb]Tb-DOTATATE formulation; and c synthesized [161 Tb]Tb-PSMA-617

Radiochemical purity of [¹⁶¹ Tb]TbCl₃, [¹⁶¹ Tb]Tb-DOTATATE and [.¹⁶¹ Tb]Tb-PSMA-617

Analytical reversed-phase radio-HPLC profiles recorded on the C18 column showed high baseline resolution separating uncomplexed radiometal from the chelated peptides. Due to its highly hydrophilic nature, free uncomplexed [¹⁶¹ Tb]TbCl₃ eluted rapidly near the void volume t_R = 2.0.–0.3.0 min. In contrast, the fully complexed target radiopharmaceuticals exhibited extended retention windows matching their specific hydrophobic surface behaviors. The [¹⁶¹ Tb]Tb-PSMA-617tracer eluted as a sharp, symmetrical peak at t_R = 15.0–17.0 min (Fig. 6b), while the structural peptide variant[¹⁶¹ Tb]Tb-DOTATATE eluted at t_R = 11.0–13.0 min (Fig. 6c). Both finished formulations exhibited single, highly symmetrical radio-peaks with a complete absence of secondary degradative radiolysis fragments or free radiometal components, confirming a final radiochemical purity exceeding 99.00%

Fig. 6.

Analytical reversed-phase radio-HPLC chromatograms illustrating radiochemical purities and peak symmetry: a uncomplexed hydrophilic [¹⁶¹ Tb]TbCl₃ control; b formulated [¹⁶¹ Tb]Tb-DOTATAE therapeutic complex; and c formulated [¹⁶¹ Tb]Tb-PSMA-617 therapeutic complex

Discussion

The experimental production yields of terbium-161 obtained from the neutron activation of highly enriched gadolinium-160 targets at the PARR-I facility demonstrated strong agreement with theoretical models derived from deterministic activation mathematics. The decay-corrected specific activities achieved across the validation batches (34.25–7 9.01 MBq/mg Gd, as detailed in Table 3, reveal that target macro-morphology governs initial activation kinetics.

The thin-layer [¹⁶⁰Gd]GdCl₃, matrices demonstrated an average 2.3-fold increase in specific activity compared to the bulk [¹⁶⁰Gd]Gd₂O₃) powder pellet. This enhancement is directly attributed to the spatial mitigation of neutron self-shielding. Within the dense bulk oxide matrix, the high macroscopic thermal neutron capture cross-section of gadolinium induces severe local flux depression toward the center of the target volume, shielding internal atoms from core activation.

Conversely, the thin-layer configuration minimizes structural depth, enabling an unperturbed neutron flux envelope to penetrate uniformly across the target matrix. The minor yield variation observed between Sample II and Sample III is attributed to subtle micro-structural thickness non-uniformities occurring during manual chemical coating over the inner quartz walls. This variance can be readily minimized through automated automated film deposition technologies during scale-up. These bench-scale validation parameters provide a solid foundation for the engineering of automated radiochemical processing modules designed to safely isolate high-activity campaigns.

The raw radionuclidic composition profiles confirmed the dominance of terbium-161 across all targets, exceeding 90% of total target activity at early post-irradiation cooling points. Short-lived gadolinium-159 (T_1/2 = 18.48 h initially presents distinct photopeaks, but it decays rapidly during the 24-h cooling window. The trace co-induced isotopic impurity terbium-160 T_1/2 = 72.3 d cannot be accurately quantified via gamma spectrometry during initial timeframes due to intensive photopeak masking by the high-flux gamma emissions of terbium-161. However, following the decay of intermediate activation matrices over extended tracking intervals, the characteristic high-energy photopeaks of terbium-160 become detectable and can be cleanly quantified below a 3σ standard error threshold as shown in Fig. 2b This observed profile matches standard neutron activation pathways and cross-section parameters governing macro-lanthanide irradiation.

The evaluated Dowex 50W. × 0.8 cation exchange system successfully resolved terbium-161 from the macro-gadolinium target mass, leveraging differences in the thermodynamic stability of their respective α -hydroxyisobutyric acid (α -HIBA) coordination structures at pH 4.5. During mobile phase transition, both elements form trivalent species represented as [Tb-IBA]₃⁺ and [Gd-IBA]₃⁺. Trivalent terbium possesses a smaller ionic radius (lanthanide contraction), allowing it to exhibit a higher charge density and form a more stable, energetically favorable coordination chelate with α -HIBA than gadolinium. Consequently, terbium-161 is stripped from the sulfonic acid resin sites significantly earlier than the macro-gadolinium fractions, as displayed in Fig. 3a.

Although this classical pathway provides adequate resolution for analytical profiling, the observed.77.93% chemical recovery reflects localized mass retention during initial column loading and interstitial cell washing phases. Furthermore, this workflow has limited utility for routine therapeutic production because breaking down the stable α -HIBA complex requires challenging post-elution back-extraction steps to yield a carrier-free product.

In the LN-3 resin extraction system utilizing a nitric acid mobile phase, the opposite elution sequence was observed; the macro-gadolinium target mass cleared the column matrix first at a weak acid strength of 0.20 M HNO₃ (Fig. 3b). Terbium-161 was strongly retained by the organophosphorus extractant layer and was recovered within a higher concentration step gradient of 0.75.–0.1.00 M HNO₃\). This separation factor (alpha = 3.35\)) is consistent with values reported by Zowltoska et al. (2025).

However, the alternative hydrochloric acid mobile phase scheme on the LN-3 matrix exhibited superior chromatographic behavior. Terbium-161 was recovered as a sharp, high-intensity peak concentrated within a narrow 0.50 M HCl zone, while the macro-gadolinium mass remained strongly bound, eluting only when the acid threshold reached 0.75 M HC (Fig. 3c). This distinct behavior indicates that the choice of coordinating anion Cl⁻ versus NO₃⁻ modifies the extraction equilibria and interfacial mass-transfer kinetics, as documented by Monroy-Guzman et al. (2020) and Furkina et al. (2026).

he extraction equilibria governing the partition chemistry within the dimeric di-(2-ethylhexyl)phosphoric acid ([HDEHP]₂) matrix can be represented by the following equations The extraction equilibria in dimeric form can be represented as:

where M represents the trivalent lanthanide ions (Tb³⁺ or. Gd³⁺ and X denotes the specific mineral acid counter-anion Cl⁻ or NO₃^..

The LN-3/HCl process layout demonstrates clear advantages over recent literature methods. While both Gizawy et al. (2024) and Furkina et al. (2026) achieved high chemical recoveries approximately 85–90%, their elution profiles yielded a final product in a nitrate form, requiring an arduous evaporation-to-dryness step to reformulate the product into a chloride salt (Talip et al. 2020; Borgna et al. 2021) In contrast, our direct LN-3/HCl separation route achieves an excellent radiochemical recovery of 91.40 ± 1.30% directly in a high-purity chloride form [¹⁶¹ Tb]TbCl₃This completely eliminates post-separation chemical conversion steps, reducing processing times and minimizing radiation doses to the laboratory technician. High-resolution gamma spectrometry confirmed that this process yields an absolute radionuclidic purity of 99.99 ± 0.003% with a long-lived terbium-160 fraction below 0.008% at the end of chemical processing.

Analytical radio-TLC and radio-HPLC quality control protocols confirmed the high chemical accessible purity of the final chloride solution. On the iTLC-SG matrix developed with a sodium citrate mobile phase, free uncomplexed [¹⁶¹ Tb]TbCl₃ migrated rapidly with the solvent front R_f = 0.70–0.80\)), confirming a baseline radiochemical purity of 99.00 ± 0.10%. This high ionic accessibility enabled efficient downstream labeling of therapeutic peptides (DOTATATE and PSMA-617), consistently exceeding a radiochemical purity of 95.00 ± 1.20%

During radio-TLC monitoring of the labeling reactions, the fully chelated complexes remained at the origin (R_f = 0.0–0.30 while any unbound metal migrated to the front, showing a complete absence of unreacted radiometal peaks. These metrics were confirmed via analytical radio-HPLC, where free hydrophilic terbium eluted rapidly t_R = 2.0.– 3.0 min while the highly symmetrical therapeutic product peaks resolved at longer retention intervals t_R = 11.0.– 13.0 min for [¹⁶¹ Tb]Tb-DOTATATE t_R = 15.0.—17.0.00 [¹⁶¹ Tb]Tb-PSMA-617. The complete absence of radiolytic degradation fragments confirms that the sodium ascorbate buffer effectively protects the peptide backbones from radiation damage during synthesis.

The high yield radiolabeling of [¹⁶¹ Tb]Tb-DOTATATE and [¹⁶¹ Tb]Tb-PSMA-617 demonstrates the practical suitability of the purified terbium-161 for radiopharmaceutical applications. Similar studies by Skalova et al. (2026) and Gracheva et al. (2023) demonstrated efficient chelation of reactor-produced terbium-161 without reporting comprehensive DOTA titration studies. Another study by Ntihabose et al. (2025) for evaluation of challenges associated with terbium-161 base radiopharmaceuticals also evaluated radionuclide quality through radiochemical purity, radiolabeling performance, and stability of the resulting radiopharmaceuticals without assessment of molar activity. Consistent with these studies, the high radiolabeling yields (> 95%) and radiochemical purities (> 99%) obtained in the present study, together with the absence of detectable ¹⁵⁹Gd breakthrough, support the suitability of the purified terbium-161 produced by the proposed chloride-based separation method for efficient radiolabeling with DOTA-based ligands.

Conclusions

Terbium -161 is a promising therapeutic radionuclide for targeted radionuclide therapy owing to its favorable decay characteristics and coordination chemistry, which enable radiolabeling with established DOTA-based ligands. In this study, an integrated production workflow was developed to improve terbium-161 production and chemical processing under resource-limited conditions.

The proposed thin-layer gadolinium (III) chloride target configuration minimized neutron self-shielding, achieving specific activities of up to 79.10 MBq mg⁻¹ Gd. In addition, the optimized hydrochloric acid elution protocol using LN₃ resin provided a radiochemical recovery of 91.40 ± 1.30% while directly producing terbium-161 in chloride form, thereby eliminating the need for nitrate-to-chloride conversion prior to radiolabeling. The purified terbium-161 exhibited a radionuclidic purity of 99.99 ± 0.003%, with non detectable gadolinium-159 activity by HPGe gamma spectrometry, and demonstrated high radiolabeling efficiencies (> 95%) with DOTATATE and PSMA-617.

These findings demonstrate that the proposed production and separation methodology provides a reproducible approach for high yield production of terbium-161 suitable for radiolabeling with DOTA-based ligands.

Although further analytical characterization, including ICP-MS determination of elemental impurities, apparent molar activity measurements, and DOTA titration studies, is required before preclinical or clinical translation, the present work establishes a practical platform for terbium-161production and separation that may facilitate future radiopharmaceutical research, particularly in laboratories with limited radiochemical infrastructure.

Abbreviations

α-HIBA

α-Hydroxyisobutyric acid

DOTA

1,4,7,10-Tetraazacyclododecane-1,4,7,10-Tetraacetic acid

DOTATATE

DOTA-[Tyr3]-Octreotate

EOB

End of bombardment

Gd

Gadolinium

Gd₂O₃

Gadolinium oxide

GdCl₃

Gadolinium chloride

HCl

Hydrochloric acid

HNO₃

Nitric acid

HPGe

High-purity germanium

HPLC

High-performance liquid chromatography

ICP-MS

Inductively coupled plasma–mass spectrometry

ITLC

Instant thin-layer chromatography

LN Resin

Lanthanide resin

MBq

Megabecquerel

PARR-I

Pakistan atomic research reactor-1

PRRT

Peptide receptor radionuclide therapy

PSMA

Prostate-specific membrane antigen

RCP

Radiochemical purity

RCY

Radiochemical yield

RLT

Radioligand therapy

SA

Specific activity

SPECT

Single-photon emission computed tomography

Tb

Terbium

TRT

Targeted radionuclide therapy

Author contributions

SK Conceptualization, project supervision, target fabrication methodology development, radiochemical separation system design, column layout engineering, manuscript critical review, and finalization. AJ Execution of chemical separations, target matrix processing, validation campaigns, radioanalytical quality control monitoring, data aggregation, and initial manuscript drafting. QK Execution of chemical separations, target matrix processing, validation campaigns, radioanalytical quality control monitoring, data aggregation, and initial manuscript drafting. KA analyzed the data and performed, assisted with methodology development, and manuscript revision. conception and design.

Funding

The authors declare that no funds, grants, or structural financial support were received from any public or commercial funding agency during the preparation of this manuscript.

Data availability

The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

Not Applicable. This study did not involve human participants, human data, or animals.

Consent for publication

Not applicable. This manuscript does not contain any individual person’s data in any form (including individual details, images, or videos).

Competing interests

The authors have no relevant financial or non-financial interests to disclose. All authors explicitly declare that there are no potential conflicts of interest with respect to the experimental research findings, authorship, or publication of this article.