A Radionuclide Generator of High-Purity Bi-213 for Instant Labeling

Abstract

A new two-column ²²⁵Ac/²¹³Bi generator was developed specifically for using ²²⁵Ac containing an impurity of long lived ²²⁷Ac. The parent ²²⁵Ac was retained on the first Actinide Resin column, while ²¹³Bi was accumulated on the second column filled with AG MP-50 resin via continuous elution and decay of intermediate ²²¹Fr. The ²¹³Bi accumulation was realized in circulation mode which allowed a compact generator design. It was demonstrated that ²¹³Bi could be quickly and effectively extracted from AG MP-50 in form of complexes with various chelating agents including DTPA and DOTA. The performance of the generator presented and a conventional single-column generator on the base of AG MP-50 was tested and both generators were loaded with ²²⁵Ac containing ²²⁷Ac impurity. The ²¹³Bi generation efficiencies were comparable and greater than 70%, whereas the developed generator provided a deeper degree of purification of ²¹³Bi from Ac isotopes and decay products of ²²⁷Ac.

1. Introduction

The radionuclide, ²¹³Bi, is considered as one of the promising radionuclides for targeted therapy of cancer [1,2]. Radiopharmaceuticals containing ²¹³Bi are successfully passing clinical trials for the treatment of leukemia [3], NHL [4], carcinoma [5], neuroendocrine tumor [6], glioma [7], and melanoma [8]. In many cases, radiopharmaceuticals ²¹³Bi-DOTATOC and ²¹³Bi-PSMA-617 have shown high efficacy even after treatment with conventional therapies has failed [6,9].

The radionuclide, ²¹³Bi, is a decay product of another promising alpha emitter ²²⁵Ac, which is used by itself [10,11] or as a parent radionuclide for ²²⁵Ac/²¹³Bi generators. A single-column “direct” generator developed at the Institute of Transuranic Elements (ITU) and based on a strongly acidic cation-exchange sorbent AG MP-50 [12] is most commonly used. Other generators based on extraction chromatographic [13,14], ion exchange [15], and inorganic sorbents [16,17] are also available.

The radionuclide, ²¹³Bi, has a relatively short half-life (46 min) and, therefore, it is preferable to use low molecular weight peptides and antibodies with fast pharmacokinetics as vectors for its delivery. For the same reason, significantly high activities of ²¹³Bi are required to achieve a therapeutic effect. Depending on the vector molecules, a possible range of ²¹³Bi administered dose may vary within 5–10 GBq [1].

Carrier-free ²²⁵Ac is applied as a source of ²¹³Bi for conducting clinical trials of ²¹³Bi-containing radiopharmaceuticals. It is obtained by the generator method from ²²⁹Th (T_1/2 = 7.340 y) and the stock supplies are extremely limited. The world production of ²²⁵Ac by this method is no more than 70 GBq per year [2]. The main alternative method of ²²⁵Ac production is the irradiation of natural thorium with medium-energy protons. This method is being actively developed at INR RAS (Moscow-Troitsk, Russia) [18,19,20,21], TRIUMPH (Canada) [22], and LANL-BNL-ORNL (tri-lab, USA) [23]. The routine production of actinium will be launched at the accelerators of these scientific centers in the coming years, which makes it possible to produce several curies of ²²⁵Ac in one irradiation session (7–10 days). ²²⁵Ac from irradiated thorium contains a chemically inseparable impurity of long-lived ²²⁷Ac (~0.2% at the end of bombardment, EOB) [19]. Furthermore, although it was shown that such actinium can be used for therapy along with those that are generator-produced without any side effects [24], this imposes additional requirements on the ²²⁵Ac/²¹³Bi generator. The generator system should not only provide a high yield of ²¹³Bi in its rapid production and possess high radiation resistance [25] but also ensure a high degree of purification from both actinium isotopes and ²²⁷Ac decay products (²²⁷Th and ²²³Ra).

The article presents and investigates an original model of ²²⁵Ac/²¹³Bi generator intended for utilizing the parent ²²⁵Ac with ²²⁷Ac impurity. The generator provides a fast production of ²¹³Bi deeply purified from actinium isotopes ²²⁷Th and ²²³Ra. It consists of two chromatographic columns; ²²⁵Ac is firmly fixed on the first column and, as a result of mobile phase circulation, ²¹³Bi is separated from ²²⁵Ac via continuous elution and decay of intermediate ²²¹Fr and is concentrated on the second column. ²¹³Bi can be efficiently and quickly stripped off by complexing agents (DTPA, DOTA, etc.), which greatly simplifies the labeling procedure.

2. Materials and Methods

All chemicals were of p.a. (pro analysis) quality or higher, obtained from Merck (Darmstadt, Germany), and used without additional purifications. All experiments were carried out using de-ionized “Milli-Q” water (18 MΩ·cm⁻¹). Actinide Resin (P,P′-di(2-ethylhexyl)methanediphosphonic acid as an extracting agent on a silica support) with 100–150 μm particle size was obtained from Triskem, France. Biotechnology grade AG MP-50 (strong-acid cation exchange resin composed of sulfonic acid functional groups attached to a styrene divinylbenzene copolymer lattice) with 106–250 μm particle size was obtained from BioRad, Hercules, CA, USA.

γ-Ray spectroscopy with high-purity Ge-detector (HPGe) GR3818 (Canberra Industries, Inc., Meriden, CT, USA) was used for radionuclide determination. γ-Ray spectra were analyzed using software Genie 2000 (Canberra Industries, Inc., Meriden, CT, USA) and BNL-database [26].

The experiments were carried out at the temperature of 21 ± 2 °С.

2.1. Radionuclides

²²⁵Ac and ²²³Ra were produced at the linear proton accelerator of the Institute for Nuclear Research of the Russian Academy of Sciences (INR RAS, Troitsk-Moscow, Russia) by irradiation of thorium plates (1–2 mm thick) with protons of initial energy 120 MeV and isolated by liquid–liquid extraction, ion-exchange, and extraction chromatography in accordance with the reported procedures [20,27]. Impurity of ²²⁷Ac in actinium was determined as ~0.2% of ²²⁵Ac activity calculated at EOB. The yield and purity of the obtained radionuclides were controlled by γ-ray spectroscopy. The γ-lines 218.1 keV (abundance 11.4%), 440.5 keV (abundance 25.9%), 236.0 keV (abundance 12.9%), and 154.2 keV (abundance 5.7%) were chosen for ²²¹Fr (T_1/2 = 4.9 min), ²²⁵Ac (T_1/2 = 9.9 d, through ²²¹Fr), ²¹³Bi (T_1/2 = 46 min), ²²⁷Th (T_1/2 = 18.7 d), and ²²³Ra (T_1/2 = 11.43 min) quantification, respectively.

The stock solution of ²⁰⁷Bi (T_1/2 = 31.55 y) in 1 M HCl (0.1 MBq/mL) was purchased from JSC Cyclotron (Russia). The γ-line 569.7 keV (abundance 97.7%) was used for ²⁰⁷Bi determination.

2.2. Batch Adsorption of Ra(II) and Bi(III) onto AG MP-50 Resin

Since AG MP-50 could contain impurities of calcium [28], it was previously purified. The resin was packed into large columns (1 cm inner diameter) and the calcium was eluted with 5 M HNO₃, until the eluate was free from calcium, rinsed with distilled water, and dried at 60 °C to constant weight.

The adsorption of ²⁰⁷Bi and ²²³Ra from nitric and hydrochloric acid solutions onto AG MP-50 was investigated by mixing 5 mL of the acid solution with 0.1 mL of ²⁰⁷Bi or ²²³Ra containing spike and 0.05 g of the sorbent in plastic tubes. The samples were prepared with various acid concentrations (0.05–1 M), taking into account the content of nitric or hydrochloric acid in the spike solution. Tubes were shaken at room temperature for 40 min. According to the preliminary kinetic studies, this time period was enough to reach the sorption equilibrium. The amount of 1.5 mL aliquots of the aqueous solutions was taken for γ-ray spectroscopy after equilibration. Mass distribution ratios (D_m), mL/g, were calculated using the well-known equation [29].

Measured activities of ²²³Ra were corrected for decay. Uncertainties of activity measurements did not exceed 10%.

2.3. Column Measurement of Capacity Factors of Fr(I) onto Actinide Resin

²²¹Fr was separated from ²²⁵Ac adsorbed onto the Actinide Resin column (0.3 or 0.4 mL) by passing neutral (pH ~6) solutions of NaCl or NH₄Cl in a concentration range of 0.0017–0.1 M. The flow rate controlled by a peristaltic pump varied from 0.2 to 1 mL/min. The eluate was collected by portions during 10–20 min. In each portion, the ²²¹Fr activity was measured immediately and decay-corrected to the end of portion sampling. Capacity factors (k’) were estimated according to the procedure reported [30].

2.4. Column Measurement of Capacity Factors of Fr(I) and Bi(III) onto AG MP-50 Resin

Four columns were filled with AG MP-50 resin of volume 0.1 mL each. The columns were connected in series and attached to the exit of ²²⁵Ac-loaded Actinide Resin column. Solution of 0.25 M HNO₃ or 0.017 M NaCl was passed through the column assembly for no less than 4 h. The flow rate controlled by a peristaltic pump varied from 0.3 to 2.8 mL/min. Having finished the elution, the columns were dismounted and the ²²¹Fr and ²¹³Bi activities in each column were measured and decay-corrected to the end of elution. Capacity factors (k’) were estimated according to the procedure reported [30].

2.5. Preparation and Milking ²²⁵Ac/²¹³Bi Generator “Afrabis”

The amount of 0.1 MBq of ²²⁵Ac (with the ²²⁷Ac impurity) was dissolved in 5 mL of 0.1 M HNO₃ and loaded with the help of a peristaltic pump slowly on the pre-packed and conditioned column containing 0.4 mL of Actinide Resin. The second column was filled with 0.4 mL of AG MP-50 resin and both columns were connected to form a closed-loop circuit as shown in Figure 1. The Actinide Resin column was scanned on a γ-ray spectrometer through a 1.5 mm wide slit between the lead blocks immediately after preparation and after milking.

One cycle of a possible procedure for ²¹³Bi generation was as follows:

• The first column was washed from the accumulated ²²³Ra with 15–20 mL of 0.25 M HNO₃;
• Then, continuous separation of the intermediate daughter radionuclide ²²¹Fr from ²²⁵Ac for ~4 h was occurring circularly (Figure 1) at flow rate 1–1.5 mL/min;
• ²¹³Bi was desorbed from the AG MP-50 column with six 0.5 mL portions of a 0.1 M HCl/0.1 M KI solution at flow rate 1 mL/min.

The desorption of ²¹³Bi from an AG MP-50 column with DTPA solutions of various concentrations and pH values was studied. An acetate buffer solution was prepared: 3.33 g of sodium hydroxide was mixed with 5.7 mL of glacial acetic acid and the total volume was adjusted to 100 mL with distilled water (pH 5.3). DTPA was dissolved in the resulting solution to the desired concentration (10⁻²–10⁻⁵ M). A solution with the desired pH value (2–5.3) was obtained by adding solutions of sodium hydroxide and nitric acid. The amount of 5 mL of 0.25 M HNO₃ containing ²⁰⁷Bi spike was slowly loaded with the help of a peristaltic pump on the pre-packed and conditioned column containing 0.4 mL of AG MP-50. Bi was eluted from the column with 5 mL of DTPA solution in an acetate buffer with DTPA concentration and pH value in the investigated range. After that, the column was washed with 5 mL 6 M HCl for desorption of residue Bi and pre-conditioned with 0.25 M HNO₃ for the next Bi sorption.

In order to test desorption of ²⁰⁷Bi with a DOTA solution, the AG MP-50 resin with ²⁰⁷Bi adsorbed on it was removed from the column, mixed with 3 mL of 10⁻⁵ M DOTA, and placed in a thermostat at 90 °C with stirring for 5 or 10 min. The solution was decanted through a quartz wool filter and measured by γ-ray spectroscopy.

2.6. Preparation and Milking A Single-Column ²²⁵Ac/²¹³Bi Generator with AG MP-50

The amount of 0.1 MBq of ²²⁵Ac (with the ²²⁷Ac impurity) was dissolved in 2.8 mL of 4 M HNO₃ and loaded slowly with the help of a peristaltic pump on a pre-packed and conditioned column filled with 0.33 mL of AG MP-50 resin. Afterwards, the column was washed with 1 mL 0.05 M HNO₃, 2 mL 2 M HCl, and finally with 0.01 M HCl [12,31]. As a result, ²²⁵Ac was distributed within the first half of the column. The column was scanned on a γ-ray spectroscopy through a 1.5 mm wide slit between the lead blocks.

The ²¹³Bi was eluted with a mixture of 0.1 M NaJ/HCl solution followed by washing with 0.01 M HCl. The resulting ²¹³Bi solution with a total volume of 3 mL was measured on a γ-ray spectrometer immediately after milking and again 6 h later. The latter measurement was conducted after the complete decay of ²¹³Bi to determine the impurity of long-lived radionuclides—²²⁵Ac, ²²⁷Th, and ²²³Ra.The generator was stored wet in the 0.01 M HCl until the next use. Seven elutions of ²¹³Bi were carried out during 1 month of the generator exploitation.

3. Results and Discussion

The ever-growing application of radionuclide generators in nuclear medicine [32] stems from the fact that a short-lived daughter radionuclide can be repeatedly reproduced and used for a long period of time; this is mainly dependent on the half-life of the parent radionuclide. Operation of most generators comprises two basic stages: accumulation and separation. First a daughter radionuclide accumulates up to transient equilibrium with the parent. Usually, both radionuclides reside together in a generator at this stage. Then the daughter is separated and recovered from the generator as fast as possible to reduce decay losses and the next generation cycle begins.

Operation mode of the generator presented here and named “Afrabis” (acronym: A—actinium-225, fra—francium-221, and bis—bismuth-213) is different. Formation and concentration of ²¹³Bi take place in the course of permanent separation of intermediate ²²¹Fr from ²²⁵Ac fixed on a mother column (Figure 1) and its decay.

As a result of dynamic accumulation arranged in circulation mode, ²¹³Bi proves to already be removed from the mother column and concentrated on the second column to the moment when it reaches the transient equilibrium with ²²⁵Ac (it takes ~5–6 half-lives of ²¹³Bi). Finally, ²¹³Bi is extracted in a small volume of appropriate solution. Overall, ²¹³Bi yield consists of the efficiency of single steps, namely, ²²¹Fr elution, ²¹³Bi concentration, and subsequent extraction from the generator.

3.1. Elution of ²²¹Fr from the Column Containing ²²⁵Ac

Strong retention of ²²⁵Ac is necessary for long-term elution of ²²¹Fr; this is why the extraction chromatographic sorbent Actinide Resin demonstrating high affinity to Ac(III) in mineral acid media [33,34] was chosen for a mother column. The behavior of ²²¹Fr on Actinide Resin was studied via relatively fast elution by portions and the activity of ²²¹Fr in each portion is corrected towards the end of portion sampling. The typical shape of ²²¹Fr elution curve is shown in Figure 2.

The first part of the curve represents, mainly, the elution of ²²¹Fr, which was in transient equilibrium with ²²⁵Ac at the start of elution. This part of ²²¹Fr was stripped off in a small bolus-like volume of eluate. The volume V_max corresponding to maximum of bolus peak (Figure 2) was used for evaluation of capacity factor k’ of Fr(I):

$$k ’=\frac{V_{\max }-V_c}{V_c}=\frac{1-\raisebox{1ex}{$q_2$}\!\left/ \!\raisebox{-1ex}{$Q$}\right.}{\raisebox{1ex}{$q_2$}\!\left/ \!\raisebox{-1ex}{$Q$}\right.}$$

(1)

where V_c denotes the free volume of sorbent packing accessible for mobile phase (eluent), Q denotes flow rate of eluent, and q₂ denotes the rate of ²²¹Fr movement through the sorbent.

Then, the elution curve reached a plateau caused by the amount of ²²¹Fr produced during the elution and immediately washed out. It is the ²²¹Fr that is responsible for ²¹³Bi production in the generator “Afrabis”. The activity of ²²¹Fr in a portion of plateau-part of the elution curve depends on the residence time V_c/q₂ of ²²¹Fr in the chromatographic column [30]. The values of q₂ obtained from the plateau-part of curve also served for k’ Fr(I) evaluation. Normally, the k’ values determined from both bolus and plateau parts of elution curve were close.

²²¹Fr was easily separated from ²²⁵Ac adsorbed onto Actinide Resin with diluted solutions of mineral acids [30]. Elution of ²²¹Fr from a mother column filled with inorganic sorbent on the base of TiO₂·xH₂O was carried out with neutral NH₄Cl solutions [35]. In this work, we report data on capacity factor k’ of Fr(I) onto Actinide Resin in neutral (pH ~6) NaCl and NH₄Cl solutions (Figure 3).

The efficiency of ²²¹Fr elution with HNO₃ and salt solutions was found to be comparable. This means that we are able to use neutral diluted NaCl solution, e.g., physiological saline, in the generator “Afrabis” at the stage of ²¹³Bi accumulation, which reduces radiolytic impact on the resin.

Depending on the time of residence in column, some part of ²²¹Fr decays into ²¹³Bi, for which its retention on Actinide Resin is rather strong. For example, the k’ value of Bi(III) in 0.25 M HNO₃ was estimated at 4·10³ [30]. Therefore, it seems reasonable to use the flow rate of mobile phase as high as possible in order to diminish ²¹³Bi losses on the mother column during the accumulation stage, as it is shown in Figure 4.

However, the increase of flow rate results in the growth of solution volume passed through the mother column because the duration of the accumulation stage cannot be significantly shortened and is determined by the time required for attaining the transient equilibrium of ²¹³Bi to ²²⁵Ac (~5–6 half-lives of ²¹³Bi). The large solution volume may, in turn, promote the breakthrough of ²²⁵Ac and ²²⁷Ac/²²⁷Th adsorbed on Actinide Resin (data on the distribution of long-lived radionuclides along the Actinide Resin column is reported below). Thus, the choice of optimal flow rate for the accumulation stage is a compromise of two opposite factors.

3.2. Concentrating ²¹³Bi Apart from ²²⁵Ac

The next point after separation of ²²¹Fr from the mother column consists in selecting the conditions for concentrated ²¹³Bi accumulation. ²²¹Fr may be allowed to move long enough in a certain reservoir, for example, in form of tube in order to decay into ²¹³Bi before the latter is captured [30]. Another approach is related to the retarding ²²¹Fr in the appropriate chromatographic medium specific to ions of heavy alkali metals. A number of sorbents including AMP-PAN (Triskem Int.), Dowex 50 × 8 [30], and inorganic sorbent modified with nickel-potassium hexacyanoferrate(II) (sorbent T-35 manufactured by “Termoxid” company, Russia) [35] were tested. The present work focuses on the investigation of macroreticular cation exchange resin AG MP-50, which is also promising for ²¹³Bi retention and consecutive extraction from the generator.

Adsorption of ²²¹Fr and ²¹³Bi onto AG MP-50 was studied according to the procedure described earlier in detail [30]. The mother ²²⁵Ac column was connected to four successive columns with AG MP-50 of a small volume (0.1 mL). The solution of 0.25 M HNO₃ or 0.017 M NaCl was passed through the chromatographic system for no less than 4 h to attain transient equilibrium in the ²²⁵Ac → ²²¹Fr → ²¹³Bi → chain. Then the system was dismounted and γ-ray spectroscopic measurements of each column were performed. ²¹³Bi distribution between four AG MP-50 columns as a function of flow rate of 0.25 M HNO₃ solution is shown in Figure 5. Similarly, the ²¹³Bi distribution for 0.017 M NaCl solution was obtained.

Following the procedure [30], the experimental data were used for estimation of capacity factors k’ of Fr(I) onto AG MP-50 resin from 0.25 M HNO₃ and 0.017 M NaCl solutions. Predictably, adsorption of ²¹³Bi onto AG MP-50 was high and so it was only possible to set an upper limit for k’ of Bi(III). The results are presented in

Table 1. Capacity factors k’ of Fr(I) and Bi(III) adsorbed onto various sorbents.

Stationary Phase (Sorbent)	Mobile Phase (Solution)	k’ Fr(I)	k’ Bi(III)
AMP-PAN (Triskem) 1	0.25 M HNO3	2.5·102	>3·104
Dowex 50 × 8 (Dow) 1	0.25 M HNO3	102	>6·104
T-35 (Termoxid) 2	1 M NH4Cl, pH 6.8	5·102	>5·104
AG MP-50 (BioRad)	0.25 M HNO3	4·102	>5·104
AG MP-50 (BioRad)	0.017 M NaCl, pH ~6	103	>5·104

Literature data: ¹ [30], ² [35].

in comparison with the data reported earlier.

According to the results obtained, AG MP-50 can serve as a sorbent of second column for ²¹³Bi accumulation. A small amount of AG MP-50 within 0.3–0.4 mL is sufficient for significant retention of ²¹³Bi and this means that ²¹³Bi can be further recovered from the generator in a more concentrated form.

Comparison of ²²¹Fr sorption on two cation exchange resins Dowex 50 × 8 and AG MP-50 from 0.25 M HNO₃ solution reveals (Table 1) that the k’ values of Fr(I) onto AG MP-50 is four times higher. Considering relatively equal chromatographic behavior of alkali metal ions in nitric and hydrochloric solutions [36,37,38], a similar tendency is observed for ions of other alkali metals (Figure 6).

Capacity factor k’ was transformed into the mass distribution ratio D_m via the known relation: $D_m=k^{\prime }\frac{\epsilon }{{\rho }_{app}}$, where $\epsilon$ denotes the fraction of sorbent free volume; ${\rho }_{app}$, denotes apparent density of sorbent. The manufacturer’s data for both Dowex and AG resin types are close and the values $\epsilon$ = 0.38 and ${\rho }_{app}$ = 0.8 g/mL may be used for a first approximation [39].

Considering that the alkali metal ions except Fr(I) were studied in batch experiments [28,38], the data consistency is quite satisfactory and allows us to conclude that the heavier the alkali metal ion, the more difference in its adsorption onto normal and macroreticular cation exchange resins is observed.

3.3. Extraction of ²¹³Bi

The advantage of a two-column generator is the ability to perform desorption of the product with any of the most convenient complexing agents. In the case of a single-column generator, the eluent should be selected in such a manner as to efficiently transfer the daughter radionuclide into the solution and minimize the breakthrough of the parent radionuclide as well. If the parent and daughter radionuclides are kept in equilibrium and are simultaneously separated in space, for example, they are held by different columns, then there is no such limitation. Desorption of ²¹³Bi can be carried out directly by a chelator or even by a chelator–protein conjugate, in which case the labeling takes place in the column and a ready-to-use radiopharmaceutical is obtained in the eluate. After desorption of ²¹³Bi, the second column is washed with the solution used at the stage of its accumulation or it is replaced with a new one and the generator is ready for the next cycle.

A 0.1M HCl/0.1M KI solution is used for ²¹³Bi desorption in a single-column generator. This solution is convenient because Bi(III) forms strong complexes with halide ions, in particular, stable complexes ${\mathrm{BiI}}_4^{-}/{\mathrm{BiI}}_5^{2-}$ are formed with iodides [40], while Ac(III) does not form such strong complexes. The low acid content in the resulting eluate allows the quick creation of the medium suitable for labeling (pH 5–7) by adding a small volume of buffer. The desorption of ²¹³Bi from a second column containing AG MP-50 resin with a 0.1M HCl/0.1M KI solution was studied. The overall efficiency of the "Afrabis" generator system was evaluated, provided that the accumulation stage was carried out by circulating 0.25 M HNO₃ solution for ~4 h and ²¹³Bi was stripped off with 0.1M HCl/0.1M KI solution. The efficiency of the generator “Afrabis” that depends on the flow rate of the eluent during circulation is shown in Figure 7. The efficiency values for the first portion of eluate (0.5 mL) and for the whole fraction (3 mL) are given. The desorption rate of the eluent was constant at 1 mL/min. It can be seen that the ²¹³Bi yield increases with an increase of the flow rate during the accumulation stage and after 1 mL/min it reaches a plateau approaching 75% in the first 0.5 mL of the eluate and 90% in 3 mL.

A series of elutions with a constant flow rate at the accumulation stage and subsequent desorption of the product (1 mL/min) was performed to study the stability of the system. The average yield of ²¹³Bi was 73 ± 2% in the first 0.5 mL of the eluate.

The elution of ²¹³Bi can also be carried out with 1 M HCl solution. The advantage of this approach is the high stability of a 1 M HCl solution unlike 0.1M HCl/0.1M KI and a small volume of eluent (0.5–1.0 mL) can be quickly evaporated using a special heater. The mass distribution ratios D_m, mL/g, of Bi(III) upon sorption onto AG MP-50 as a function of HCl concentrations are given in Figure 8. It can be seen that D_m of Bi(III) becomes negligible for a HCl concentration higher than 0.5 M. At the same time, the retention of parent Ac in 1 M HCl is lower than in 0.1M HCl/0.1M KI, which can affect the service life of a single-column generator. In the case of a two-column generator, there is no such a problem and the efficiency for desorption with 1 M HCl is as high as for a 0.1M HCl/0.1M KI solution.

1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and diethylenetriaminepentaacetic acid (DTPA) are the most often used in clinical medicine, although much of the new chelating ligands for Bi(III) are being synthesized and presently actively studied [41]. That is why these compounds were taken as test chelators for Bi desorption from the second column of the generator “Afrabis”.

The DTPA ligand forms a stable complex with Bi(III) (lgK (Bi-DTPA) = 35.2 [42]), which is formed in a few minutes and is already at room temperature. Due to its fast kinetics, DTPA is well suited for dynamic chelation, i.e., for desorption of ²¹³Bi from the second column of the generator “Afrabis” in contrast to static chelation performed in batch conditions. The elution efficiencies of Bi from the column filled with AG MP-50 depending on the DTPA concentration and pH of the eluent are presented in Figure 9. Even at a concentration of 10⁻⁵ M, the desorption efficiency is close to 90% (²¹³Bi distribution in other parts of the chromatographic system is not taken into account) and increases with an increase in the concentration of the ligand. The desorption efficiency also slightly increases with an increase of the pH value of solution. This effect is probably related to the kinetics of complex formation. At a constant DTPA concentration, the rate of complex formation decreases as pH rises. A similar dependence is observed for the kinetics of the formation of DTPA complexes with lanthanides [43].

Integral (% of activity in the eluate) and differential (%/0.1 mL) ²¹³Bi elution curves are shown in Figure 10. The typical elution was conducted with 10⁻⁵ M DTPA with a pH 5.3. About 85% of ²¹³Bi is eluted with the first 1.0 mL of the solution. For comparison, the same figure shows the elution curves of ²¹³Bi with 0.1 M HCl/0.1 M HI for the conventional single-column generator based on AG MP-50 (discussed in 3.5). It can be observed that, in comparison with iodide complexes, the kinetics of Bi-DTPA complex formation is slower, but the overall desorption efficiency with DTPA solution is higher than with 0.1M HCl/0.1M KI.

Another widely used ligand is DOTA, an azacrown compound that is often referred to as the "gold standard" for many cations. It forms stable complexes (lgK (Bi-DOTA) = 30.3 [44]), while its main disadvantage is a slow rate of binding with most radionuclides. Thus, according to the literature data, the complexation with Bi must be carried out at heating and at 60–100 °C it will take 5–15 min [45]. For this reason, it is unsuitable to carry out the desorption of Bi(III) with a DOTA solution in a dynamic condition. However, the AG MP-50 sorbent can be quickly removed from the column and DOTA is labeled in a static regime by mixing the sorbent with the ligand solution at heating. It is found that the desorption with 3 mL 10⁻⁵ M DOTA solution (pH 5.3) under static conditions at a temperature of 90 ± 3 °С results in the binding of 80 ± 3% ²⁰⁷Bi in 5 min. In this case, there is no need to separate the non-complexed part of the radionuclide, since it remains bound on the cation-exchange sorbent.

3.4. Radionuclide Purity of ²¹³Bi Extracted from the Generator “Afrabis”

It is a matter for the near future when the part of ²²⁵Ac applied as a source for ²¹³Bi generation is produced with the admixture of long-lived ²²⁷Ac. More stringent requirements will be imposed on the radionuclide purity of the ²¹³Bi eluate in this case. In the generator “Afrabis”, due to the non-aggressive mobile phase (weakly acidic or neutral saline solution) used for the ²¹³Bi accumulation stage, ^225,227Ac radioisotopes are strongly bound onto the Actinide Resin column. Less than 0.2% of actinium was reported to be washed out of the mother column after one month of daily four hour generation cycles [46]. The actinium breakthrough was then effectively fixed on the second column preventing the final ²¹³Bi from the impurity of actinium radioisotopes.

The influence of relatively long-lived decay products of ²²⁷Ac, namely ²²⁷Th and ²²³Ra, on the radionuclidic purity of ²¹³Bi eluate is also important. Starting from the initial ²²⁷Ac/²²⁵Ac ratio ~0.2% at EOB and assuming null presence of ²²⁷Th and ²²³Ra at the moment of generator loading, the content of ²²⁷Ac, ²²⁷Th, and ²²³Ra grows up to 4.2%, 2.8%, and 1.7%, respectively, by the end-of-month generator operation. ²²⁷Th is retained by the sorbents of both columns no less firmly than actinium [33]. In contrast, the adsorption of ²²³Ra depends, to a greater extent, on solution acidity. For the example of 0.25 M HNO₃ solution circulating in the system at the stage of ²¹³Bi accumulation, the k’ value of Ra(II) on Actinide Resin falls within 20–30 [33], i.e., ²²³Ra is quite easily eluted from the mother column.

Scans of total activity produced by ^225,227Ac and ²²⁷Th permanently adsorbed on the Actinide Resin column as a function of number of elutions or of a total volume of 0.25 M HNO₃ solution passed through the column are shown in Figure 11. It can be observed that the initial location of ^225,227Ac and ²²⁷Th in the first third of the column is slightly displaced after passing 6 L of the solution.

Since ²²³Ra is being removed in every generation cycle from the Actinide Resin column, the chromatographic system does not work as a double trap, which is the case of actinium and thorium radioisotopes. ²²³Ra is concentrated together with ²¹³Bi on the second AG MP-50 column. According to the results presented in Figure 8, the D_m value of Ra(II) in 0.25 M HNO₃ solution reaches 10⁵ mL/g, while the D_m Ra(II) in 0.1 M HCl/0.1 M HI solution used for ²¹³Bi elution decreases down to 1.2∙10⁴ mL/g. Although the adsorption of radium on AG MP-50 is rather strong, some ²²³Ra breakthrough into the ²¹³Bi eluate is possible, especially when ²¹³Bi is eluted with certain ligand solutions such as DTPA.

There were two opportunities considered for the periodic elimination of ²²³Ra from the generator. The first method was to replace the second AG MP-50 column with a new one after each generation cycle and, hence, ²²³Ra produced from ²²⁷Th between the cycles will be transferred to the second column. Otherwise, the mother column can be preconditioned before a generation cycle as it is shown in Figure 1, i.e., it can be preliminary washed with 0.25 M HNO₃ or saline solution for removing ²²³Ra from the system. In this case, ²²³Ra produced from ²²⁷Th during only a single cycle is concentrated on the second column and accumulated from one cycle to another. Finally, we can combine the two opportunities. Figure 12 illustrates estimations of maximal ²²³Ra amount residing in the generator “Afrabis” in the course of a months work proceeding from the premises that ²¹³Bi is generated once a day and one cycle’s duration is four hours.

Obviously, the least content of ²²³Ra is achieved by both preconditioning the mother column and replacing the second column. However, the generator maintenance becomes rather tedious so far and that is why the scheme including only the preconditioning was chosen for experimental trial. As a result of a months generator operation following the procedure given in Section 2.5, it was found that the impurities of ²²⁵Ac, ²²⁷Th, and ²²³Ra in 0.5 mL of the ²¹³Bi eluate did not exceed 10⁻⁶% (detection limit), while the impurity of ²²⁷Ac estimated from the initial ²²⁷Ac/²²⁵Ac ratio was less than 10⁻⁸%.

3.5. Single-Column ²²⁵Ac/²¹³Bi Generator with AG MP-50

Single-column "direct" ²²⁵Ac/²¹³Bi generator with AG MP-50 resin [12] is the most common for ²¹³Bi production. Clinical tests are carried out using such generators. The parent ²²⁵Ac is firmly retained by the sorbent and ²¹³Bi is eluted with a complexing agent. The milking protocol was proposed at the Joint Research Centre in Karlsruhe (JRC, Germany). According to the protocol, ²¹³Bi is eluted with 0.6 mL of 0.1 M NaI/0.1 M HCl solution from a column containing 0.33 mL of AG MP-50. The elution efficiency is reported to be 76 ± 3%, ²²⁵Ac breakthrough is less than 2·10⁻⁵%. The possible disadvantage of this generator is low radiation resistance of the organic sorbent [25]. Moreover, there are no data on the application of this generator with an accelerator-produced ²²⁵Ac containing an impurity of ²²⁷Ac and ²²⁷Ac decay products—²²⁷Th and ²²³Ra.

A generator based on AG MP-50 was prepared in order to compare its performance with the generator system “Afrabis”. The generator was loaded, stored, and milked according to the procedure described [12] with the only difference being that ²²⁵Ac that is obtained by irradiation of thorium with protons (~0.2% ²²⁷Ac at the EOB) is applied. As a result of loading of ²²⁵Ac in a small volume of 4 M HNO₃, the activity deposits on the top two-third part of the column. This approach was proposed by JRC to reduce the influence of radiation impact on the sorbent. Initial distribution of ²²⁵Ac in the AG MP-50 column of ²²⁵Ac/²¹³Bi generator is shown in Figure A1. It can be seen that the distribution of activity is consistent with the literature data [12].

The results of the generator investigation carried out during 1 month are given in

Table 2. Results of ²¹³Bi elution from the single-column ²²⁵Ac/²¹³Bi generator with AG MP-50 resin.

225Ac/213Bi Generator	Efficiency of 213Bi Elution, %/in the First 0.5 mL of Eluate	Impurity in the First Eluate Portion of 0.5 mL, %
		225Ac	227Ac	227Th	223Ra
“Afrabis”	73 ± 2	<10−6	<10−8 **	<10−6	<10−6
JRC (Karlsruhe, Germany)	67 ± 2 76 ± 3 [10] *	<3.5·10−5 2·10−5 [10] *	<10−6 **	<10−6	10−4–10−3

* For first 0.6 mL of eluate; ** (estimated by ²²⁵Ac).

in comparison with literature data and the generator “Afrabis” performance.

The typical integral and differential ²¹³Bi elution curves with 0.1M HCl/0.1M KI from the single-column generator with AG MP-50 are shown in Figure 10. The elution efficiency of ²¹³Bi for the single-column generator (67 ± 2% in 0.5 mL) is slightly lower than for the generator “Afrabis” (73 ± 2% in 0.5 mL). At the same time, the estimate of the breakthrough of the parent ²²⁵Ac (<3.5 × 10⁻⁵%) is consistent with the literature data, which was obtained for higher loaded ²²⁵Ac activity. The resulting ²²⁵Ac content limit for the generator system “Afrabis” is noticeably lower (<10⁻⁶%).

The difference in the degree of purification from ²²³Ra, which is a decay product of ²²⁷Ac, is significant; for a single-column generator, ²²³Ra activity concentration in the first portion of ²¹³Bi eluate is 10^−4–10⁻³%, while for the generator “Afrabis” the corresponding value is <10⁻⁶%. The obtained results are in accordance with theoretical views. Ac(III), Th(IV), and Ra(II) do not form strong iodide and chloride complexes, unlike Bi(III) [40]. Therefore, the retention of these ions by a strongly acidic cation exchange sorbent from the 0.1 M NaI/0.1 M HCl solution increases with their charges in the order Ra(II) < Ac(III) < Th(IV) [39].

It can be concluded that the developed generator “Afrabis” provides ²¹³Bi with a higher radionuclide purity than compared to the conventional generator. The content of the parent radionuclide in ²¹³Bi solution is at least one order of a magnitude lower and ²²³Ra is at least two orders of a magnitude lower. At the same time, the efficiencies of ²¹³Bi extraction from both generators are similar.

4. Conclusions

A new two-column ²²⁵Ac/²¹³Bi generator intended for using the parent ²²⁵Ac with ²²⁷Ac impurity is proposed and investigated. The formation and concentration of ²¹³Bi on the second column is realized by continuous separation of intermediate ²²¹Fr from ²²⁵Ac fixed on the first column. The generator is compact due to circulation of the mobile phase in a closed-loop circuit. In comparison with the generators described in literature, this provides a high product yield with a low breakthrough of actinium isotopes and ²²⁷Ac decay products. The concentration of ²¹³Bi on a separate column allows us to desorb it with any convenient complexing agent, including chelation and labeling directly on the column.