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

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.

The radionuclide, 213 Bi, is a decay product of another promising alpha emitter 225 Ac, which is used by itself [10,11] or as a parent radionuclide for 225 Ac/ 213 Bi 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, 213 Bi, 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 213 Bi are required to achieve a therapeutic effect. Depending on the vector molecules, a possible range of 213 Bi administered dose may vary within 5-10 GBq [1].
Carrier-free 225 Ac is applied as a source of 213 Bi for conducting clinical trials of 213 Bicontaining radiopharmaceuticals. It is obtained by the generator method from 229 Th (T 1/2 = 7.340 y) and the stock supplies are extremely limited. The world production of 225 Ac by this method is no more than 70 GBq per year [2]. The main alternative method of 225 Ac production is the irradiation of natural thorium with medium-energy protons. This

Preparation and Milking 225 Ac/ 213 Bi Generator "Afrabis"
The amount of 0.1 MBq of 225 Ac (with the 227 Ac impurity) was dissolved in 5 mL of 0.1 M HNO 3 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 213 Bi generation was as follows: 1.
The first column was washed from the accumulated 223 Ra with 15-20 mL of 0.25 M HNO 3 ; 2.
Then, continuous separation of the intermediate daughter radionuclide 221 Fr from 225 Ac for~4 h was occurring circularly (Figure 1) at flow rate 1-1.5 mL/min; 3. 213 Bi 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 213 Bi 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 207 Bi 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 207 Bi with a DOTA solution, the AG MP-50 resin with 207 Bi 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.

Preparation and Milking A Single-Column 225 Ac/ 213 Bi Generator with AG MP-50
The amount of 0.1 MBq of 225 Ac (with the 227 Ac 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 The desorption of 213 Bi 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 HNO 3 containing 207 Bi 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 HNO 3 for the next Bi sorption.
In order to test desorption of 207 Bi with a DOTA solution, the AG MP-50 resin with 207 Bi 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.

Preparation and Milking A Single-Column 225 Ac/ 213 Bi Generator with AG MP-50
The amount of 0.1 MBq of 225 Ac (with the 227 Ac impurity) was dissolved in 2.8 mL of 4 M HNO 3 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 HNO 3 , 2 mL 2 M HCl, and finally with 0.01 M HCl [12,31]. As a result, 225 Ac 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 213 Bi was eluted with a mixture of 0.1 M NaJ/HCl solution followed by washing with 0.01 M HCl. The resulting 213 Bi 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 213 Bi to determine the impurity of long-lived radionuclides-225 Ac, 227 Th, and 223 Ra.The generator was stored wet in the 0.01 M HCl until the next use. Seven elutions of 213 Bi were carried out during 1 month of the generator exploitation.

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 213 Bi take place in the course of permanent separation of intermediate 221 Fr from 225 Ac fixed on a mother column ( Figure 1) and its decay.
As a result of dynamic accumulation arranged in circulation mode, 213 Bi 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 225 Ac (it takes~5-6 half-lives of 213 Bi). Finally, 213 Bi is extracted in a small volume of appropriate solution. Overall, 213 Bi yield consists of the efficiency of single steps, namely, 221 Fr elution, 213 Bi concentration, and subsequent extraction from the generator.

Elution of 221 Fr from the Column Containing 225 Ac
Strong retention of 225 Ac is necessary for long-term elution of 221 Fr; 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 221 Fr on Actinide Resin was studied via relatively fast elution by portions and the activity of 221 Fr in each portion is corrected towards the end of portion sampling. The typical shape of 221 Fr elution curve is shown in Figure 2.  The first part of the curve represents, mainly, the elution of 221 Fr, which was in transient equilibrium with 225 Ac at the start of elution. This part of 221 Fr was stripped off in a small bolus-like volume of eluate. The volume Vmax corresponding to maximum of bolus  The first part of the curve represents, mainly, the elution of 221 Fr, which was in transient equilibrium with 225 Ac at the start of elution. This part of 221 Fr 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): where V c denotes the free volume of sorbent packing accessible for mobile phase (eluent), Q denotes flow rate of eluent, and q 2 denotes the rate of 221 Fr movement through the sorbent. Then, the elution curve reached a plateau caused by the amount of 221 Fr produced during the elution and immediately washed out. It is the 221 Fr that is responsible for 213 Bi production in the generator "Afrabis". The activity of 221 Fr in a portion of plateau-part of the elution curve depends on the residence time V c /q 2 of 221 Fr in the chromatographic column [30]. The values of q 2 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. 221 Fr was easily separated from 225 Ac adsorbed onto Actinide Resin with diluted solutions of mineral acids [30]. Elution of 221 Fr from a mother column filled with inorganic sorbent on the base of TiO 2 ·xH 2 O was carried out with neutral NH 4 Cl solutions [35]. In this work, we report data on capacity factor k' of Fr(I) onto Actinide Resin in neutral (pH~6) NaCl and NH 4 Cl solutions ( Figure 3).  The efficiency of 221 Fr 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 213 Bi accumulation, which reduces radiolytic impact on the resin.
Depending on the time of residence in column, some part of 221 Fr decays into 213 Bi, 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 4·10 3 [30]. Therefore, it seems reasonable to use the flow rate of mobile phase as high as possible in order to diminish 213 Bi 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 213 Bi to 225 Ac (~5-6 half-lives of 213 Bi). The large solution volume may, in The efficiency of 221 Fr elution with HNO 3 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 213 Bi accumulation, which reduces radiolytic impact on the resin.
Depending on the time of residence in column, some part of 221 Fr decays into 213 Bi, for which its retention on Actinide Resin is rather strong. For example, the k' value of Bi(III) in 0.25 M HNO 3 was estimated at 4·10 3 [30]. Therefore, it seems reasonable to use the flow rate of mobile phase as high as possible in order to diminish 213 Bi losses on the mother column during the accumulation stage, as it is shown in Figure 4.

Concentrating 213 Bi Apart from 225 Ac
The next point after separation of 221 Fr from the mother column consists in selecting the conditions for concentrated 213 Bi accumulation. 221 Fr may be allowed to move long enough in a certain reservoir, for example, in form of tube in order to decay into 213 Bi before the latter is captured [30]. Another approach is related to the retarding 221 Fr 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 213 Bi retention and consecutive extraction from the generator.
Adsorption of 221 Fr and 213 Bi onto AG MP-50 was studied according to the procedure described earlier in detail [30]. The mother 225 Ac 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 225 Ac → 221 Fr → 213 Bi → chain. Then the system was dismounted and γ-ray spectroscopic measurements of each column were performed. 213 Bi 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 213 Bi distribution for 0.017 M NaCl solution was obtained. 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 213 Bi to 225 Ac (~5-6 half-lives of 213 Bi). The large solution volume may, in turn, promote the breakthrough of 225 Ac and 227 Ac/ 227 Th 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.

Concentrating 213 Bi Apart from 225 Ac
The next point after separation of 221 Fr from the mother column consists in selecting the conditions for concentrated 213 Bi accumulation. 221 Fr may be allowed to move long enough in a certain reservoir, for example, in form of tube in order to decay into 213 Bi before the latter is captured [30]. Another approach is related to the retarding 221 Fr 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 213 Bi retention and consecutive extraction from the generator.
Adsorption of 221 Fr and 213 Bi onto AG MP-50 was studied according to the procedure described earlier in detail [30]. The mother 225 Ac column was connected to four successive columns with AG MP-50 of a small volume (0.1 mL). The solution of 0.25 M HNO 3 or 0.017 M NaCl was passed through the chromatographic system for no less than 4 h to attain transient equilibrium in the 225 Ac → 221 Fr → 213 Bi → chain. Then the system was dismounted and γ-ray spectroscopic measurements of each column were performed. 213 Bi distribution between four AG MP-50 columns as a function of flow rate of 0.25 M HNO 3 solution is shown in Figure 5. Similarly, the 213 Bi 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 213 Bi 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 in comparison with the data reported earlier. Literature data: 1 [30], 2 [35].
According to the results obtained, AG MP-50 can serve as a sorbent of second column for 213 Bi accumulation. A small amount of AG MP-50 within 0.3-0.4 mL is sufficient for significant retention of 213 Bi and this means that 213 Bi can be further recovered from the generator in a more concentrated form.
Comparison of 221 Fr 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). 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 HNO 3 and 0.017 M NaCl solutions. Predictably, adsorption of 213 Bi 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 in comparison with the data reported earlier. Literature data: 1 [30], 2 [35].
According to the results obtained, AG MP-50 can serve as a sorbent of second column for 213 Bi accumulation. A small amount of AG MP-50 within 0.3-0.4 mL is sufficient for significant retention of 213 Bi and this means that 213 Bi can be further recovered from the generator in a more concentrated form.
Comparison of 221 Fr sorption on two cation exchange resins Dowex 50 × 8 and AG MP-50 from 0.25 M HNO 3 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).  [28,38] are shown by empty circles and squares.
Capacity factor k' was transformed into the mass distribution ratio Dm via the known relation: = ′ , where denotes the fraction of sorbent free volume; , denotes apparent density of sorbent. The manufacturer's data for both Dowex and AG resin types are close and the values = 0.38 and = 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.

Extraction of 213 Bi
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 213 Bi 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 213 Bi, 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 213 Bi desorption in a single-column generator. This solution is convenient because Bi(III) forms strong complexes with halide ions, in particular, stable complexes BiI 4 − /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 213 Bi 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 circulat- Figure 6. The mass distribution ratios D m , mg/mL, of alkali metal ions upon sorption onto Dowex 50 × 8 and AG MP-50 from 0.25 M nitric or hydrochloric acid. Literature data [28,38] are shown by empty circles and squares.
Capacity factor k' was transformed into the mass distribution ratio D m via the known relation: D m = k ε ρ app , where ε denotes the fraction of sorbent free volume; ρ app , denotes apparent density of sorbent. The manufacturer's data for both Dowex and AG resin types are close and the values ε = 0.38 and ρ 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.

Extraction of 213 Bi
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 213 Bi 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 213 Bi, 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 213 Bi desorption in a single-column generator. This solution is convenient because Bi(III) forms strong complexes with halide ions, in particular, stable complexes BiI − 4 /BiI 2− 5 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 213 Bi 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 HNO 3 solution for~4 h and 213 Bi 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 213 Bi 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.
Pharmaceutics 2021, 13, x FOR PEER REVIEW 11 of 20 ing 0.25 M HNO3 solution for ~4 h and 213 Bi 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 213 Bi 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 213 Bi was 73 ± 2% in the first 0.5 mL of the eluate.
The elution of 213 Bi 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 Dm, 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 Dm 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. 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 213 Bi was 73 ± 2% in the first 0.5 mL of the eluate.
The elution of 213 Bi 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".
Pharmaceutics 2021, 13, x FOR PEER REVIEW 13 of 21 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 213 Bi 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% ( 213 Bi 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) 213 Bi elution curves are shown in Figure 10  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 213 Bi 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% ( 213 Bi 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) 213 Bi 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 213 Bi is eluted with the first 1.0 mL of the solution. For comparison, the same figure shows the elution curves of 213 Bi 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. 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 • C results in the binding of 80 ± 3% 207 Bi 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.

Radionuclide Purity of 213 Bi Extracted from the Generator "Afrabis"
It is a matter for the near future when the part of 225 Ac applied as a source for 213 Bi generation is produced with the admixture of long-lived 227 Ac. More stringent requirements will be imposed on the radionuclide purity of the 213 Bi eluate in this case. In the generator "Afrabis", due to the non-aggressive mobile phase (weakly acidic or neutral saline solution) used for the 213 Bi accumulation stage, 225,227 Ac 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 213 Bi from the impurity of actinium radioisotopes.
The influence of relatively long-lived decay products of 227 Ac, namely 227 Th and 223 Ra, on the radionuclidic purity of 213 Bi eluate is also important. Starting from the initial 227 Ac/ 225 Ac ratio~0.2% at EOB and assuming null presence of 227 Th and 223 Ra at the moment of generator loading, the content of 227 Ac, 227 Th, and 223 Ra grows up to 4.2%, 2.8%, and 1.7%, respectively, by the end-of-month generator operation. 227 Th is retained by the sorbents of both columns no less firmly than actinium [33]. In contrast, the adsorption of 223 Ra depends, to a greater extent, on solution acidity. For the example of 0.25 M HNO 3 solution circulating in the system at the stage of 213 Bi accumulation, the k' value of Ra(II) on Actinide Resin falls within 20-30 [33], i.e., 223 Ra is quite easily eluted from the mother column.
Scans of total activity produced by 225,227 Ac and 227 Th permanently adsorbed on the Actinide Resin column as a function of number of elutions or of a total volume of 0.25 M HNO 3 solution passed through the column are shown in Figure 11. It can be observed that the initial location of 225,227 Ac and 227 Th in the first third of the column is slightly displaced after passing 6 L of the solution. There were two opportunities considered for the periodic elimination of 223 Ra 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, 223 Ra produced from 227 Th 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 223 Ra from the system. In this case, 223 Ra produced from 227 Th 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 223 Ra amount residing in the generator "Afrabis" in the course of a months work proceeding from the premises that 213 Bi is generated once a day and one cycle's duration is four hours. Since 223 Ra 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. 223 Ra is concentrated together with 213 Bi 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 HNO 3 solution reaches 10 5 mL/g, while the D m Ra(II) in 0.1 M HCl/0.1 M HI solution used for 213 Bi elution decreases down to 1.2·10 4 mL/g. Although the adsorption of radium on AG MP-50 is rather strong, some 223 Ra breakthrough into the 213 Bi eluate is possible, especially when 213 Bi is eluted with certain ligand solutions such as DTPA.
There were two opportunities considered for the periodic elimination of 223 Ra 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, 223 Ra produced from 227 Th 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 HNO 3 or saline solution for removing 223 Ra from the system. In this case, 223 Ra produced from 227 Th 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 223 Ra amount residing in the generator "Afrabis" in the course of a months work proceeding from the premises that 213 Bi is generated once a day and one cycle's duration is four hours.
There were two opportunities considered for the periodic elimination of 223 Ra 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, 223 Ra produced from 227 Th 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 223 Ra from the system. In this case, 223 Ra produced from 227 Th 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 223 Ra amount residing in the generator "Afrabis" in the course of a months work proceeding from the premises that 213 Bi is generated once a day and one cycle's duration is four hours. Figure 12. Estimation of maximal 223 Ra amount in the generator "Afrabis" producing 213 Bi once a day in a four hour cycle: 1-without preconditioning the mother column or replacing the second Figure 12. Estimation of maximal 223 Ra amount in the generator "Afrabis" producing 213 Bi once a day in a four hour cycle: 1-without preconditioning the mother column or replacing the second column (black line); 2-with replacing the second column after each cycle (red line); 3-with preconditioning the mother column before each cycle (blue line); 4-with preconditioning the mother column and replacing the second column every time (green line).
Obviously, the least content of 223 Ra 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 225 Ac, 227 Th, and 223 Ra in 0.5 mL of the 213 Bi eluate did not exceed 10 −6 % (detection limit), while the impurity of 227 Ac estimated from the initial 227 Ac/ 225 Ac ratio was less than 10 −8 %.

Single-Column 225 Ac/ 213 Bi Generator with AG MP-50
Single-column "direct" 225 Ac/ 213 Bi generator with AG MP-50 resin [12] is the most common for 213 Bi production. Clinical tests are carried out using such generators. The parent 225 Ac is firmly retained by the sorbent and 213 Bi is eluted with a complexing agent. The milking protocol was proposed at the Joint Research Centre in Karlsruhe (JRC, Germany). According to the protocol, 213 Bi 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%, 225 Ac 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 225 Ac containing an impurity of 227 Ac and 227 Ac decay products-227 Th and 223 Ra.
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 225 Ac that is obtained by irradiation of thorium with protons (~0.2% 227 Ac at the EOB) is applied. As a result of loading of 225 Ac in a small volume of 4 M HNO 3 , 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 225 Ac in the AG MP-50 column of 225 Ac/ 213 Bi 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 in comparison with literature data and the generator "Afrabis" performance. The typical integral and differential 213 Bi 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 213 Bi 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 225 Ac (<3.5 × 10 −5 %) is consistent with the literature data, which was obtained for higher loaded 225 Ac activity. The resulting 225 Ac content limit for the generator system "Afrabis" is noticeably lower (<10 −6 %).
The difference in the degree of purification from 223 Ra, which is a decay product of 227 Ac, is significant; for a single-column generator, 223 Ra activity concentration in the first portion of 213 Bi 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 213 Bi with a higher radionuclide purity than compared to the conventional generator. The content of the parent radionuclide in 213 Bi solution is at least one order of a magnitude lower and 223 Ra is at least two orders of a magnitude lower. At the same time, the efficiencies of 213 Bi extraction from both generators are similar.

Conclusions
A new two-column 225 Ac/ 213 Bi generator intended for using the parent 225 Ac with 227 Ac impurity is proposed and investigated. The formation and concentration of 213 Bi on the second column is realized by continuous separation of intermediate 221 Fr from 225 Ac 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 227 Ac decay products. The concentration of 213 Bi on a separate column allows us to desorb it with any convenient complexing agent, including chelation and labeling directly on the column.