Investigation of a tellurium-packed column for isolation of astatine-211 from irradiated bismuth targets and demonstration of a semi-automated system

Astatine-211 is an attractive radionuclide for use in targeted alpha therapy of blood-borne diseases and micrometastatic diseases. Efficient isolation methods that can be adapted to robust automated 211At isolation systems are of high interest for improving the availability of 211At. Based on the early studies of Bochvarova and co-workers involving isolation of 211At from irradiated thorium targets, we developed a method for 211At isolation from bismuth targets using tellurium-packed columns. Dissolution of irradiated bismuth targets is accomplished using HNO3; however, 211At is not captured on the Te column material in this matrix. Our method involves slow addition of aqueous NH2OH·HCl to the Bi target dissolved in HNO3 to convert to a HCl matrix. The amount of NH2OH·HCl was optimized because (1) the quantity of NH2OH·HCl used appears to affect the radiolabeling yield of phenethyl-closo-decaborate(2-) (B10)-conjugated antibodies and (2) reducing the volume of NH2OH·HCl solution can effectively shorten the overall isolation time. A proof-of-concept semi-automated process has been demonstrated using targets containing ~0.96 GBq (~26 mCi) of 211At. High isolation yields (88–95%) were obtained. Radiochemical purity of the isolated 211At was assessed by radio-HPLC. Concentrations of Bi and Te contaminants in the 211At and the astatinated antibodies were evaluated using ICP-MS.

is removed by distillation and 8 M HCl is used to re-dissolve the Bi(NO 3 ) 3 salt residue. Liquid-liquid extractions are then performed to isolate 211 At from the 211 At/Bi 3+ mixture and to remove Bi 3+ salts using DIPE and 8 M HCl. We have investigated automation of this wet chemistry, liquid-liquid exaction method for 211 At isolation, and although it has been technically challenging, we have had some success in automation 18 . Unfortunately, thus far we have not been able to decrease the time required using the automated "wet chemistry" 211 At isolation process from that achieved in the manual separation procedure (unpublished data). In an effort to simplify the isolation process and decrease the time to obtain the isolated 211 At, we looked for alternative isolation methods.
During the separation process in the wet chemistry method, astatine undergoes several changes in its oxidation state, possibly from At(+5) to At(+3), then to At(0), and finally to At(−1) 14 . Along with the change in oxidation state there are likely different chemical species produced. In addition to astatide, four other unknown astatine species have been observed at different times by anion exchange radio-HPLC 12 . The inconsistency in the radiochemical purity of the 211 At isolated using the DIPE extraction method can result in poor radiolabeling yields, which can be a major problem in fulfilling prescribed doses in the clinical setting.
Here we report a new 211 At-isolation approach based on a tellurium-packed column (Te column) previously described in the literature. Bochvarova et al. reported a method of using two tellurium metal packed columns to effectively isolate 211 At from 660 MeV proton beam irradiated thorium targets 19 . Astatine-211 can be rapidly absorbed on metallic tellurium in HCl in the presence of SnCl 2 and eluted by a solution of 1-2 M NaOH. In order to adapt the Te column method to irradiated bismuth targets, we used NH 2 OH⋅HCl to convert the HNO 3 solution containing the dissolved target to a solution of HCl. We also demonstrated this new method can be readily automated and can provide [ 211 At]NaAt of consistent and high radiochemical purity.

Results
At-211 isolation using Te columns. Figure 1 summarizes the steps involved in the process of isolating 211 At from irradiated Bi targets using Te columns.
Step 1 involves dissolution of the bismuth target using 10 M HNO 3 . This is the same initial step used in the automation of the wet chemistry isolation process 18 .
Step 2 involves addition of NH 2 OH·HCl to the HNO 3 solution containing dissolved Bi dropwise until complete cessation of bubbling is noted. Steps 3-6 are conducted as shown in Fig. 1. However, in our initial studies steps 3 and 5 were slightly different, as a reductant, SnCl 2 was used to assure that the 211 At was in the astatide form. The steps initially used were as follows: In step 3, 0.1 M SnCl 2 in 6 M HCl was used instead of 1.5 M HCl and in step 5, the column was washed by 0.1 M SnCl 2 in 6 M HCl, 6 M HCl and deionized (D.I.) H 2 O sequentially. As NH 2 OH·HCl is a strong reducing agent, the use of 0.1 M SnCl 2 seemed to be redundant but we thought it would be best to evaluate isolation yields with and without having SnCl 2 present. Table 1 shows that the astatine adsorption kinetics of the Te column is very fast and efficient, irrespective of whether SnCl 2 was used or not. Essentially all of the 211 At was absorbed by the Te column from 1.5 M HCl even at a high flow rate of 5-10 mL/min. Moreover, very little activity was found in the washes (<0.1%) and isolation yields ~75% were obtained when the 211 At/Bi mixture was adjusted to 0.1 M SnCl 2 in 6 M HCl. Comparable or even higher 211 At isolation yields were obtained without the use of SnCl 2 (Table 1). Therefore, the later 211 At isolation experiments were conducted without SnCl 2 , as outlined in Fig. 1.   Semi-automated 211 At isolation runs. The results of the manual 211 At isolation runs were very encouraging, so the procedure was adapted to a semi-automated system to demonstrate potential application of the Te column method for routine 211 At isolation. Three proof-of-concept semi-automated 211 At isolation runs were conducted using irradiated bismuth targets following the procedure outlined in Fig. 1. The schematic of the semi-automated process is shown in Fig. 2. A picture of our prototype is shown in Fig. S1. Results of the semi-automated runs are summarized in Table 2. The irradiated bismuth targets each contained about 962 MBq (26 mCi) and the overall 211 At isolation process run time was 90-100 minutes. On average, the system was able to recover 93±4% of the 211 At in 2 mL of 1 M NaOH. For the 1 st and 2 nd semi-automated runs, 80 mL of 35% aqueous NH 2 OH⋅HCl was used and the columns were washed with 20 mL of 1.5 M HCl, followed by 20 mL of deionized (D.I.) H 2 O. For the 3 rd semi-automated run, the volume of 35% NH 2 OH⋅HCl was reduced by 15 mL which appears to be enough to destroy all of the nitrate. Decreasing the volume of aqueous NH 2 OH⋅HCl reduced the overall run time, even though an additional 20 mL of 1.5 M HCl and D.I. H 2 O was used for washing the column in the 3 rd semi-automated run.
optimization of the amount of nH 2 oH·Hcl. Although the 211 At isolation yields were encouraging when an excess of NH 2 OH·HCl was used, we obtained inconsistent radiolabeling yields, ranging from 10.4% to 94.7%, when using the isolated 211 At solutions to label isothiocyanato-phenethyl-closo-decaborate (2-) (B10-NCS)-conjugated monoclonal antibodies (MAbs) at room temperature in the absence of an oxidant. We also observed that addition of the oxidant chloramine-T improved the MAb-B10 radiolabeling yield, suggesting that there might be reductive impurities in the isolated 211 At solution.
We hypothesized optimizing the amount of NH 2 OH·HCl might mitigate this problem. Experiments were conducted to determine the minimal volume of 35% NH 2 OH·HCl required to destroy all the NO 3 − (V min ) in the HNO 3 solution containing the dissolved Bi target, as it seemed to be impossible to accurately assign defined stoichiometry to the reaction between NH 2 OH·HCl and HNO 3 . Minimally, about 52 mL of 35% NH 2 OH·HCl is needed for a Bi target that is dissolved in 15 mL of 10 M HNO 3 . A series of manual 211 At isolations were conducted using different percentages of V min . Table 3 shows that although the 211 At isolation yield decreased as the volume of 35% NH 2 OH·HCl was reduced, the radiolabeling yields for B10-conjugated MAb increased significantly. Reducing the amount of NH 2 OH·HCl to 52% of V min appears to be optimal, as the radiolabeling yield increased to 82.5%, equivalent to the normal radiolabeling yield achieved with 211 At isolated using the DIPE method. And at 52% of V min , an isolation yield of about 80% could still be obtained.   At decayed. Calibration curves were generated using Bi and Te standard solutions at concentrations of 1, 10, 50 100 ppb, with R 2 ≥0.9995. Recoveries of the internal standards (ISTDs) were very close to 100% for all samples including calibration standards. On average, the concentration of residual Bi in the isolated 211 At solution was 3.0 ppm, which is slightly higher than that of the 211 At isolated using the DIPE method ( Table 4). The concentration of the Te contaminant in the isolated 211 At solution is rather high, about 32.8 ppm on average. However, it should be noted that 211 At-labeled MAb prepared from Te column isolated 211 At and purified by a size-exclusion (PD-10) column had significantly reduced Bi and Te concentrations of about 0.05 and 0.04 ppm, respectively (Table 4).
Anion exchange radio-HPLC analyses were performed to evaluate the radiochemical purity of the isolated 211 At. A representative radio-chromatogram is shown in Fig. 3. Only one radiopeak at 9.5 min has been observed for several 211 At solutions purified using the Te column method (n > 10), which suggests radiochemical purity >99% can be consistently achieved using the Te column method.

Discussion
Astatine-211 can be rapidly absorbed on metallic Te in HCl in the presence of SnCl 2 and eluted by a solution of 1-2 M NaOH. The high affinity of At − to elemental Te in HCl might be the result of the formation of a coordination bond between the surface Te and the highly polarizable At − 19 . In the literature, Te columns were used for separation of 211 At from irradiated thorium targets 19 or polonium impurities, when 211 At is produced via high energy proton induced spallation of thorium 19 or the 209 Bi( 7 Li, 5n) 211 Rn → 211 At route 20 , respectively. In those scenarios, the solutions containing 211 At/impurities do not contain large amounts of HNO 3 and can be readily diluted and constituted to approximately 0.1 M SnCl 2 in 6 M HCl prior to loading onto the Te column. However, dissolution of our Bi targets which contain 4-5 grams of Bi metal requires the use of 15-17 mL of 10 M or concentrated HNO 3 . Hydroxylamine can reduce HNO 3 to HNO 2 which further reacts with NH 3 OH + and produce gaseous N 2 O (g), and N 2 (g), so it is used to convert the nitrate matrix to HCl 21,22 . The addition of NH 2 OH⋅HCl also eliminates the need for using SnCl 2 in the solution transferred onto the Te column. It is likely that NH 2 OH⋅HCl reduces astatine in other oxidation states to astatide, the astatine species that might be required for the Te column method to work properly.   www.nature.com/scientificreports www.nature.com/scientificreports/ Compared to the DIPE extraction method, using NH 2 OH⋅HCl for converting the HNO 3 solution containing the dissolved Bi target to a HCl matrix is not only easier to automate, but can be faster than distilling the HNO 3 to dryness. It takes ~25 min to completely destroy the nitrate, adding the NH 2 OH⋅HCl solution (V min = 52 mL) using the semi-automated system, which is comparable to the time it takes to remove the HNO 3 by distillation (~30 min) 12 . However, we found that not all of the nitrate needs to be destroyed to obtain good isolation yields. In fact, adding 52% of V min provides high 211 At isolation yields as well as high B10-conjugated MAb labeling yields ( Table 3).
The overall run times of the semi-automated 211 At isolation experiments are 20-30 min shorter than those of the DIPE extraction process ( Table 2). The high affinity of At − to elemental Te allows adsorption of 211 At onto Te columns and washing the Te columns with HCl and H 2 O at high flow rates of 6 mL/min. Also, the elution of 211 At from the Te column using NaOH is very efficient, averaging 93% of the 211 At in 2 mL volume at a flow rate of 60 mL/min. It should be noted the NaOH back extraction step in the DIPE extraction method can takes 10-20 min to finish 12 . The fast flow rates used in the Te column isolation process are critical for achieving good 211 At isolation yields in a reasonable amount of time, especially, considering the volume of 211 At solution passing over the Te columns is rather large.
Astatine-211 solutions obtained using the DIPE liquid-liquid extraction method can have multiple astatine species which can lead to low radiochemical purity 12 . In contrast, 211 At solutions isolated using Te columns consistently provide only astatide in a radiochemical purity >99%. This might be due to 211 At being reduced to astatide by NH 2 OH⋅HCl before being transferred onto the column. However, it must be noted that a small amount of tellurium metal is dissolved in 1 M NaOH as 211 At is eluted off the column and Na 2 TeO 3 is a weak reducing agent. While untested at this time, the presence of Na 2 TeO 3 might cause problems for astatine labeling, especially electrophilic astatination reactions. Thus, methods for purifying 211 At from the Te (and possibly Bi) impurities need to be evaluated for applications that require higher purity.
In conclusion, Te columns provide an alternative method for efficiently isolating 211 At from irradiated Bi targets. The isolated 211 At solution is of high radiochemical purity and is suitable for B10-conjugated MAb labeling. A semi-automated process based on the Te column method has been demonstrated. Studies to evaluate the influence of the mesh size of the Te powder used on the 211 At isolation yield are on-going. As future work, the geometry of the Te column, the volumes of reagents including 1.5 M HCl, D.I. H 2 O and 1 M NaOH need to be optimized to minimize the overall run time and to reduce the volume of the final product to 0.5 mL or less.

Methods
Reagents and general procedures. The chemicals and reagents used were purchased from VWR International (Radnor, PA), Sigma Aldrich (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA), and were used without further purification unless otherwise specified. Empty Mini Spe-ed column cartridges were obtained from Applied Separations (Allentown, PA). ICP-MS tuning solution and 10 μg/mL standard solutions of Bi and Te were obtained from Inorganic Ventures (Christiansburg, VA). Monoclonal antibodies were obtained from the Fred Hutchinson Cancer Research Center Biologics Production Facility and isothiocyanato-phenethyl-closo-decaborate (2-) (B10-NCS)-conjugated MAbs were prepared in house as previously described 23 . Astatine-211 was produced by irradiation of Bi metal on an aluminum target support with 29 MeV α-particles using the Scanditronix MC50 cyclotron as previously described 24 . Various solutions containing 211 At generated before and after Te column separation were measured in a Capintec CRC-55tR dose calibrator using the calibration setting number 44. Astatination of the B10-conjugated MAbs with 211 At isolated using Te columns were conducted as previously described 25 . Determine the minimal volume of nH 2 oH·Hcl. Determining the production rate of each of the products generated by the HNO 3 and NH 2 OH·HCl redox reaction was not attempted. It seemed that accurately assigning a defined stoichiometry to this reaction was not possible, thus the minimal volume of 35% NH 2 OH·HCl required to destroy all the nitrate ions in the dissolved Bi target solution (V min ) was determined experimentally.
To mimic the semi-automated isolation process, 4.25 g of Bi metal was dissolved in 15 mL of 10 M HNO 3 . The resultant solution was split into three 5-mL fractions and 35% aqueous NH 2 OH·HCl was added dropwise at a flow rate of approximately 2 mL/min using a 25-mL burette. The completion of the NH 2 OH·HCl and nitric acid reaction was observed as a cessation of bubbling. Nitrate/nitrite test paper (EMD Millipore TM ) was used to verify the nitrate ion was below the detection limit (10 ppm). An irradiated Bi target containing ~0.96 GBq (~26 mCi) of 211 At was placed Bi face down in a plastic container. A total of 17 mL of concentrated HNO 3 was manually added to dissolve the Bi and 211 At. Because 10 M HNO 3 rather than concentrated HNO 3 would be used for target dissolution in the semi-automated 211 At isolation process, 1 g of high purity (99.999% trace metal basis) Bi beads were dissolved in 3 mL of 10 M nitric acid to mimic the Bi 3+ and NO 3 − concentration in the dissolved Bi target solution obtained using the semi-automated process. The resultant solution was spiked with 0.5-1 mL concentrated HNO 3 of dissolved irradiated Bi targets. Depending on the chromatographic conditions to be evaluated, the obtained solution was adjusted accordingly.