Quality Control of 225Ac and associated Radiopharmaceuticals: 221Fr to be or not to be?

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Miguel Toro Gonzalez, Luke Wheeless, Vijai Kumar Reddy Tangadanchu, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7095997/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Jan, 2026 Read the published version in EJNMMI Radiopharmacy and Chemistry → Version 1 posted 4 You are reading this latest preprint version Abstract Background: Actinium-225 radiopharmaceuticals have drawn great interest in cancer therapy due to their tumor-specific delivery of cytotoxic alpha-particles. Detection and quality control (QC) are critical for these potent agents. There is currently no consensus for best practice of 225 Ac QC. Detection of 225 Ac (T 1/2 = 9.92 days) is challenging; however, the gamma-emitting progenies 221 Fr (T 1/2 = 4.8 min, 218 keV) and 213 Bi (T 1/2 = 45 min, 440 keV) facilitate the indirect measurement of 225 Ac. Using several instruments, we compared multiple analytical methods for 225 Ac limit of quantification and the radiopharmaceutical radiochemical purity (RCP%). The RCP% of 225 Ac-radiopharmaceutical was evaluated at both 221 Fr and 213 Bi secular equilibrium. RCP% was measured using two TLC plate readers: a gas-filled proportional counter, under mixed emission and alpha specific parameters; and a plastic silicon photomultiplier detector and compared. Chromatographic strips were also analyzed using LS, HPGe, and NaI(Tl) gamma well counting. We correlated these RCP% to HPLC results measured at both equilibria utilizing multiple energy windows. Finally, examining accuracy and precision for each instrument, free 225 Ac spiking assays were conducted on the radiopharmaceutical and measured. Results: The TLC plate-reader using the plastic silicon detector resulted in significant RCP% differences between 30 min and >5 hr readings, whereas the gas-filled proportional counter showed no-significant differences between alpha-specific (30 min) and the mixed-isotope setting (>5 hr). HPGe-TLC demonstrated RCP% equivalence at 221 Fr and 213 Bi equilibrium. NaI(Tl) and LS measurements significantly underestimated the RCP% at 30 min. Finally, gamma well counting of HPLC fractions resulted in 3-5 % RCP% underestimation at 221 Fr equilibrium. Conclusion: Five instruments have been tested for their accuracy, sensitivity, linearity and specificity response to 225 Ac quantification using 221 Fr and 213 Bi measurements. 221 Fr secular equilibrium was deemed acceptable for TLC RCP% analyses using HPGe and gas-filled proportional counter under adequate parameters. LS, gamma well counting and TLC-scanner with plastic silicon detector required a reading at 213 Bi equilibrium for accurate RCP% characterization. Low content radioimpurity detection was most accurately measured using TLC rather than gamma well counting of HPLC fraction collection due to gamma counting limitations. 225Ac quality control Liquid Scintillation High Purity Germanium Gamma well counting Thin Layer Chromatography HPLC Figures Figure 1 Figure 2 Figure 3 Introduction Actinium-225 radiopharmaceuticals have drawn a great interest in cancer therapy due to their tumor-specific delivery of cytotoxic alpha-particles. To date, > 20 clinical trials have been launched world-wide using 225 Ac radiopharmaceuticals, including > 10 clinical trials actively recruiting in phase 2–3 [ 1 ]. While 225 Ac radiopharmaceutical development has exponentially grown over the last 10 years, several complications have hindered the progression to clinic. Particularly, the global 225 Ac isotope supply is currently insufficient, however manufacturing initiatives are in progress to support the needs of research and clinical trials. Precluded access to this isotope has led to expedited studies and little understanding of its specific detection. Consequently, there is currently no consensus for best practice in quality control (QC) of 225 Ac and associated radiopharmaceuticals. This is a critical issue hampering the development of these potent agents. The vast majority of nuclear medicine drug products are diagnostics radiopharmaceuticals. Unlike PET or SPECT isotopes, 225 Ac (T 1/2 = 9.92 days) gamma-ray energies are too weak to be accurately detected with commonly used radiopharmacy equipment. Alternatively, the gamma-emitting progenies 221 Fr (T 1/2 = 4.8 min, 218 keV, %I : 11.44) and 213 Bi (T 1/2 = 45 min, 440 keV, %I : 25.9) have been adopted as the isotopes of choice to quantify 225 Ac at their respective secular equilibrium (Fig. 1 ). Following purification or separation of 225 Ac or labelled substance, secular equilibria of the progenies 221 Fr or 213 Bi are disturbed and require time to reestablish equilibrium in order to accurately quantify 225 Ac. To determine the 225 Ac activity, quantification by 221 Fr may be undertaken as early as 30 min post-separation and quantification by 213 Bi as early as 5 hr. Previous studies reported 225 Ac quantification based on the 221 Fr gamma ray (218 keV) within 30 min post-separation [ 2 – 5 ]. This strategy was applied to radiochemical purity (RCP%) evaluation of 225 Ac drug products (DPs) using High Performance Liquid Chromatography (HPLC) fractions which were gamma counted within 30 min of the chromatographic separation [ 4 ]. Using this method, the development of [ 225 Ac]AcPSMA-I&T, indicated significant differences in RCP% (7–9%) when comparing Thin Layer Chromatography (TLC) scanner results to those of HPLC or TLC counted using HPGe [ 2 ]. This inter-instrument discrepancy poses challenges in accuracy. A difference of > 5% in RCP% is problematic for the DP release, typically, RCP values of ≥ 95% are required for release. These discrepancies are likely elicited by the varying detection sensitivity across instruments and whether quantification is affected through detection of 221 Fr, 213 Bi, or both at secular equilibrium. In contrast, 225 Ac radiopharmaceutical QC exclusively based on 213 Bi secular equilibrium quantification using both TLC and HPLC analyses were reported within a < 5% difference between the two techniques [ 6 ]. While this is an improvement to the 221 Fr only detection method, a difference of 2–5% may still not satisfy accuracy and precision criteria for radiopharmaceuticals as recommended [ 7 – 8 ]. In the present investigation, multiple analytical methods are compared for 225 Ac quantification using 221 Fr (at 30 min) and 213 Bi (> 5 hr) detection. Linearity and limit of quantifications (LOQs) of 225 Ac in solution were defined utilizing: liquid scintillation (LS), high purity germanium (HPGe), and NaI(Tl) gamma well counting. TLC evaluations were conducted using TLC scanners equipped with a gas-filled proportional counter or a plastic silicon photomultiplier detector as well as a “cut and count” strategy using LS, HPGe, or NaI(Tl) gamma well counting. Once the instrument capabilities were defined with respect to the 225 Ac detection limit, drug products RCP% were assessed using 221 Fr or 213 Bi in equilibrium. Determinations based on 221 Fr equilibrium were compared with those of 213 Bi and evaluated for accuracy and precision. Additional attributes were examined, focusing on the specificity and sensitivity of free 225 Ac quantification in the DP. Recommendations for radiopharmaceutical QC indicate a detection down to 0.5% impurities (7). In previous investigations of 225 Ac-radiopharmaceuticals, reports suggested a detection limit as low as 1% of free 225 Ac impurities [ 3 ]. The low radioactive concentration of an 225 Ac-radiopharmaceutical confounds achieving an adequately low limit of quantification. In this report, free 225 Ac spiking assays were conducted, and accuracy was evaluated for each instrument. Altogether, the examination of each instrument quantification limits, the RCP% based on 221 Fr (at 30 min) versus 213 Bi (> 5 hr) detection and the quantification of low-level impurity are meant to better support the understanding of 225 Ac radiopharmaceutical. Results Five instruments were utilized to measure the radiochemical purity of 225 Ac-labeled drug preparations using either TLC or HPLC. Investigations using TLC scanners were undertaken comparing the performance of a plastic silicon detector to that of a gas-filled proportional counter. While the plastic silicon detector is mostly designed to detect beta- and gamma-emitting isotopes, the gas-filled proportional counter enables a high voltage modulation for alpha specific detection at 1,000 V and a beta/gamma specific detection at 1,500V [ 9 , 10 ]. Other detectors were considered for TLC analysis using a cut and counting approach. Liquid scintillation (LS) counting was undertaken to measure 225 Ac spotted on TLC strips by immersing each half of the strip in scintillating cocktail for detection of alpha and beta-emitting isotopes. The LS parameters were selected to exclusively count the alpha-pulse window. Spectroscopy using High Purity Germanium (HPGe) resulted in high resolution and adequate quantification of gamma rays to precisely measure 221 Fr at 218 keV and 213 Bi at 440 keV. The radiochemical purity was determined by using TLC combined with HPGe and evaluated for both 221 Fr and 213 Bi at equilibrium. Gamma well counting with a NaI(Tl) detector was also used with both HPLC fractions and TLC strips. Linearity and limit of quantifications (LOQs) The performance of each instrument was assessed, evaluating linearity and LOQs for 225 Ac in solution or spotted on TLC strips. The 225 Ac activity in solution ranged from 0.185 Bq to 37 kBq (5 pCi to 1000 nCi) and were measured for linearity, demonstrating R 2 > 0.99 for all instruments (Suppl. Figure 1). The LOQ for 225 Ac in solution was defined as 2 Bq (55 pCi) for LS; 30 Bq (810 pCi) for HPGe; and 555–740 Bq (15–20 nCi) for gamma well counting, depending on the energy window (Table 1 , Suppl Fig. 1). Table 1 LOQ for 225 Ac in solution, measured using LS, HPGe and gamma well counting. Gamma-well counting was acquired under 221 Fr (195–240 keV); 213 Bi (350–530 keV) or open energy window (15–530 keV). Solution LSC HPGe (keV) Gamma well counter (keV) Detection alpha 218 440 195–240 350–530 15–530 LOQ (Bq) 2 30 30 773 644 566 A similar evaluation was conducted with activity spotted on non-developed TLC strips from 0.37 to 55.5 kBq (0.01 to 1500 nCi). The LOQs were measured as low as 1.1 Bq (30 pCi) for LS; between 14.8 Bq (400 pCi) and 36.3 Bq (0.98 nCi) for HPGe at 440 keV and 218 keV, respectively; and 63–66 Bq (1.7–1.8 nCi) for gamma-well counting (Table 2 , Suppl Fig. 2). The gas-filled proportional counter TLC scanner outperformed the silicon detector with a LOQ defined at 11.1 Bq (300 pCi) at 1,000 V. Table 2 LOQ for 225 Ac spotted on non-developed TLC strips measured using LS, HPGe, gamma-well counting and TLC scanners equipped with plastic silicon or gas-filled proportional detectors. TLC LSC HPGe (keV) Gamma well counter (keV) TLC scanner TLC scanner Detection alpha 218 440 195–240 350–530 15–530 silicon proportional LOQ (Bq) 1.1 36.3 14.8 62 67 64.7 4366 11.1 Overall, these results place LS, HPGe, and the gas-filled proportional counter TLC scanner as the most sensitive tools to detect < 37 Bq (1 nCi) of free 225 Ac in drug products whether in solution or spotted on TLC. The limits of quantification for 225 Ac in solution resulted in higher values than those spotted on TLC strips, suggesting a possible geometry effect for all instruments. Francium-221 versus Bismuth-213 secular equilibrium Suitability of the various instruments and techniques for 221 Fr acquired at 30 minutes versus 213 Bi acquired at > 5 hr post TLC development was evaluated. Drug products formulated with radioactive concentrations ranging from 0.185 to 1.48 kBq/ µL (5 to 40 nCi/µL) (Suppl. Table 2) were examined for each detection time post-TLC development. TLC scanners were each investigated for their response to 221 Fr versus 213 Bi at equilibrium. The plastic silicon detector (Fig. 2 B) displayed significant differences (P < 0.0001) in RCP% for the same TLC strip, 61 ± 5% evaluated at 30 min post-development, versus 99 ± 0.1% measured at 213 Bi equilibrium. Using a different radiolabeled molecule (RCP%: 74 ± 1.0%), the gas-filled proportional counter did not show any significant differences between the alpha-specific acquisition (1,000 V, 30 min post-development) versus beta- and gamma-specific (1,500 V at > 5 hr). It indicated a mean difference of 1.2% between the two tested parameters (n = 5, P = 0.0712, t-test, Fig. 2 A). This demonstrates a capability to specifically discriminate alpha emissions from the beta when adjusting the high voltage settings. This was further confirmed comparing acquisitions at only 1,000 V for the same TLC strip at 30 min and > 5 hrs post-development. These showed significant differences in RCP % (P = 0.0002, n = 5); and similarly, under 1,500 V with P < 0.0001 (Suppl. Figure 5). A TLC cut and count strategy was adopted to evaluate LS, HPGe and gamma-well counting. Prior to counting, distinct separation of the radiolabeled-molecule with the free 225 Ac was confirmed using a TLC scanner (Rf = 0 vs Rf = 1, respectively). The TLC strips were cut in half and characterized using LS, gamma-well counting and HPGe for RCP% determination (Fig. 2 C, D, E). Examining the detection at 30 min versus > 5 hrs post-TLC strip development, HPGe at 218 keV demonstrated at 30 min an exact RCP% match with that of 440 keV at > 5 hrs for the same sample (n = 6, RSD = 0.14–0.22%). In contrast, LS under alpha specific settings, and gamma-well counting regardless of the energy window (Suppl. Figure 6), both failed to demonstrate equivalence of RCP% between the two time points. Similarly, when the drug product RCP% was evaluated using HPLC combined with gamma-well counting of collected fractions, the RCP% resulted in discrepancies ranging from 2–4% differences between the 30 min and > 5 hr counting of fractions (Fig. 2 F). However, negligeable differences of RCP% were found between TLC scanner and HPLC gamma counting at 213 Bi secular equilibrium (Suppl Info Fig. 4). In light of these results, 221 Fr equilibrium does not adequately fulfill precision and accuracy criteria for RCP% determination using HPLC and gamma-well counting. Accuracy with spiking of free 225 Ac in the pure drug product Both TLC and HPLC methods were evaluated for accuracy when analyzing DP spiked with known amounts of free 225 Ac (Fig. 3 ). Each technique was investigated for determining the recovery of free 225 Ac compared to the expected value. The gas-filled proportional counter TLC scanners generated an excellent response and accurately reported the expected free 225 Ac activity (Fig. 3 A). Measurements of impurity % demonstrated a recovery of 104 and 91% for both acquisition at 30 min (1,000 V) and > 5 hr (1,500 V), respectively. In contrast, the silicon detector acquired at 213 Bi secular equilibrium failed to accurately measure the expected free 225 Ac (recoveries: 87%) at similar radioactive concentration (Fig. 3B1). Since all free 225 Ac spikes spotted on TLC were below the LOQ of the instrument, the silicon detector failed to accurately measure 225 Ac in the drug product (Fig. 3B1-3). TLC cut and count measurement strategies using LS, gamma-well counting, and HPGe demonstrated acceptable recoveries. Liquid Scintillation and gamma-well counting were both acquired at > 5 hr post-TLC development, and both resulted in adequate recoveries (90–110%) (Fig. 3 C, E). HPGe demonstrated similar successful results regardless of the secular equilibrium (Fig. 3 D), except for one sample for which the free 225 Ac spike activity approached the LOQ (Fig. 3 D2). Finally, evaluation of HPLC collected fractions using gamma-well counting at 213 Bi equilibrium (Fig. 3 F) passed all recoveries. RCP% correlation between TLC and HPLC HPLC fraction collection combined with gamma-well counting, was completed in a number of DP samples (n = 17). The RCP% evaluated using HPLC/gamma-well counting radiochromatogram (Suppl. Figure 3), were correlated to that of TLC scanner (silicon) measured at 213 Bi secular equilibrium using extended acquisition times in order to obtain sufficient counts for accuracy (> 40 min). The cross-evaluation resulted in an R 2 value of > 0.99, with less than 1% difference in RCP% between the two techniques (Suppl. Figure 4). Discussion The development of molecularly-targeted radiopharmaceutical therapy has undergone a tremendous progression over the last 10 years, launching an increasing number of first-in-human clinical trials for treatments of various cancers. In parallel, the characterization of these drugs is not trivial with tests spanning across several expertises including chemical, radiochemical and biological evaluations. For 225 Ac radiopharmaceuticals, the path to complete QC is rather challenging due to the nature of this rare isotope. As of today, there is no consensus on the best practices for 225 Ac quality control. Actinium-225 is an alpha-emitting which decays to stability with the emission of four alpha-emitting (and two beta-emitting) progeny including 221 Fr and 213 Bi. 225 Ac is more readily quantifiable through indirect progeny detection due to the abundance of their gamma-rays, emitted at 218 keV ( 221 Fr) and 440 keV ( 213 Bi). Historically, 225 Ac quantification has been described using 213 Bi detection, at secular equilibrium > 5 h after separation, at this time > 99% of the 213 Bi activity content equals that of 225 Ac [ 8 , 12 , 13 , 14 ]. From the standpoint of drug production manufacturing, a 5 h hold after each processing step due to equilibrium disruption is a major impediment, imposing lead times of several days. Alternatively, 221 Fr secular equilibrium reached after 30 min from the time of separation may offer a compelling solution to a time sensitive process. In the present study, accuracy and precision of 225 Ac quantification, using 221 Fr or 213 Bi at secular equilibrium, was evaluated across 5 instruments. Each instrument was first verified for linearity and LOQ using 225 Ac in secular equilibrium with its progeny. The LOQ determined in solution (Table 1 ) was found to be higher than the LOQ of material spotted on TLC strips (Table 2 ). A 20-fold higher sensitivity was observed for TLC using NaI(Tl) gamma-well counting and twice as high for LS than that in solution. This may be explained by the geometry, for which large homogeneously dispersed radioactive solution resulted in a higher LOQ to that of more concentrated spotted activity on chromatographic strips. A similar effect was observed when decreasing the standard solution volume from 750 to 20 µL, the LOQ was found to decrease (Suppl. Figure 1C-D). Among all instruments tested, LS was identified as the most sensitive technique to quantify 225 Ac with a limit of quantification as low as 1 Bq (~ 27 picocuries). This technique was unsuitable for radionuclidic identification and indicated significant RCP% differences between 30 min and > 5 hr acquisition. At 30 min post-TLC development, 221 Fr has reached > 99% of its equilibrium activity (compared to 225 Ac activity), whereas 213 Bi ingrowth has only reached 29% of its equilibrium activity considering no prior 213 Bi contribution from the radiolabeling preparation. Radiolabeled samples were let decayed overnight to allow for all 213 Bi-labeled molecule to disappear. For LS detection, the beta emission from 213 Bi, 209 Tl and 209 Pb strongly interfered with the pule shape of the alphas from the decay of 225 Ac, 221 Fr, 217 At and 213 Po. The latest alpha-emitters may be attenuated by the embedment in the chromatographic strip, diminishing the interaction with the cocktail. As a result, LS detection for TLC was found unfit to read RCP% before 213 Bi secular equilibrium, this would not be the case if the source was in solution. HPGe demonstrated LOQs slightly higher than that of LS, < 37 Bq ranges (nanocuries). In contrast to LS, HPGe enables a high radionuclidic resolution with identification and quantification of 221 Fr (218 keV, 11.4%) as early as 30 min post-TLC development, and 213 Bi quantification (440 keV, 25.9%) after > 5 hr. This radionuclide-specific evaluation demonstrated negligible differences between the RCP% measurements at 30 min and > 5 hr. HPGe was found adequate to evaluate RCP% at both 221 Fr and 213 Bi secular equilibrium. NaI(Tl) gamma-well counting is known for its modest gamma resolution with high background due to Compton scattering and the photoelectric effects in the energy range of < 200 keV. The scattering elicited by 213 Bi beta-contribution has been reported to interfere in the 221 Fr detection (11). Exempt of scattering correction, the RCP% evaluation at 221 Fr secular equilibrium indicated lower accuracy compared to that of 213 Bi at equilibration. This is mostly due to limitations with the NaI(Tl) detector to accurately quantify the 221 Fr peak from background and other interferences. Examining the RCP% determined using HPLC fraction collection coupled with gamma-well counting with that of TLC results showed the quantification at 221 Fr secular equilibrium was unfit for accuracy. Inter instrument TLC and HPLC agreement was only successful when comparing RCP% at 213 Bi secular equilibrium (Suppl. Figure 5). In contrast, the TLC scanner equipped with the gas-filled proportional counter, operated at 1,000 V (alpha-specific setting), demonstrated an acceptable RCP% accuracy at 30 min, compared to measurements conducted at > 5 hr at 1,500 V. At 30 min post-development, the ionization emitted by 225 Ac, 221 Fr and 217 At alphas were substantial contributors at 1,000V [ 15 ]. After 5 hr, the equilibrated RCP% was evaluated using mostly the beta contributions delivered by 213 Bi and other progeny detected at 1,500V (Fig. 2 A). The same measurement conducted at 1,000 V after 5 hr, however, did not result in accurate RCP% (Suppl. Figure 5A). In light of the detection sensitivity and LOQ defined for the gamma-well counter, measurement of radioimpurities using HPLC fractions may be limited to 1% of the total content. Actinium-225-drug product (1.48–2.22 kBq/µL; 40–60 nCi/µL) analyzed at the end-of-synthesis, generally leads to a total HPLC injection of 2 to 3 µCi. Detecting 0.5% impurity would require a detection limit of 10–15 nCi, spread across several fractions. This is at or below the gamma-well counter LOQ (0.555–0.740 Bq or 15–20 nCi in solution, Table 1 ). When the impurity specification is raised to 1%, a minimum detection of 0.74–1.11 Bq (20–30 nCi) could be achieved, which is above the measured LOQ. Considering the LOQ of the instrument and the low radioactive concentration of a typical 225 Ac radiopharmaceutical, gamma-well counting of fractionated HPLC samples may be suitable to measure as low as 1% radio impurity. HPGe and LS of fractions would meet 0.5% impurity detection; however, both techniques require labor-intensive sample transfer associated with a higher risk of error. When spotting a DP on TLC (5–10 µL), the total activity spotted ranges from 14.8–22.2 kBq (400–600 nCi). Detecting 0.5% impurity requires a TLC scanner or cut/count technique to measure down to 74–111 Bq (2–3 nCi). In light of the TLC-LOQ reported for all instruments (Table 2 ), all techniques except the silicon detector are suitable to measure 0.5% impurity, fulfilling the recommended criteria for radiopharmaceutical QC (7). Linearity, accuracy, specificity and repeatability have been demonstrated utilizing 5 different instruments, all fit for 225 Ac detection when measured at 213 Bi secular equilibrium. However, when using 221 Fr at secular equilibrium for 225 Ac quantification, only the HPGe and gas-filled proportional counter were adequate for radiopharmaceutical drug product release testing. Conclusion Five instruments have been tested for their response to 225 Ac measurement in accuracy, sensitivity, linearity and specificity. Liquid scintillation counting and HPGe were found to be the most sensitive techniques to detect 225 Ac progeny. Analysis and quantification of RCP% at 221 Fr secular equilibrium is acceptable for HPGe and the gas-filled proportional counter using the right parameters. Liquid scintillation counting, gamma-well counting and the TLC-scanner plastic silicon detector require 213 Bi equilibrium for accurate RCP% characterization of the DP. Low content radioimpurity detection was found best measured using TLC rather than rather than gamma-well counting of HPLC fraction collection. Abbreviations QC: quality Control DP: Drug Product LS: Liquid Scintillation HPGe: High Purity Germanium γ: Gamma counting TLC: Thin Layer Chromatography HPLC: High Performance Liquid Chromatography LOQ: Limit of Quantification RCP%: Radiochemical Purity % E: Expected M: Measured n/a: non assigned Declarations Acknowledgements . Some or all of the 225 Ac used in this research was supplied by the U.S. Department of Energy Isotope Program managed by the Office of Isotope R&D and Production. Material and Methods are described in the Supplemental information. Ethics approval and consent to participate: not applicable Consent for publication: not applicable Authors contributions: MT Gonzalez : conceptualization, investigation, analysis, interpretation; L Wheeless : synthesis, investigation, analysis and interpretation; VKR Tangadanchu : analysis and interpretation. C. Hawkins : analysis and interpretation; S. Provo : analysis. W. Smith : analysis. M. Hommen : analysis; D. De Vries : reviewing and editing; J. Harvey : reviewing and editing; T. Drum : reviewing, editing and supervision; D.S. Abou: conceptualization, synthesis, investigation, method development, analysis, interpretation, original draft writing, supervision. Availability of data and material: all methods and data are available and reported in supplemental information. Competing Interest: all authors listed are full-time employees of NorthStar Medical Radioisotope., LLC. The corresponding author D.S. Abou is a guest editor at EJNMMI, Radiopharmacy and Chemistry, collection: Radiopharmaceutical quality assurance. Funding: this research was funded by NorthStar Medical Radioisotopes, LLC. Corresponding Author Information: Diane S. Abou, PhD; [email protected] References https://clinicaltrials.gov/ Hooijman, E.L.; Chalashkan, Y.; Ling, S.W.; Kahyargil, F.F.; Segbers, M.; Bruchertseifer, F.; Morgenstern, A.; Seimbille, Y.; Koolen, S.L.W.; Brabander, T.; et al. Development of [225Ac]Ac-PSMA-I&T for Targeted Alpha Therapy According to GMP Guidelines for Treatment of mCRPC. Pharmaceutics 2021, 13, 715. Hooijman, E.L., de Jong, J.R., Ntihabose, C.M. et al. Ac-225 radiochemistry through the lens of [225Ac]Ac-DOTA-TATE. EJNMMI radiopharm. chem. 10, 9 (2025). Hooijman, E.L., Radchenko, V., Ling, S.W. et al. Implementing Ac-225 labelled radiopharmaceuticals: practical considerations and (pre-)clinical perspectives. EJNMMI radiopharm. chem. 9, 9 (2024). 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Pretze, M.; Wendrich, J.; Hartmann, H.; Freudenberg, R.; Bundschuh, R.A.; Kotzerke, J.; Michler, E. Comparison of ZnS(Ag) Scintillator and Proportional Counter Tube for Alpha Detection in Thin-Layer Chromatography. Pharmaceuticals 2025, 18, 26. Supplementary Files EJNMMIRPQASupplementalInformation.docx Cite Share Download PDF Status: Published Journal Publication published 17 Jan, 2026 Read the published version in EJNMMI Radiopharmacy and Chemistry → Version 1 posted Reviewers agreed at journal 18 Jul, 2025 Reviewers invited by journal 18 Jul, 2025 Editor assigned by journal 16 Jul, 2025 First submitted to journal 16 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7095997","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":487535509,"identity":"508d29f6-dadd-40b3-9c37-793c9b5207ea","order_by":0,"name":"Miguel Toro Gonzalez","email":"","orcid":"","institution":"NorthStar Medical Radioisotopes, LLC","correspondingAuthor":false,"prefix":"","firstName":"Miguel","middleName":"Toro","lastName":"Gonzalez","suffix":""},{"id":487535510,"identity":"2facc49e-dd42-42c1-b3c4-7c17bb54ae9f","order_by":1,"name":"Luke Wheeless","email":"","orcid":"","institution":"NorthStar Medical Radioisotopes, LLC","correspondingAuthor":false,"prefix":"","firstName":"Luke","middleName":"","lastName":"Wheeless","suffix":""},{"id":487535511,"identity":"9b86a02f-a54f-4e6b-bda1-3eb08e5b3a0c","order_by":2,"name":"Vijai Kumar Reddy Tangadanchu","email":"","orcid":"","institution":"NorthStar Medical Radioisotopes, LLC","correspondingAuthor":false,"prefix":"","firstName":"Vijai","middleName":"Kumar Reddy","lastName":"Tangadanchu","suffix":""},{"id":487535512,"identity":"1a46dfdb-cc88-48cd-b8ac-e1d37c5a6532","order_by":3,"name":"Cory Hawkins","email":"","orcid":"","institution":"NorthStar Medical Radioisotopes, LLC","correspondingAuthor":false,"prefix":"","firstName":"Cory","middleName":"","lastName":"Hawkins","suffix":""},{"id":487535513,"identity":"e8d73198-b51b-422e-82c5-788acb747363","order_by":4,"name":"Shannon Provo","email":"","orcid":"","institution":"NorthStar Medical Radioisotopes, LLC","correspondingAuthor":false,"prefix":"","firstName":"Shannon","middleName":"","lastName":"Provo","suffix":""},{"id":487535514,"identity":"9bd33c08-a0d1-4d15-ba60-5c064b9edbe8","order_by":5,"name":"William Smith","email":"","orcid":"","institution":"NorthStar Medical Radioisotopes, LLC","correspondingAuthor":false,"prefix":"","firstName":"William","middleName":"","lastName":"Smith","suffix":""},{"id":487535515,"identity":"8cabc23a-d6f3-442c-8256-0f32ca3ce47d","order_by":6,"name":"Michael Hommen","email":"","orcid":"","institution":"NorthStar Medical Radioisotopes, LLC","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Hommen","suffix":""},{"id":487535516,"identity":"15b4afc3-8300-481d-9614-992b6d38b241","order_by":7,"name":"Dan De Vries","email":"","orcid":"","institution":"NorthStar Medical Radioisotopes, LLC","correspondingAuthor":false,"prefix":"","firstName":"Dan","middleName":"","lastName":"De Vries","suffix":""},{"id":487535517,"identity":"d3742632-6782-483e-8514-5318b1b28c08","order_by":8,"name":"Jim Harvey","email":"","orcid":"","institution":"NorthStar Medical Radioisotopes, LLC","correspondingAuthor":false,"prefix":"","firstName":"Jim","middleName":"","lastName":"Harvey","suffix":""},{"id":487535518,"identity":"5a4e2b88-5459-4d66-9815-d95bb507affc","order_by":9,"name":"Tyler Drum","email":"","orcid":"","institution":"NorthStar Medical Radioisotopes, LLC","correspondingAuthor":false,"prefix":"","firstName":"Tyler","middleName":"","lastName":"Drum","suffix":""},{"id":487535519,"identity":"a3632825-de80-4715-925a-6344c730bbdc","order_by":10,"name":"Diane S. Abou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0UlEQVRIiWNgGAWjYDCCAzxAogLE4iFJyxkgZgMyDhCthbGNFC18x88efMw7746c+fzew58/1NzL021gfvjoBh4tkmfyko15tz0zljnGlyZx4FhxsdkBNmPjHDxaDA7kmEnzbjucOIONx4zhAFtC4rYDPGzSeLWcfwPUMgesxfjDgX/EaLkBsqUBrMVA4mAbEVokb7xLNpxz7LCxBFuOmcTZPqCWwwT8wnc+9+CDNzWH5SSYzxh/qPgG1HK8+eFjfFqwAGbSlI+CUTAKRsEowAIAWkpP8QDTQN8AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-3005-9623","institution":"NorthStar Medical Radioisotopes, LLC","correspondingAuthor":true,"prefix":"","firstName":"Diane","middleName":"S.","lastName":"Abou","suffix":""}],"badges":[],"createdAt":"2025-07-10 20:34:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7095997/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7095997/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s41181-025-00419-7","type":"published","date":"2026-01-17T16:30:16+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":87317004,"identity":"d1b50dd7-348e-41f1-b2a5-d04bfcf8bed3","added_by":"auto","created_at":"2025-07-22 16:02:17","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":20315,"visible":true,"origin":"","legend":"\u003cp\u003eActinium-225 decay and secular equilibrium of \u003csup\u003e221\u003c/sup\u003eFr reached at \u0026gt; 30 min (\u0026gt; 99 %) and \u003csup\u003e213\u003c/sup\u003eBi at 5 hr (\u0026gt; 99 %)\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7095997/v1/1ef564127226758f7b780d13.jpg"},{"id":87317015,"identity":"1ca3a751-659c-4205-8c89-1bed727fa548","added_by":"auto","created_at":"2025-07-22 16:02:17","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":87986,"visible":true,"origin":"","legend":"\u003cp\u003eComparative RCP% determined using TLC either detected at \u003csup\u003e221\u003c/sup\u003eFr (30 min) or \u003csup\u003e213\u003c/sup\u003eBi equilibrium (\u0026gt;5 hr) using a TLC scanner equipped with (A) a gas-filled proportional counter; (B) a silicon detector; or using a TLC cut and count using (C) LS; (D) HPGe; (E) gamma well counting. (F) RCP% of 3 different samples (2F1, 2F2 and 2F3) determined using HPLC-fraction collection and gamma counting at 30 min (218 keV) or \u0026gt; 5 h (440 keV) and compared to TLC at \u003csup\u003e213\u003c/sup\u003eBi equilibrium. Sample numbering and identifiers are listed in Suppl. Table 2B.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7095997/v1/b3bedab095d94e68e23a3bb3.jpg"},{"id":87320386,"identity":"b7c6a142-bc75-4d55-b9ce-ce397f7bdd9d","added_by":"auto","created_at":"2025-07-22 16:26:17","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":97896,"visible":true,"origin":"","legend":"\u003cp\u003eExpected “E” \u003csup\u003e225\u003c/sup\u003eAc impurity % in the drug product compared with measured impurity % “M” using: TLC scanners equipped with (A) a gas-filled proportional counter at 30 min (1000 V) or \u0026gt; 5 hr (1500 V) post-TLC development; (B) a silicon detector TLC scanner at \u003csup\u003e213\u003c/sup\u003eBi secular equilibrium. TLC cut and count measured using (C) LS (\u0026gt; 5 hr); (D) HPGe at 218 keV (30 min-pink) and 440 keV (\u0026gt;5 h-purple); and (E) Gamma well counting (\u0026gt; 5 hr). (F) Impurity % determination using HPLC fraction collection combined with gamma well counting (440 keV at \u0026gt; 5 hr). Recoveries are reported in % on top of the columns. Sample numbering and identifiers are listed in Suppl. Table 2B.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7095997/v1/64e4b2a2461740c2906f5481.jpg"},{"id":100614794,"identity":"e5932051-9f35-464d-9200-3fead422f4df","added_by":"auto","created_at":"2026-01-19 17:25:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":805943,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7095997/v1/c09ce8b2-f189-4756-8948-7e02220efa5e.pdf"},{"id":87317773,"identity":"167b66ab-1701-429b-b5e2-7f9c2bae2cae","added_by":"auto","created_at":"2025-07-22 16:10:18","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":209750,"visible":true,"origin":"","legend":"","description":"","filename":"EJNMMIRPQASupplementalInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7095997/v1/be583042e9da736555d2e8be.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eQuality Control of \u003csup\u003e225\u003c/sup\u003eAc and associated Radiopharmaceuticals: \u003csup\u003e221\u003c/sup\u003eFr to be or not to be?\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eActinium-225 radiopharmaceuticals have drawn a great interest in cancer therapy due to their tumor-specific delivery of cytotoxic alpha-particles. To date, \u0026gt;\u0026thinsp;20 clinical trials have been launched world-wide using \u003csup\u003e225\u003c/sup\u003eAc radiopharmaceuticals, including\u0026thinsp;\u0026gt;\u0026thinsp;10 clinical trials actively recruiting in phase 2\u0026ndash;3 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. While \u003csup\u003e225\u003c/sup\u003eAc radiopharmaceutical development has exponentially grown over the last 10 years, several complications have hindered the progression to clinic. Particularly, the global \u003csup\u003e225\u003c/sup\u003eAc isotope supply is currently insufficient, however manufacturing initiatives are in progress to support the needs of research and clinical trials. Precluded access to this isotope has led to expedited studies and little understanding of its specific detection. Consequently, there is currently no consensus for best practice in quality control (QC) of \u003csup\u003e225\u003c/sup\u003eAc and associated radiopharmaceuticals. This is a critical issue hampering the development of these potent agents.\u003c/p\u003e\u003cp\u003eThe vast majority of nuclear medicine drug products are diagnostics radiopharmaceuticals. Unlike PET or SPECT isotopes, \u003csup\u003e225\u003c/sup\u003eAc (T\u003csub\u003e1/2\u003c/sub\u003e = 9.92 days) gamma-ray energies are too weak to be accurately detected with commonly used radiopharmacy equipment. Alternatively, the gamma-emitting progenies \u003csup\u003e221\u003c/sup\u003eFr (T\u003csub\u003e1/2\u003c/sub\u003e = 4.8 min, 218 keV, \u003cem\u003e%I\u003c/em\u003e: 11.44) and \u003csup\u003e213\u003c/sup\u003eBi (T\u003csub\u003e1/2\u003c/sub\u003e = 45 min, 440 keV, \u003cem\u003e%I\u003c/em\u003e: 25.9) have been adopted as the isotopes of choice to quantify \u003csup\u003e225\u003c/sup\u003eAc at their respective secular equilibrium (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFollowing purification or separation of \u003csup\u003e225\u003c/sup\u003eAc or labelled substance, secular equilibria of the progenies \u003csup\u003e221\u003c/sup\u003eFr or \u003csup\u003e213\u003c/sup\u003eBi are disturbed and require time to reestablish equilibrium in order to accurately quantify \u003csup\u003e225\u003c/sup\u003eAc. To determine the \u003csup\u003e225\u003c/sup\u003eAc activity, quantification by \u003csup\u003e221\u003c/sup\u003eFr may be undertaken as early as 30 min post-separation and quantification by \u003csup\u003e213\u003c/sup\u003eBi as early as 5 hr. Previous studies reported \u003csup\u003e225\u003c/sup\u003eAc quantification based on the \u003csup\u003e221\u003c/sup\u003eFr gamma ray (218 keV) within 30 min post-separation [\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. This strategy was applied to radiochemical purity (RCP%) evaluation of \u003csup\u003e225\u003c/sup\u003eAc drug products (DPs) using High Performance Liquid Chromatography (HPLC) fractions which were gamma counted within 30 min of the chromatographic separation [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Using this method, the development of [\u003csup\u003e225\u003c/sup\u003eAc]AcPSMA-I\u0026amp;T, indicated significant differences in RCP% (7\u0026ndash;9%) when comparing Thin Layer Chromatography (TLC) scanner results to those of HPLC or TLC counted using HPGe [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This inter-instrument discrepancy poses challenges in accuracy. A difference of \u0026gt;\u0026thinsp;5% in RCP% is problematic for the DP release, typically, RCP values of \u0026ge;\u0026thinsp;95% are required for release. These discrepancies are likely elicited by the varying detection sensitivity across instruments and whether quantification is affected through detection of \u003csup\u003e221\u003c/sup\u003eFr, \u003csup\u003e213\u003c/sup\u003eBi, or both at secular equilibrium.\u003c/p\u003e\u003cp\u003eIn contrast, \u003csup\u003e225\u003c/sup\u003eAc radiopharmaceutical QC exclusively based on \u003csup\u003e213\u003c/sup\u003eBi secular equilibrium quantification using both TLC and HPLC analyses were reported within a\u0026thinsp;\u0026lt;\u0026thinsp;5% difference between the two techniques [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. While this is an improvement to the \u003csup\u003e221\u003c/sup\u003eFr only detection method, a difference of 2\u0026ndash;5% may still not satisfy accuracy and precision criteria for radiopharmaceuticals as recommended [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn the present investigation, multiple analytical methods are compared for \u003csup\u003e225\u003c/sup\u003eAc quantification using \u003csup\u003e221\u003c/sup\u003eFr (at 30 min) and \u003csup\u003e213\u003c/sup\u003eBi (\u0026gt;\u0026thinsp;5 hr) detection. Linearity and limit of quantifications (LOQs) of \u003csup\u003e225\u003c/sup\u003eAc in solution were defined utilizing: liquid scintillation (LS), high purity germanium (HPGe), and NaI(Tl) gamma well counting. TLC evaluations were conducted using TLC scanners equipped with a gas-filled proportional counter or a plastic silicon photomultiplier detector as well as a \u0026ldquo;cut and count\u0026rdquo; strategy using LS, HPGe, or NaI(Tl) gamma well counting.\u003c/p\u003e\u003cp\u003eOnce the instrument capabilities were defined with respect to the \u003csup\u003e225\u003c/sup\u003eAc detection limit, drug products RCP% were assessed using \u003csup\u003e221\u003c/sup\u003eFr or \u003csup\u003e213\u003c/sup\u003eBi in equilibrium. Determinations based on \u003csup\u003e221\u003c/sup\u003eFr equilibrium were compared with those of \u003csup\u003e213\u003c/sup\u003eBi and evaluated for accuracy and precision.\u003c/p\u003e\u003cp\u003eAdditional attributes were examined, focusing on the specificity and sensitivity of free \u003csup\u003e225\u003c/sup\u003eAc quantification in the DP. Recommendations for radiopharmaceutical QC indicate a detection down to 0.5% impurities (7). In previous investigations of \u003csup\u003e225\u003c/sup\u003eAc-radiopharmaceuticals, reports suggested a detection limit as low as 1% of free \u003csup\u003e225\u003c/sup\u003eAc impurities [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The low radioactive concentration of an \u003csup\u003e225\u003c/sup\u003eAc-radiopharmaceutical confounds achieving an adequately low limit of quantification. In this report, free \u003csup\u003e225\u003c/sup\u003eAc spiking assays were conducted, and accuracy was evaluated for each instrument.\u003c/p\u003e\u003cp\u003eAltogether, the examination of each instrument quantification limits, the RCP% based on \u003csup\u003e221\u003c/sup\u003eFr (at 30 min) versus \u003csup\u003e213\u003c/sup\u003eBi (\u0026gt;\u0026thinsp;5 hr) detection and the quantification of low-level impurity are meant to better support the understanding of \u003csup\u003e225\u003c/sup\u003eAc radiopharmaceutical.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eFive instruments were utilized to measure the radiochemical purity of \u003csup\u003e225\u003c/sup\u003eAc-labeled drug preparations using either TLC or HPLC. Investigations using TLC scanners were undertaken comparing the performance of a plastic silicon detector to that of a gas-filled proportional counter. While the plastic silicon detector is mostly designed to detect beta- and gamma-emitting isotopes, the gas-filled proportional counter enables a high voltage modulation for alpha specific detection at 1,000 V and a beta/gamma specific detection at 1,500V [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOther detectors were considered for TLC analysis using a cut and counting approach. Liquid scintillation (LS) counting was undertaken to measure \u003csup\u003e225\u003c/sup\u003eAc spotted on TLC strips by immersing each half of the strip in scintillating cocktail for detection of alpha and beta-emitting isotopes. The LS parameters were selected to exclusively count the alpha-pulse window. Spectroscopy using High Purity Germanium (HPGe) resulted in high resolution and adequate quantification of gamma rays to precisely measure \u003csup\u003e221\u003c/sup\u003eFr at 218 keV and \u003csup\u003e213\u003c/sup\u003eBi at 440 keV. The radiochemical purity was determined by using TLC combined with HPGe and evaluated for both \u003csup\u003e221\u003c/sup\u003eFr and \u003csup\u003e213\u003c/sup\u003eBi at equilibrium. Gamma well counting with a NaI(Tl) detector was also used with both HPLC fractions and TLC strips.\u003c/p\u003e\u003cp\u003e\u003cem\u003eLinearity and limit of quantifications (LOQs)\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe performance of each instrument was assessed, evaluating linearity and LOQs for \u003csup\u003e225\u003c/sup\u003eAc in solution or spotted on TLC strips. The \u003csup\u003e225\u003c/sup\u003eAc activity in solution ranged from 0.185 Bq to 37 kBq (5 pCi to 1000 nCi) and were measured for linearity, demonstrating R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.99 for all instruments (Suppl. Figure\u0026nbsp;1). The LOQ for \u003csup\u003e225\u003c/sup\u003eAc in solution was defined as 2 Bq (55 pCi) for LS; 30 Bq (810 pCi) for HPGe; and 555\u0026ndash;740 Bq (15\u0026ndash;20 nCi) for gamma well counting, depending on the energy window (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Suppl Fig.\u0026nbsp;1).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eLOQ for \u003csup\u003e225\u003c/sup\u003eAc in solution, measured using LS, HPGe and gamma well counting. Gamma-well counting was acquired under \u003csup\u003e221\u003c/sup\u003eFr (195\u0026ndash;240 keV); \u003csup\u003e213\u003c/sup\u003eBi (350\u0026ndash;530 keV) or open energy window (15\u0026ndash;530 keV).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSolution\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLSC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eHPGe (keV)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e\u003cp\u003eGamma well counter (keV)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDetection\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ealpha\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e218\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e440\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e195\u0026ndash;240\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e350\u0026ndash;530\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e15\u0026ndash;530\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLOQ (Bq)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e773\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e644\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e566\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eA similar evaluation was conducted with activity spotted on non-developed TLC strips from 0.37 to 55.5 kBq (0.01 to 1500 nCi). The LOQs were measured as low as 1.1 Bq (30 pCi) for LS; between 14.8 Bq (400 pCi) and 36.3 Bq (0.98 nCi) for HPGe at 440 keV and 218 keV, respectively; and 63\u0026ndash;66 Bq (1.7\u0026ndash;1.8 nCi) for gamma-well counting (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Suppl Fig.\u0026nbsp;2). The gas-filled proportional counter TLC scanner outperformed the silicon detector with a LOQ defined at 11.1 Bq (300 pCi) at 1,000 V.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eLOQ for \u003csup\u003e225\u003c/sup\u003eAc spotted on non-developed TLC strips measured using LS, HPGe, gamma-well counting and TLC scanners equipped with plastic silicon or gas-filled proportional detectors.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTLC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLSC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eHPGe (keV)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e\u003cp\u003eGamma well counter (keV)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eTLC\u003c/p\u003e\u003cp\u003escanner\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eTLC\u003c/p\u003e\u003cp\u003escanner\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDetection\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ealpha\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e218\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e440\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e195\u0026ndash;240\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e350\u0026ndash;530\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e15\u0026ndash;530\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003esilicon\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eproportional\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLOQ (Bq)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e36.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e14.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e64.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e4366\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e11.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eOverall, these results place LS, HPGe, and the gas-filled proportional counter TLC scanner as the most sensitive tools to detect\u0026thinsp;\u0026lt;\u0026thinsp;37 Bq (1 nCi) of free \u003csup\u003e225\u003c/sup\u003eAc in drug products whether in solution or spotted on TLC. The limits of quantification for \u003csup\u003e225\u003c/sup\u003eAc in solution resulted in higher values than those spotted on TLC strips, suggesting a possible geometry effect for all instruments.\u003c/p\u003e\u003cp\u003e\u003cem\u003eFrancium-221 versus Bismuth-213 secular equilibrium\u003c/em\u003e\u003c/p\u003e\u003cp\u003eSuitability of the various instruments and techniques for \u003csup\u003e221\u003c/sup\u003eFr acquired at 30 minutes versus \u003csup\u003e213\u003c/sup\u003eBi acquired at \u0026gt;\u0026thinsp;5 hr post TLC development was evaluated. Drug products formulated with radioactive concentrations ranging from 0.185 to 1.48 kBq/ \u0026micro;L (5 to 40 nCi/\u0026micro;L) (Suppl. Table\u0026nbsp;2) were examined for each detection time post-TLC development.\u003c/p\u003e\u003cp\u003eTLC scanners were each investigated for their response to \u003csup\u003e221\u003c/sup\u003eFr versus \u003csup\u003e213\u003c/sup\u003eBi at equilibrium. The plastic silicon detector (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) displayed significant differences (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) in RCP% for the same TLC strip, 61\u0026thinsp;\u0026plusmn;\u0026thinsp;5% evaluated at 30 min post-development, versus 99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1% measured at \u003csup\u003e213\u003c/sup\u003eBi equilibrium. Using a different radiolabeled molecule (RCP%: 74\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0%), the gas-filled proportional counter did not show any significant differences between the alpha-specific acquisition (1,000 V, 30 min post-development) versus beta- and gamma-specific (1,500 V at \u0026gt;\u0026thinsp;5 hr). It indicated a mean difference of 1.2% between the two tested parameters (n\u0026thinsp;=\u0026thinsp;5, P\u0026thinsp;=\u0026thinsp;0.0712, t-test, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). This demonstrates a capability to specifically discriminate alpha emissions from the beta when adjusting the high voltage settings. This was further confirmed comparing acquisitions at only 1,000 V for the same TLC strip at 30 min and \u0026gt;\u0026thinsp;5 hrs post-development. These showed significant differences in RCP % (P\u0026thinsp;=\u0026thinsp;0.0002, n\u0026thinsp;=\u0026thinsp;5); and similarly, under 1,500 V with P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 (Suppl. Figure\u0026nbsp;5).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eA TLC cut and count strategy was adopted to evaluate LS, HPGe and gamma-well counting. Prior to counting, distinct separation of the radiolabeled-molecule with the free \u003csup\u003e225\u003c/sup\u003eAc was confirmed using a TLC scanner (Rf\u0026thinsp;=\u0026thinsp;0 vs Rf\u0026thinsp;=\u0026thinsp;1, respectively). The TLC strips were cut in half and characterized using LS, gamma-well counting and HPGe for RCP% determination (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D, E). Examining the detection at 30 min versus \u0026gt;\u0026thinsp;5 hrs post-TLC strip development, HPGe at 218 keV demonstrated at 30 min an exact RCP% match with that of 440 keV at \u0026gt;\u0026thinsp;5 hrs for the same sample (n\u0026thinsp;=\u0026thinsp;6, RSD\u0026thinsp;=\u0026thinsp;0.14\u0026ndash;0.22%). In contrast, LS under alpha specific settings, and gamma-well counting regardless of the energy window (Suppl. Figure\u0026nbsp;6), both failed to demonstrate equivalence of RCP% between the two time points.\u003c/p\u003e\u003cp\u003eSimilarly, when the drug product RCP% was evaluated using HPLC combined with gamma-well counting of collected fractions, the RCP% resulted in discrepancies ranging from 2\u0026ndash;4% differences between the 30 min and \u0026gt;\u0026thinsp;5 hr counting of fractions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). However, negligeable differences of RCP% were found between TLC scanner and HPLC gamma counting at \u003csup\u003e213\u003c/sup\u003eBi secular equilibrium (Suppl Info Fig.\u0026nbsp;4). In light of these results, \u003csup\u003e221\u003c/sup\u003eFr equilibrium does not adequately fulfill precision and accuracy criteria for RCP% determination using HPLC and gamma-well counting.\u003c/p\u003e\u003cp\u003e\u003cem\u003eAccuracy with spiking of free\u003c/em\u003e \u003csup\u003e\u003cem\u003e225\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eAc in the pure drug product\u003c/em\u003e\u003c/p\u003e\u003cp\u003eBoth TLC and HPLC methods were evaluated for accuracy when analyzing DP spiked with known amounts of free \u003csup\u003e225\u003c/sup\u003eAc (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Each technique was investigated for determining the recovery of free \u003csup\u003e225\u003c/sup\u003eAc compared to the expected value.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe gas-filled proportional counter TLC scanners generated an excellent response and accurately reported the expected free \u003csup\u003e225\u003c/sup\u003eAc activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Measurements of impurity % demonstrated a recovery of 104 and 91% for both acquisition at 30 min (1,000 V) and \u0026gt;\u0026thinsp;5 hr (1,500 V), respectively. In contrast, the silicon detector acquired at \u003csup\u003e213\u003c/sup\u003eBi secular equilibrium failed to accurately measure the expected free \u003csup\u003e225\u003c/sup\u003eAc (recoveries: 87%) at similar radioactive concentration (Fig.\u0026nbsp;3B1). Since all free \u003csup\u003e225\u003c/sup\u003eAc spikes spotted on TLC were below the LOQ of the instrument, the silicon detector failed to accurately measure \u003csup\u003e225\u003c/sup\u003eAc in the drug product (Fig.\u0026nbsp;3B1-3).\u003c/p\u003e\u003cp\u003eTLC cut and count measurement strategies using LS, gamma-well counting, and HPGe demonstrated acceptable recoveries. Liquid Scintillation and gamma-well counting were both acquired at \u0026gt;\u0026thinsp;5 hr post-TLC development, and both resulted in adequate recoveries (90\u0026ndash;110%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, E). HPGe demonstrated similar successful results regardless of the secular equilibrium (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), except for one sample for which the free \u003csup\u003e225\u003c/sup\u003eAc spike activity approached the LOQ (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eD2).\u003c/p\u003e\u003cp\u003eFinally, evaluation of HPLC collected fractions using gamma-well counting at \u003csup\u003e213\u003c/sup\u003eBi equilibrium (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eF) passed all recoveries.\u003c/p\u003e\u003cp\u003e\u003cem\u003eRCP% correlation between TLC and HPLC\u003c/em\u003e\u003c/p\u003e\u003cp\u003eHPLC fraction collection combined with gamma-well counting, was completed in a number of DP samples (n\u0026thinsp;=\u0026thinsp;17). The RCP% evaluated using HPLC/gamma-well counting radiochromatogram (Suppl. Figure\u0026nbsp;3), were correlated to that of TLC scanner (silicon) measured at \u003csup\u003e213\u003c/sup\u003eBi secular equilibrium using extended acquisition times in order to obtain sufficient counts for accuracy (\u0026gt;\u0026thinsp;40 min). The cross-evaluation resulted in an R\u003csup\u003e2\u003c/sup\u003e value of \u0026gt;\u0026thinsp;0.99, with less than 1% difference in RCP% between the two techniques (Suppl. Figure\u0026nbsp;4).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe development of molecularly-targeted radiopharmaceutical therapy has undergone a tremendous progression over the last 10 years, launching an increasing number of first-in-human clinical trials for treatments of various cancers. In parallel, the characterization of these drugs is not trivial with tests spanning across several expertises including chemical, radiochemical and biological evaluations. For \u003csup\u003e225\u003c/sup\u003eAc radiopharmaceuticals, the path to complete QC is rather challenging due to the nature of this rare isotope. As of today, there is no consensus on the best practices for \u003csup\u003e225\u003c/sup\u003eAc quality control. Actinium-225 is an alpha-emitting which decays to stability with the emission of four alpha-emitting (and two beta-emitting) progeny including \u003csup\u003e221\u003c/sup\u003eFr and \u003csup\u003e213\u003c/sup\u003eBi. \u003csup\u003e225\u003c/sup\u003eAc is more readily quantifiable through indirect progeny detection due to the abundance of their gamma-rays, emitted at 218 keV (\u003csup\u003e221\u003c/sup\u003eFr) and 440 keV (\u003csup\u003e213\u003c/sup\u003eBi). Historically, \u003csup\u003e225\u003c/sup\u003eAc quantification has been described using \u003csup\u003e213\u003c/sup\u003eBi detection, at secular equilibrium\u0026thinsp;\u0026gt;\u0026thinsp;5 h after separation, at this time\u0026thinsp;\u0026gt;\u0026thinsp;99% of the \u003csup\u003e213\u003c/sup\u003eBi activity content equals that of \u003csup\u003e225\u003c/sup\u003eAc [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. From the standpoint of drug production manufacturing, a 5 h hold after each processing step due to equilibrium disruption is a major impediment, imposing lead times of several days. Alternatively, \u003csup\u003e221\u003c/sup\u003eFr secular equilibrium reached after 30 min from the time of separation may offer a compelling solution to a time sensitive process.\u003c/p\u003e\u003cp\u003eIn the present study, accuracy and precision of \u003csup\u003e225\u003c/sup\u003eAc quantification, using \u003csup\u003e221\u003c/sup\u003eFr or \u003csup\u003e213\u003c/sup\u003eBi at secular equilibrium, was evaluated across 5 instruments. Each instrument was first verified for linearity and LOQ using \u003csup\u003e225\u003c/sup\u003eAc in secular equilibrium with its progeny.\u003c/p\u003e\u003cp\u003eThe LOQ determined in solution (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) was found to be higher than the LOQ of material spotted on TLC strips (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). A 20-fold higher sensitivity was observed for TLC using NaI(Tl) gamma-well counting and twice as high for LS than that in solution. This may be explained by the geometry, for which large homogeneously dispersed radioactive solution resulted in a higher LOQ to that of more concentrated spotted activity on chromatographic strips. A similar effect was observed when decreasing the standard solution volume from 750 to 20 \u0026micro;L, the LOQ was found to decrease (Suppl. Figure\u0026nbsp;1C-D).\u003c/p\u003e\u003cp\u003eAmong all instruments tested, LS was identified as the most sensitive technique to quantify \u003csup\u003e225\u003c/sup\u003eAc with a limit of quantification as low as 1 Bq (~\u0026thinsp;27 picocuries). This technique was unsuitable for radionuclidic identification and indicated significant RCP% differences between 30 min and \u0026gt;\u0026thinsp;5 hr acquisition. At 30 min post-TLC development, \u003csup\u003e221\u003c/sup\u003eFr has reached\u0026thinsp;\u0026gt;\u0026thinsp;99% of its equilibrium activity (compared to \u003csup\u003e225\u003c/sup\u003eAc activity), whereas \u003csup\u003e213\u003c/sup\u003eBi ingrowth has only reached 29% of its equilibrium activity considering no prior \u003csup\u003e213\u003c/sup\u003eBi contribution from the radiolabeling preparation. Radiolabeled samples were let decayed overnight to allow for all \u003csup\u003e213\u003c/sup\u003eBi-labeled molecule to disappear. For LS detection, the beta emission from \u003csup\u003e213\u003c/sup\u003eBi, \u003csup\u003e209\u003c/sup\u003eTl and \u003csup\u003e209\u003c/sup\u003ePb strongly interfered with the pule shape of the alphas from the decay of \u003csup\u003e225\u003c/sup\u003eAc, \u003csup\u003e221\u003c/sup\u003eFr, \u003csup\u003e217\u003c/sup\u003eAt and \u003csup\u003e213\u003c/sup\u003ePo. The latest alpha-emitters may be attenuated by the embedment in the chromatographic strip, diminishing the interaction with the cocktail. As a result, LS detection for TLC was found unfit to read RCP% before \u003csup\u003e213\u003c/sup\u003eBi secular equilibrium, this would not be the case if the source was in solution.\u003c/p\u003e\u003cp\u003eHPGe demonstrated LOQs slightly higher than that of LS, \u0026lt;\u0026thinsp;37 Bq ranges (nanocuries). In contrast to LS, HPGe enables a high radionuclidic resolution with identification and quantification of \u003csup\u003e221\u003c/sup\u003eFr (218 keV, 11.4%) as early as 30 min post-TLC development, and \u003csup\u003e213\u003c/sup\u003eBi quantification (440 keV, 25.9%) after \u0026gt;\u0026thinsp;5 hr. This radionuclide-specific evaluation demonstrated negligible differences between the RCP% measurements at 30 min and \u0026gt;\u0026thinsp;5 hr. HPGe was found adequate to evaluate RCP% at both \u003csup\u003e221\u003c/sup\u003eFr and \u003csup\u003e213\u003c/sup\u003eBi secular equilibrium.\u003c/p\u003e\u003cp\u003eNaI(Tl) gamma-well counting is known for its modest gamma resolution with high background due to Compton scattering and the photoelectric effects in the energy range of \u0026lt;\u0026thinsp;200 keV. The scattering elicited by \u003csup\u003e213\u003c/sup\u003eBi beta-contribution has been reported to interfere in the \u003csup\u003e221\u003c/sup\u003eFr detection (11). Exempt of scattering correction, the RCP% evaluation at \u003csup\u003e221\u003c/sup\u003eFr secular equilibrium indicated lower accuracy compared to that of \u003csup\u003e213\u003c/sup\u003eBi at equilibration. This is mostly due to limitations with the NaI(Tl) detector to accurately quantify the \u003csup\u003e221\u003c/sup\u003eFr peak from background and other interferences. Examining the RCP% determined using HPLC fraction collection coupled with gamma-well counting with that of TLC results showed the quantification at \u003csup\u003e221\u003c/sup\u003eFr secular equilibrium was unfit for accuracy. Inter instrument TLC and HPLC agreement was only successful when comparing RCP% at \u003csup\u003e213\u003c/sup\u003eBi secular equilibrium (Suppl. Figure\u0026nbsp;5).\u003c/p\u003e\u003cp\u003eIn contrast, the TLC scanner equipped with the gas-filled proportional counter, operated at 1,000 V (alpha-specific setting), demonstrated an acceptable RCP% accuracy at 30 min, compared to measurements conducted at \u0026gt;\u0026thinsp;5 hr at 1,500 V. At 30 min post-development, the ionization emitted by \u003csup\u003e225\u003c/sup\u003eAc, \u003csup\u003e221\u003c/sup\u003eFr and \u003csup\u003e217\u003c/sup\u003eAt alphas were substantial contributors at 1,000V [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. After 5 hr, the equilibrated RCP% was evaluated using mostly the beta contributions delivered by \u003csup\u003e213\u003c/sup\u003eBi and other progeny detected at 1,500V (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The same measurement conducted at 1,000 V after 5 hr, however, did not result in accurate RCP% (Suppl. Figure\u0026nbsp;5A).\u003c/p\u003e\u003cp\u003eIn light of the detection sensitivity and LOQ defined for the gamma-well counter, measurement of radioimpurities using HPLC fractions may be limited to 1% of the total content. Actinium-225-drug product (1.48\u0026ndash;2.22 kBq/\u0026micro;L; 40\u0026ndash;60 nCi/\u0026micro;L) analyzed at the end-of-synthesis, generally leads to a total HPLC injection of 2 to 3 \u0026micro;Ci. Detecting 0.5% impurity would require a detection limit of 10\u0026ndash;15 nCi, spread across several fractions. This is at or below the gamma-well counter LOQ (0.555\u0026ndash;0.740 Bq or 15\u0026ndash;20 nCi in solution, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). When the impurity specification is raised to 1%, a minimum detection of 0.74\u0026ndash;1.11 Bq (20\u0026ndash;30 nCi) could be achieved, which is above the measured LOQ. Considering the LOQ of the instrument and the low radioactive concentration of a typical \u003csup\u003e225\u003c/sup\u003eAc radiopharmaceutical, gamma-well counting of fractionated HPLC samples may be suitable to measure as low as 1% radio impurity. HPGe and LS of fractions would meet 0.5% impurity detection; however, both techniques require labor-intensive sample transfer associated with a higher risk of error.\u003c/p\u003e\u003cp\u003eWhen spotting a DP on TLC (5\u0026ndash;10 \u0026micro;L), the total activity spotted ranges from 14.8\u0026ndash;22.2 kBq (400\u0026ndash;600 nCi). Detecting 0.5% impurity requires a TLC scanner or cut/count technique to measure down to 74\u0026ndash;111 Bq (2\u0026ndash;3 nCi). In light of the TLC-LOQ reported for all instruments (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), all techniques except the silicon detector are suitable to measure 0.5% impurity, fulfilling the recommended criteria for radiopharmaceutical QC (7).\u003c/p\u003e\u003cp\u003eLinearity, accuracy, specificity and repeatability have been demonstrated utilizing 5 different instruments, all fit for \u003csup\u003e225\u003c/sup\u003eAc detection when measured at \u003csup\u003e213\u003c/sup\u003eBi secular equilibrium. However, when using \u003csup\u003e221\u003c/sup\u003eFr at secular equilibrium for \u003csup\u003e225\u003c/sup\u003eAc quantification, only the HPGe and gas-filled proportional counter were adequate for radiopharmaceutical drug product release testing.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eFive instruments have been tested for their response to \u003csup\u003e225\u003c/sup\u003eAc measurement in accuracy, sensitivity, linearity and specificity. Liquid scintillation counting and HPGe were found to be the most sensitive techniques to detect \u003csup\u003e225\u003c/sup\u003eAc progeny. Analysis and quantification of RCP% at \u003csup\u003e221\u003c/sup\u003eFr secular equilibrium is acceptable for HPGe and the gas-filled proportional counter using the right parameters. Liquid scintillation counting, gamma-well counting and the TLC-scanner plastic silicon detector require \u003csup\u003e213\u003c/sup\u003eBi equilibrium for accurate RCP% characterization of the DP. Low content radioimpurity detection was found best measured using TLC rather than rather than gamma-well counting of HPLC fraction collection.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eQC: quality Control\u003c/p\u003e\n\u003cp\u003eDP: Drug Product\u003c/p\u003e\n\u003cp\u003eLS: Liquid Scintillation\u003c/p\u003e\n\u003cp\u003eHPGe: High Purity Germanium\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026gamma;: Gamma counting\u003c/p\u003e\n\u003cp\u003eTLC: Thin Layer Chromatography\u003c/p\u003e\n\u003cp\u003eHPLC: High Performance Liquid Chromatography\u003c/p\u003e\n\u003cp\u003eLOQ: Limit of Quantification\u003c/p\u003e\n\u003cp\u003eRCP%: Radiochemical Purity %\u003c/p\u003e\n\u003cp\u003eE: Expected\u003c/p\u003e\n\u003cp\u003eM: Measured\u003c/p\u003e\n\u003cp\u003en/a: non assigned\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eSome or all of the \u003csup\u003e225\u003c/sup\u003eAc used in this research was supplied by the U.S. Department of Energy Isotope Program managed by the Office of Isotope R\u0026amp;D and Production.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterial and Methods\u003c/strong\u003e are described in the Supplemental information.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEthics approval and consent to participate:\u003c/em\u003e not applicable\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConsent for publication:\u003c/em\u003e not applicable\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAuthors contributions:\u0026nbsp;\u003c/em\u003e\u003cstrong\u003eMT Gonzalez\u003c/strong\u003e: conceptualization, investigation, analysis, interpretation;\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003cstrong\u003eL Wheeless\u003c/strong\u003e: synthesis, investigation, analysis and interpretation; \u003cstrong\u003eVKR Tangadanchu\u003c/strong\u003e: analysis and interpretation. \u003cstrong\u003eC. Hawkins\u003c/strong\u003e: analysis and interpretation; \u003cstrong\u003eS. Provo\u003c/strong\u003e: analysis. \u003cstrong\u003eW. Smith\u003c/strong\u003e: analysis. \u003cstrong\u003eM. Hommen\u003c/strong\u003e: analysis; \u003cstrong\u003eD. De Vries\u003c/strong\u003e: reviewing and editing; \u003cstrong\u003eJ. Harvey\u003c/strong\u003e: reviewing and editing; \u003cstrong\u003eT. Drum\u003c/strong\u003e: reviewing, editing and supervision; \u003cstrong\u003eD.S. Abou:\u003c/strong\u003e conceptualization, synthesis, investigation, method development, analysis, interpretation, original draft writing, supervision.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAvailability of data and material:\u003c/em\u003e all methods and data are available and reported in supplemental information.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCompeting Interest:\u003c/em\u003e all authors listed are full-time employees of NorthStar Medical Radioisotope., LLC. The corresponding author D.S. Abou is a guest editor at EJNMMI, Radiopharmacy and Chemistry, collection: Radiopharmaceutical quality assurance.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFunding:\u0026nbsp;\u003c/em\u003ethis research was funded by NorthStar Medical Radioisotopes, LLC.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCorresponding Author Information:\u0026nbsp;\u003c/em\u003eDiane S. Abou, PhD; [email protected]\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ehttps://clinicaltrials.gov/ \u003c/li\u003e\n\u003cli\u003eHooijman, E.L.; Chalashkan, Y.; Ling, S.W.; Kahyargil, F.F.; Segbers, M.; Bruchertseifer, F.; Morgenstern, A.; Seimbille, Y.; Koolen, S.L.W.; Brabander, T.; et al. Development of [225Ac]Ac-PSMA-I\u0026amp;T for Targeted Alpha Therapy According to GMP Guidelines for Treatment of mCRPC. Pharmaceutics 2021, 13, 715. \u003c/li\u003e\n\u003cli\u003eHooijman, E.L., de Jong, J.R., Ntihabose, C.M. et al. Ac-225 radiochemistry through the lens of [225Ac]Ac-DOTA-TATE. EJNMMI radiopharm. chem. 10, 9 (2025). \u003c/li\u003e\n\u003cli\u003eHooijman, E.L., Radchenko, V., Ling, S.W. et al. Implementing Ac-225 labelled radiopharmaceuticals: practical considerations and (pre-)clinical perspectives. EJNMMI radiopharm. chem. 9, 9 (2024). \u003c/li\u003e\n\u003cli\u003eINTERNATIONAL ATOMIC ENERGY AGENCY, Production and Quality Control of Actinium-225 Radiopharmaceuticals, IAEA-TECDOC-2057, IAEA, Vienna (2024), https://doi.org/10.61092/iaea.95h3-2ji2\u003c/li\u003e\n\u003cli\u003eAbou DS, Zerkel P, Robben J, McLaughlin M, Hazlehurst T, Morse D, Wadas TJ, Pandya DN, Oyama R, Gaehle G, Nickels ML, Thorek DLJ. Radiopharmaceutical Quality Control Considerations for Accelerator-Produced Actinium Therapies. Cancer Biother Radiopharm. 2022 Jun;37(5):355-363\u003c/li\u003e\n\u003cli\u003eGillings, N., Todde, S., Behe, M. et al. EANM guideline on the validation of analytical methods for radiopharmaceuticals. EJNMMI radiopharm. chem. 5, 7 (2020). https://doi.org/10.1186/s41181-019-0086-z\u003c/li\u003e\n\u003cli\u003eICH guideline Q2(R2) Validation of Analytical Procedures, Guidance for Industry \u003c/li\u003e\n\u003cli\u003eFriedlander, G., Kennedy, J; Macias, E; Miller, JM; Nuclear and Radiochemistry, 3\u003csup\u003erd\u003c/sup\u003e Ed., 1981, ISBN 0-471-28021-6\u003c/li\u003e\n\u003cli\u003eChase, G. Rabinowitz, JL; Principles of Radioisotope Methodology, 3\u003csup\u003erd\u003c/sup\u003e edition, SBN 8087-0308-0\u003c/li\u003e\n\u003cli\u003eCastillo Seoane, D., De Saint-Hubert, M., Ahenkorah, S. et al. Gamma counting protocols for the accurate quantification of 225Ac and 213Bi without the need for a secular equilibrium between parent and gamma-emitting daughter. EJNMMI radiopharm. chem. 7, 28 (2022).\u003c/li\u003e\n\u003cli\u003eKelly JM, Amor-Coarasa A, Sweeney E, Wilson JJ, Causey PW, Babich JW. A suitable time point for quantifying the radiochemical purity of 225Ac-labeled radiopharmaceuticals. EJNMMI Radiopharm Chem. 2021 Dec 20;6(1):38.\u003c/li\u003e\n\u003cli\u003eMcDevitt MR, Ma D, Simon J, Frank RK, Scheinberg DA. Design and synthesis of 225Ac radioimmunopharmaceuticals. Appl Radiat Isot. 2002 Dec;57(6):841-7.\u003c/li\u003e\n\u003cli\u003eMaguire WF, McDevitt MR, Smith-Jones PM, Scheinberg DA. Efficient 1-step radiolabeling of monoclonal antibodies to high specific activity with 225Ac for \u0026alpha;-particle radioimmunotherapy of cancer. J Nucl Med. 2014 Sep;55(9):1492-8.\u003c/li\u003e\n\u003cli\u003ePretze, M.; Wendrich, J.; Hartmann, H.; Freudenberg, R.; Bundschuh, R.A.; Kotzerke, J.; Michler, E. Comparison of ZnS(Ag) Scintillator and Proportional Counter Tube for Alpha Detection in Thin-Layer Chromatography. Pharmaceuticals 2025, 18, 26.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"ejnmmi-radiopharmacy-and-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"erpc","sideBox":"Learn more about [EJNMMI Radiopharmacy and Chemistry](http://ejnmmipharmchem.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/erpc/default.aspx","title":"EJNMMI Radiopharmacy and Chemistry","twitterHandle":"@officialEANM","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"225Ac quality control, Liquid Scintillation, High Purity Germanium, Gamma well counting, Thin Layer Chromatography, HPLC ","lastPublishedDoi":"10.21203/rs.3.rs-7095997/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7095997/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Actinium-225 radiopharmaceuticals have drawn great interest in cancer therapy due to their tumor-specific delivery of cytotoxic alpha-particles. Detection and quality control (QC) are critical for these potent agents. There is currently no consensus for best practice of \u003csup\u003e225\u003c/sup\u003eAc QC. Detection of \u003csup\u003e225\u003c/sup\u003eAc (T\u003csub\u003e1/2\u003c/sub\u003e = 9.92 days) is challenging; however, the gamma-emitting progenies \u003csup\u003e221\u003c/sup\u003eFr (T\u003csub\u003e1/2\u003c/sub\u003e = 4.8 min, 218 keV) and \u003csup\u003e213\u003c/sup\u003eBi (T\u003csub\u003e1/2\u003c/sub\u003e = 45 min, 440 keV) facilitate the indirect measurement of \u003csup\u003e225\u003c/sup\u003eAc. Using several instruments, we compared multiple analytical methods for \u003csup\u003e225\u003c/sup\u003eAc limit of quantification and the radiopharmaceutical radiochemical purity (RCP%). The RCP% of \u003csup\u003e225\u003c/sup\u003eAc-radiopharmaceutical was evaluated at both \u003csup\u003e221\u003c/sup\u003eFr and \u003csup\u003e213\u003c/sup\u003eBi secular equilibrium.\u003c/p\u003e\n\u003cp\u003eRCP% was measured using two TLC plate readers: a gas-filled proportional counter, under mixed emission and alpha specific parameters; and a plastic silicon photomultiplier detector and compared. Chromatographic strips were also analyzed using LS, HPGe, and NaI(Tl) gamma well counting. We correlated these RCP% to HPLC results measured at both equilibria utilizing multiple energy windows. Finally, examining accuracy and precision for each instrument, free \u003csup\u003e225\u003c/sup\u003eAc spiking assays were conducted on the radiopharmaceutical and measured.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e The TLC plate-reader using the plastic silicon detector resulted in significant RCP% differences between 30 min and \u0026gt;5 hr readings, whereas the gas-filled proportional counter showed no-significant differences between alpha-specific (30 min) and the mixed-isotope setting (\u0026gt;5 hr). HPGe-TLC demonstrated RCP% equivalence at \u003csup\u003e221\u003c/sup\u003eFr and \u003csup\u003e213\u003c/sup\u003eBi equilibrium. NaI(Tl) and LS measurements significantly underestimated the RCP% at 30 min. Finally, gamma well counting of HPLC fractions resulted in 3-5 % RCP% underestimation at \u003csup\u003e221\u003c/sup\u003eFr equilibrium.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eFive instruments have been tested for their accuracy, sensitivity, linearity and specificity response to \u003csup\u003e225\u003c/sup\u003eAc quantification using \u003csup\u003e221\u003c/sup\u003eFr and \u003csup\u003e213\u003c/sup\u003eBi measurements. \u003csup\u003e221\u003c/sup\u003eFr secular equilibrium was deemed acceptable for TLC RCP% analyses using HPGe and gas-filled proportional counter under adequate parameters. LS, gamma well counting and TLC-scanner with plastic silicon detector required a reading at \u003csup\u003e213\u003c/sup\u003eBi equilibrium for accurate RCP% characterization. Low content radioimpurity detection was most accurately measured using TLC rather than gamma well counting of HPLC fraction collection due to gamma counting limitations.\u003c/p\u003e","manuscriptTitle":"Quality Control of 225Ac and associated Radiopharmaceuticals: 221Fr to be or not to be?","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-22 16:02:13","doi":"10.21203/rs.3.rs-7095997/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-07-18T17:01:22+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-18T15:22:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-17T03:41:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"EJNMMI Radiopharmacy and Chemistry","date":"2025-07-16T10:51:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"ejnmmi-radiopharmacy-and-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"erpc","sideBox":"Learn more about [EJNMMI Radiopharmacy and Chemistry](http://ejnmmipharmchem.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/erpc/default.aspx","title":"EJNMMI Radiopharmacy and Chemistry","twitterHandle":"@officialEANM","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f50f9d2b-74a1-4cf8-ab69-49747571561c","owner":[],"postedDate":"July 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-19T16:49:43+00:00","versionOfRecord":{"articleIdentity":"rs-7095997","link":"https://doi.org/10.1186/s41181-025-00419-7","journal":{"identity":"ejnmmi-radiopharmacy-and-chemistry","isVorOnly":false,"title":"EJNMMI Radiopharmacy and Chemistry"},"publishedOn":"2026-01-17 16:30:16","publishedOnDateReadable":"January 17th, 2026"},"versionCreatedAt":"2025-07-22 16:02:13","video":"","vorDoi":"10.1186/s41181-025-00419-7","vorDoiUrl":"https://doi.org/10.1186/s41181-025-00419-7","workflowStages":[]},"version":"v1","identity":"rs-7095997","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7095997","identity":"rs-7095997","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}