Assessment of the Radiochemical Purity and Chemical Identity of [225Ac]Ac-Labeled Radiotherapeutics by TLC and RP-HPLC

Introduction: ²²⁵Ac-labeled radiotherapeutics have proven to be highly successful in the treatment of cancer patients, even after targeted radionuclide therapy with β^--emitters has failed.^a Due to four successive α- and two β-emissions in the decay chain, actinium-225 has been called atomic in vivo nanogenerator, depositing large amounts of energy at the target site.^b The same unique properties that make it an effective therapeutic radionuclide create a challenge for the assessment of the radiochemical purity (RCP) and chemical identity of ²²⁵Ac-labeled radiopharmaceuticals. The absence of detectable γ-emissions and the requirement for > 20 h^c,d to reach secular equilibrium with all daughter nuclides in the decay chain do not allow for immediate quantification of actinium-225 neither in its free form nor when bound to a biovector. Widely used quality control (QC) analyses involve radio-TLC, which is evaluated a minimum of 2 h after development.^d While in many cases an accurate measure of uncomplexed [²²⁵Ac]Ac³⁺, the chemical identity of the ²²⁵Ac-label radiotherapeutic cannot be confirmed using this method, leaving open the possibility of administering compound fragments arising during radiosynthesis or as a result of radiolysis.

Methods: In this study, the macropa-conjugated PSMA-binding peptidomimetic, [²²⁵Ac]Ac-RPS-088^d, was used as model compound. After synthesis, RCP was analyzed by radio-TLC in 50 mM citrate buffer (pH 5.0) and 1 M NH₄OAc (pH 5.5)/DMF 1:1^e. Chemical identity was confirmed by RP-HPLC by injection on a Symmetry® C18 column (3.5 µm, 4.6 x 50 mm). The radio-chromatogram was reconstructed from the collection of either 30 (20 sec) or 84 (72 x 5 sec, 12 x 20 sec) fractions and analysis by three different methods: TLC reader, phosphor imager and γ-counter. The evolution of the radio-chromatogram over time was observed on the phosphor imager to establish the minimum waiting time between the end of the HPLC run and the reconstruction. On the γ-counter, the option of using a narrow window around the 218 keV γ-rays emitted by Fr-221 was compared to the open window count after full equilibrium was reached. Finally, the separation of artificially generated fragments of [²²⁵Ac]Ac-RPS-088 by 1D or 2D radio-TLC was evaluated.

Results: Radio-TLC in 50 mM citrate buffer at pH 5.0 was confirmed to be a highly sensitive measure for free [²²⁵Ac]Ac³⁺ (Rf = 0.4-0.5) with any bound actinium-225 remaining at the point of origin. Some separation of [²²⁵Ac]Ac-RPS-088, its fragments and degradation products was observed on 1D and 2D TLCs developed in 1 M NH₄OAc/DMF 1:1 (Rf = 0.6-0.95), though this was insufficient for accurate quantification. Radio-chromatograms could be reconstructed by all three methods used. The TLC reader was the least sensitive and provided the least detailed chromatogram. Early observations of the changes in the AUCs suggest an acceptable accuracy 2 h after the end of the HPLC run, comparable to previous studies with radio-TLC.^d The chromatogram reconstructed from the 218 keV Fr-221 γ-emission alone appears to be accurate (within 5 %) 90 min after the end of the HPLC run, though acquisition of this chromatogram was the most time-consuming technique attempted.

Conclusions: In summary, using the conditions stated above, radio-TLC alone provides accurate information on the presence of uncomplexed [²²⁵Ac]Ac³⁺, but can only be a complementary measure of the chemical identity. Radio-chromatograms of peptidomimetic and small molecule ²²⁵Ac-radiotherapeutics may be reconstructed after fraction collection and used to confirm the chemical identity. The time expenditure would allow for the compound to be injected on the same day as the synthesis and the QC.

References:

^aJ Nucl Med 2016;57:1941, J Nucl Med 2018;59:795. ^bPharmaceuticals 2020;13:76. ^cSci Rep 2019;9:11671, Chem Commun (Camb) 2018;54:2599. ^dEJNMMI Radiopharmacy and Chemistry 2021;6:38. ^eEJNMMI Research 2016;6:65, Molecules 2022;27:543.