QSPTP_Abou-QC alpha.pdf

Quality Control of long-lived alpha-emitting radiopharmaceuticals Diane Abou, PhD  Quality control of long-lived alpha-emitting radiopharmaceuticals This lecture will address: Actinium-225/227 Thorium-227 and Radium-223 Why the QC of alpha-emitting isotop...

Quality Control of long-lived alpha-emitting radiopharmaceuticals Diane Abou, PhD  Quality control of long-lived alpha-emitting radiopharmaceuticals This lecture will address: Actinium-225/227 Thorium-227 and Radium-223 Why the QC of alpha-emitting isotopes is challenging? Detection methods Contaminants and progenies Applications with radiopharmaceuticals Basics: types of radiation and shielding NRC  Why Targeted Alpha Particle Therapy? Alpha particle emission is characterized by: Single strand break Long path- - high Linear Energy Transfer ( keV/μm) length ~2mm - short path-length in tissue (μm) This alpha particle energy deposition Short path- results in fatal double strand DNA damage. length ~50µm Great academic, industrial and clinical Double strand break interest in translating alpha particle therapies.  Why a challenge? Diagnostic isotopes Alpha-emitting isotopes Direct detection of gamma A direct gamma detection emissions is not always possible Decay leads to a stable The decay leads to a cascade “cold isotope’ of alpha emitting progenies Requires high activity Requires low activity amount for in vivo detection for therapy efficacy (µCis range – preclinical (nCis – preclinical research) research) Decay and storage is Decay and storage is facile challenging (tolerated up to 10 days ½ life) Actinium-225  Basics of Actinium-225 T½ life 9.92 d It can be labeled to targeting constructs Available (?) Detectable Gammas T½ lives 225Ac (9.92d) >> 213Bi (45min) > 221Fr (5min) 218 keV (9%) Secular equilibrium 440 keV is reached at (26%) 55 min with 221Fr and ~6.5h with 213Bi Actinium-225 supply and demand Research and industrial interest greatly outweighs current production capacity. Established production sites: Oak Ridge National Laboratories (US Dept. of Energy) Rosatom (Russia) Institute for Transuranium Elements (Germany) Trace sources elsewhere Ongoing efforts for novel production routes and scaling up capacity… Actinium-225 supplies: QC sheets issued by DOE 229Th generator produced Accelerator produced  CGMP automated [225Ac]Ac-DOTA-peptide RL module Using 229Th-generated material Quality control procedures (QCPs) include but not limited to: Radiochemical purity Chemical identity Radioisotipic purity - Thin Layer Chromatography - HPLC - Gamma counting of HPLC collected fractions for secular equilibrium analysis - Radioisotopic purity based on Oak Ridge certificate of analysis and previous validations. Abou, DS et al. PMID: 35695807 Quality Control of [225Ac]Ac-DOTA-peptide Using 229Th-generated material Gamma counting TLC HPLC of HLPC fractions Processed at secular equilibrium Processed at secular equilibrium >5h post production >5h post separation TLC shows RCP> 99.9% Gamma counting of collected fractions ≥90% Radioisotopic purity is validated based on previous test runs Actinium-225 supply: alternative production route Using accelerator produced 225Ac 99% ≈0.5% 235, 255 keV 154; 269 keV Detectable gammas using HPGe α 227Ac is gamma silent  Peptide QC of [225Ac]Ac-DOTA-peptide using accelerator produced material Free 225/227Ac Standard procedures were operated under identical conditions using the automated radiolabeling module HPLC – UV - TLC was read with RCP> 99.9% - Chemical identity was verified - Gamma counting RCP=94.2% Gamma Counting These techniques are not reporting the radioisotopic composition  Peptide QC of [225Ac]Ac-DOTA-peptide using accelerator produced material Free 225/227Ac Standard procedures were operated under identical conditions using HPLC – UV the automated synthesis module - TLC was read with RCP> 99.9% - Chemical identity was verified Gamma - Gamma counting RCP=94.2% Counting These techniques are not reporting the radioisotopic composition Fraction 12 was further analyzed using gamma spectrometry utilizing High Purity Germanium detector  High Purity Germanium Analysis of the peptide fraction (#12) The day of patient injection 2 days post RL 8 days post source production  HPGe analysis of peptide fraction (#12) over 100 days The day of patient injection 2 days post RL 8 days post source production Towards secular equilibrium of 227Ac>>227Th>223Ra Quantification of 227Ac in 227/225Ac-labeled agents: Bateman equations 227Ac (21.7 y) >> 227Th (18.7d) 227Th is at equilibrium with 227Ac at 100 days post-separation 227Th quantification leads to 227Ac content Abou, DS et al. PMID: 35695807 Using Actinium accelerated material source HPGe measured radionuclidic purity at the day of injection Composition of dose aliquot in purified fraction 12: 225Ac 227Ac 227Th 98 kBq 1.5 kBq 1.8 kBq Radioisotopic purity= 98.5% Radionuclidic purity= 96.7% High temperature radiolabeling conditions resulted in 3 isotopes labeled In conclusion when comparing the 2 sources…. At patient injection day, 8 days post source production: 229ThGenerator Accelerator Chemical Purity Validated Validated Radiochemical >95% 94.2% Purity Radioisotopic > 99.5% 98.5%* Purity *This value is influx… Abou, DS et al. PMID: 35695807  Alpha spectrometry of Actinium isotopic mix 227/225 Counts Pure 225Ac 225 Ac 221 Fr 217 At 213 Po Mix 225Ac/227Ac 223 Ra+227Th 10000 211 Bi 215 Po 227 Ac 8000 227 Ac 211 Po 1000000 6000 4000 100000 2000 211Po 0 225 3500 4000 4500 5000 5500 6000 6500 7000 7500 8000 8500 9000 mix Ac/227Ac 10000 keV Log 10 Counts Enriched 227Ac 225 Ac 221 Fr 217 At 213 Po 2000 227 223 Ac Ra+227Th 211 Bi 215 Po 1000 1500 225 Ac 1000 100 500 211Po 0 10 keV 3500 4000 4500 5000 5500 6000 6500 7000 7500 8000 8500 9000 3500 4000 4500 5000 5500 6000 6500 7000 7500 8000 8500 9000 keV keV 211Po is the only 227Ac daughter identified in the mix Challenges to tackle when using 225/227Ac accelerator produced material: Waste handling of extreme long lived isotopes (21.7 y 227Ac) Extensive quality control to define the in-flux radioisotopic purity: 100 days to quantify 227Ac (HPGe) Faster method to identify 227Ac: Alpha spectrometry is not easily accessible and involves tricky sample preparation, but 211Po detection progeny is possible in a mix 227/225Ac Thorium-227 and Radium-223  Why Thorium-227? 227Th(t1/2 18.7 d) Detectable Produces 5 alpha particles Gammas No supply issues 235, 255 keV First daughter is 223Ra 154; 269 keV FDA, EMA approved bone seeker Chemistry compatible with 89Zr 351 keV for a theranostic application Gamma detection is possible, 404, requiring pre-calibration measurements 427 keV  227 Th and 223Ra detection methods Detectable 227Th (18.7 d) ~ 223Ra (11.4 d) Gammas 235, 255 keV 154; 269 keV 351 keV Transient equilibrium The transient equilibrium and promiscuity of energy peaks poses challenges in detection 404, when using common radiopharmacy 427 keV equipment.  227Th/ 223Ra discrimination using High Purity Germanium detection Radium-223 269 154 145 mix Radium-223/Thorium-227 235 255 Automated measurements are needed to conduct organ distribution evaluation of radiopharmaceuticals  Can we achieve Thorium-227 exclusive detection using a gamma counter? Detection challenge when using NaI detection with 223Ra/227Th mix Pure 227Th Pure 223Ra Mix 227Th/223Ra Automated measurements are needed to conduct organ distribution evaluation of the radiopharmaceutical  Gamma counting 227Th/ 223Ra discrimination? Pure 227Th Pure 223Ra 227Th + 223Ra co-mixed Open Channel All measurements were conducted open (including 2k channels)  How can we acquire exclusive detection of Thorium-227 combining gamma counting with NaI spectral deconvolution From the 1st day of 227Th production we predicted 223Ra (and daughters) ingrowth modeled using the Bateman equations Bateman H. Proc Cambridge Phil Soc 1910;15(V):423–427 https://epa-prgs.ornl.gov/radionuclides/FINALPEAKTM.pdf 223Ra and 227Th calibrations of the gamma counter 223Ra 223Ra NIST Standards counting efficiency cpm  kBq Purified 227Th Decaying 223Ra measured from over 23 days 0 to 23 days Hasson A, et al. PMID: 36149725  Gamma counting 227Th/ 223Ra discrimination Pure 223Ra 227Th + 223Ra co-mixed Pure 227Th Open Channel 223Ra/227Th discrimination using isolated isotopic calibrations and Bateman equations Hasson A, et al. PMID: 36149725  Applications to radiopharmaceuticals Theranostic capability using 227Th / 89Zr pair Trastuzumab was labeled with 227Th or 89Zr using identical chemical precursor 227Th 89Zr Alpha emitting PET diagnostic therapy tracer  Pharmacokinetic & organ distribution in mice using 227Th / 223Ra discrimination to define in vivo 223Ra ingrowth [227Th]Th-Trastuzumab was administered iv to immunocompetent naïve female swiss/webster mice 227Th (Bq/gram) 800 24 h 700 3 days 600 7 days 227Th 500 14 days 400 300 200 100 0 t I I t Radiochemical and radionuclidic purity >95% d s r n y h le e oo ar ng e ee e ac rG G m sc fa on ro w bl he lu liv l dn er cu b ar sp ki om pe w ce m u st up lo m What is happening with 227Th progenies in vivo?  Pharmacokinetic & organ distribution in mice using 227Th / 223Ra discrimination to define in vivo 223Ra ingrowth [227Th]Th-Trastuzumab was administered iv to immunocompetent naïve female swiss/webster mice 227Th 223Ra (Bq/gram) 800 (Bq/gram) 24 h 150 24 h 3 days 700 3 days 125 7 days 600 7 days 14 days 500 14 days 100 400 75 300 50 200 100 25 0 0 d t s r n y h I I le t e ar e m fa w e n w od I y um ne e h gs t ng ac rG rt r I oo ee G sc on ro G fa G cu e liv ne ee cl dn ac a he er ro bl l ar liv n o bo lu pe b he us u r ki er c sp om ce ar d om lu l bl pe sp ce w m ki st w up m m lo m up lo st 227Th immune construct shows: elevated liver, decreasing spleen, low bone uptake 223Ra in vivo ingrowth noticeable accumulated in spleen and bones, while liver stayed low  Theranostic capability: Trastuzumab labeled 227Th or 89Zr using identical chemical precursor 227Th 89Zr %Injected Actvity/gram  SD (227Th & 89Zr) 20 24 h 227Th 24 h 89Zr 227 15 14 d Th 89 14 d Zr 10 ✱ 5 ✱ ✱ 0 e n w I od y um ne h gs rt r t I G fa G e ne ee cl ac a ro liv n o bo he us r er c ar d om lu l bl pe sp ce ki w m m up lo st  Quality Control of Alpha Emitting Isotopes Challenging? Yes… but not impossible Requires similar equipment to an imaging radiopharmaceuticals laboratory: TLC reader, dose calibrator and gamma counter HpGe: High Purity Germanium detector is needed Alpha Spectrometer (in some instances) Adjustments are required for each isotope and considerations for progenies: respecting secular/transient equilibrium, conducting calibrations and cross-instrument checks Team with multi disciplinary skills  Acknowledgements and Funding Thorek Laboratory and the Program for Quantitative Molecular Therapeutics Nadia Benabdallah, PhD Peng Lu, B.Eng. Modulation Therapeutics, Inc. Hanwen Zhang, PhD Mark McLaughlin, PhD Michael McDevitt, M.Eng., PhD Tim Hazlehurst Abbie Hasson, B.Sc., M.Eng. Bradley J. Beattie, PhD Moffit Cancer Center, FL Amanda Fears, B.Sc. John L. Humm, PhD David Morse, PhD Wen Jiang, PhD University of Iowa Zhiyao Li, B.Sc. Thad Wadas, PhD Lucy Summer, B.Sc. Darpan N. Pandya, PhD Ryan Unnerstall, B.Sc. Daniel LJ Thorek, PhD Darren Magda, PhD David Tatum, PhD Department of Chemistry – Wash. U Lee Sobotka, PhD Cyclotron Facilities Tangadanchu, Vijai Kumar Reddy Greg Gaehle, MS Reiko Oyama, RPh, MS, BCNP Patrick Zerkel, MS James Robben,BS Sally Schwarz, RPh, MS, BCNP Mike Nickels, PhD Pamela K. Woodard, MD Dr. Wahl laboratory Shim, Kyuhwan, PhD Mark Longtine, PhD Richard L. Wahl, MD DOE – PNNL: Chuck Soderquist, PhD