Quality data collection for clinical translation

Deliverable D4.2 Quality data collection for clinical translation This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 101008571 (PRISMAP). This document reflects only the view of the author(s). The Agency is not responsible for any use that may be made of the information it contains. Deliverable D4.2 ii data collection for clinical translation Project Acronym PRISMAP Project Title The European medical isotope programme: Production of high purity isotopes by mass separation Grant Agreement No. 101008571 Topic INFRAIA-02-2020: Integrating Activities for Starting Communities Project start date 01 May 2021 Nature Report Dissemination level Public Due date M51 Date of delivery M51 Lead partner MUI Contributing partners ARRONAX, CHUV, DTU, CERN, ILL, IST-ID, JRC, KULeuven, NCBJ, PSI, SCK CEN, TUM Authors Clemens Decristoforo, Hessam Roustaei Firouzabad (MUI); Renata Mikolajczak, Malgorzata Zoltowska (NCBJ); Talip Zeynep, Susanne Geistlich, Nick van der Meulen (PSI); Calogero D'Alessandria (TUM); Thierry Stora (CERN); Cécile Bourdeau, Ferid Haddad (ARRONAX); David Viertl (CHUV); Claire Deville, Clive Naidoo, Kristina Søborg Pedersen, Mikael Jensen (DTU); Ulli Köster (ILL); Lurdes Gano (IST-ID); Frank Bruchertseifer (JRC); Maarten Ooms, Michiel Van de Voorde (SCK CEN) Reviewer Frederic Cleeren (KULeuven) António Paulo (IST-ID) Point of Contact Clemens Decristoforo Institution MUI E-mail [email protected] Phone +4351250480951 © PRISMAP 2021. This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. Deliverable D4.2 iii Revision History Version Date Author Comment 0.1 16.01.2025 C. Decristoforo First draft 0.2 26.04.2025 C. Decristoforo Second draft 0.3 02.05.2025 C. Decristoforo Upload on Google Drive 0.4 29.05.2025 C. Decristoforo Removal from Google Drive, Draft for WP4 review 0.5 09.06.2025 C. Decristoforo+WP4 After final review WP4 0.6 23.06.2025 C. Decristoforo + Reviewers Reviewers input (F.Cleeren, A.Paulo) 0.7 21.07.2025 C. Decristoforo Update with new Position Paper on GMP 0.8 21.07.2025 V. Gobry Formatting 0.9 22.07.2025 K. Leufgen Review, version for review by the General Assembly 0.95 28.07.2025 C. Decristoforo Revision after review by the General Assembly 1.0 28.07.2025 K. Leufgen Final version, approved by the General Assembly Deliverable D4.2 iv Contents Abbreviations, Participant short names vi Abbreviations vi Participant short names vii List of Figures viii List of Tables viii Summary 1 1. Introduction 2 1.1 Standardisation and harmonisation of Quality Data within PRISMAP 2 1.2 Scope of deliverable 4.2 2 1.3 Deviations from the original plan 2 2. General activities related to quality of novel radionuclides 3 2.1 Changes in the pharmaceutical regulatory framework in the EU 3 2.2 The revision of the pharmaceutical Directive and the regulatory definition of novel radionuclides 3 2.3 The EMA guideline on Radiopharmaceuticals and novel radionuclides 4 2.4 An EMA Innovation Task Force meeting on the quality of novel radionuclides and radiopharmaceuticals therefrom 4 2.4.1 EMA-regulatory expert’s opinions on the definition of a novel radionuclide in a clinical trial and the requirement for Marketing authorisation 5 2.4.2 EMA-regulatory expert’s opinions on the GMP requirement of a novel radionuclide in a clinical trial 6 2.4.3 EMA-regulatory expert’s opinions on the Radionuclidic and Radiochemical Impurity levels of novel radionuclides 6 2.4.4 EMA-regulatory expert’s opinions on the criteria for a reliable and accurate determination of the amount of radioactivity in a radiopharmaceutical? 6 2.5 GMPAnnex 3 questions and answers from the EMA GMP/GDP IWG on the GMP requirements for novel radionuclides 6 2.6 Activities of PRISMAP with the IAEA 8 3. Quality Data from specific radionuclides from PRISMAP`s portfolio 8 3.1 Terbium – 161 8 3.1.1 GMP considerations in the production of radiopharmaceuticals using Terbium-161 8 3.1.2 Quality requirements for Terbium-161 – experiences from the production in the PRISMAP project 9 3.2 Copper-64 13 3.2.1 Quality considerations in the production of radiopharmaceuticals using Copper-64 13 3.2.2 Quality requirements for Cu-64 – experiences from the production in the PRISMAP project 15 3.3 Actinium-225 16 3.3.1 Quality considerations in the production of radiopharmaceuticals using Actinium-225 16 Deliverable D4.2 v 3.3.2 How to define quality specifications for Actinium-225 18 4. Conclusions 20 5. References 21 6. Annexes 23 Annex 1: Response to the European Commission`s Proposal on the revision of the EU general pharmaceuticals legislation 23 Annex 2: Letter to Members of the European Parliament regarding the revision of the EU general pharmaceuticals legislation 25 Annex 3: PRISMAP`s comments to the Concept paper on the revision of the 'Guideline on Radiopharmaceuticals-Revision 1` (Extract) 26 Annex 4: Statement PRISMAP Radionuclides and GMP-Annex 3 and EMA response 29 Annex 5: Position Paper on GMP requirements for novel radionuclides 34 Annex 6: Report from the Interlaboratory Comparison study (ILC) of 64Cu 40 Deliverable D4.2 vi Abbreviations, Participant short names Abbreviations AMA Apparent Molar (Radio)Activity API Active Pharmaceutical Ingredient CA Consortium Agreement CoA Certificate of Analysis DoA Description of Action EANM European Association of Nuclear Medicine EDQM European Directorate for the Quality of Medicines EMA European Medicines Agency ESA Effective Specific (Radio)Activity EU European Union GA Grant Agreement GMP Good Manufacturing Practices HPG High Purity Germanium (detector) IAEA International Atomic Energy Agency ICP-MS Inductively coupled plasma mass spectrometry ICP-OES Inductively coupled plasma optical emission spectroscopy ILC Interlaboratory Comparison (Akronnym for a study conducted within WP4) IMPD Investigational Medicinal Product Dossier ITF Innovation Task Force ITLC Instant Thin layer Chromatography IWP Inspectors Working Group MA Molar (Radio)Activity MAuth Marketing Authorisation Pharm Eur European Pharmacopoeia QC Quality Control QWP Quality Working Party RCP Radiochemical Purity RNP Radionuclidic Purity SOP Standard Operating Procedure TLC Thin layer Chromatography Deliverable D4.2 vii Participant short names CERN European organisation for nuclear research NPL National Physical Laboratory PSI Paul Scherrer Institut CEA Commissariat à l’énergie atomique et aux énergies alternatives IST-ID Associação do Instituto Superior Técnico para a IST-ID Investigação e Desenvolvimento DTU Danmarks Tekniske Universitet CHUV Centre hospitalier universitaire vaudois GANIL Grand Accélérateur National d’Ions Lourds SCK CEN Studiecentrum voor Kernenergie / Centre d'étude de l'énergie nucléaire ARRONAX Groupement d’intérêt public ARRONAX ESS European spallation source ERIC TUM Klinikum rechts der Isar der technischen Universität München KULeuven Katholieke Universiteit Leuven MedAustron Entwicklungsund Betriebsgesellschaft MedAustron GmbH SCIPROM SCIPROM Sàrl MUI Medizinische Universität Innsbruck ILL Institut Max von Laue - Paul Langevin JRC JRC -Joint Research CentreEuropean Commission NCBJ Narodowe Centrum Badań Jądrowych GSI GSI Helmholtzzentrum fr Schwerionenforschung GmbH LU Latvijas Universitāte INFN Istituto Nazionale di Fisica Nucleare UiO Universitetet i Oslo Deliverable D4.2 viii List of Figures Figure 1. Title page of the EMA Innovation Task Force Briefing meeting report from March 31st, 2023 5 Figure 2. Excerpt from the letter as response to PRISMAPs problem statement on GMP for the production and use of novel radionuclides 7 Figure 3. Manufacturing strategy for clinical 161Tb-radiopharmaceuticals developed and approved by the Swiss national health-authority for first clinical trials in-human 9 Figure 4. Manufacturing strategy for 64Cu-radiopharmaceuticals used for compassionate use and clinical trials developed and approved by the Danish Health Authority 14 Figure 5. Manufacturing strategy for Ac-225 from Th-232 mass separation process (from MEDICIS) 17 List of Tables Table 1. Quality Control results of Tb-161 radiolabelling solution produced at PSI for PRISMAP users 10 Table 2. TLC results of radiochemical purity testing of Tb-161 Terbium(III) produced at NCBJ for PRISMAP users 11 Table 3. Metals in Tb-161 solutions determined by ICP OES of batches produced for PRISMAP users at NCBJ. 11 Table 4. Summary quality data of relevant parameters for radiolabelling performance – outcome of Cu64 ILC study 16 Summary Radiopharmaceuticals are considered Medicinal Products, thereby they must be prepared and applied within the regulated area of pharmaceuticals. This includes radionuclides, which have seen extraordinary advancements in research and development over the last decade regarding theranostics. Over the past four years WP4 of the PRISMAP project on Harmonisation and Standardisation has analysed the regulatory landscape in relation to chemical and pharmaceutical quality aspects of the use of novel radionuclides in radiopharmaceuticals, monitored the regulatory developments in this field and tried to communicate the challenges for clinical translation. Within these activities the revision of the pharmaceutical directive was reviewed and comments from PRISMAP provided. Contact with EMA resulted in dedicated events providing the radionuclides community point of view on GMP and other pharmaceutical regulations for novel radionuclides and input in IAEA activities were given. This is described in this deliverable. In the second part of this deliverable specific quality data and consideration for three major radionuclides from the PRISMAP portfolio, Tb-161, Cu-64 and Ac-225 are described. In particular, GMP requirements in the production and use of these radionuclides are addressed, and also specific quality issues and activities are described. This includes an outcome from an interlaboratory comparison study on Cu-64, comparison of quality data for Tb161 provided within PRISMAP and specific developments on the quality of Ac-225. Overall, this deliverable provides a summary and insight into many aspects related to harmonisation and standardisation of the quality of novel radionuclides. Deliverable D4.2 8 With respect to the applicability of GMP Annex 3 to IMPs and novel radionuclides, exemptions were confirmed, however, indicating that “Member States shall make the processes set out in paragraph 5 subject to appropriate and proportionate requirements to ensure subject safety and reliability and robustness of the data generated in the clinical trial. They shall subject the processes to regular inspections” as outlined in the Clinical Trial Regulation leading to the situation that individual member states may still require compliance with GMP, therefore applicability of Annex 3 for IMP should remain. Regarding novel production methods, a general exemption from GMP is also not supported but can be considered on a case-by-case basis. Overall, the regulatory experts agreed that an update of GMP Annex 3 is needed in light of the scientific process and they will seek and welcome the views of stakeholders during this revision phase. The topic of regulatory GMP requirements in the production of novel radionuclides and the activities of the PRISMAP consortium were recently summarised in a position paper published in July 2025 (7), that is found in Annex 5. 2.6 Activities of PRISMAP with the IAEA WP4 also participated in a technical meeting on “Health and Pharmaceutical Regulations for Radiopharmaceuticals”, at the International Atomic Energy Agency in Vienna, Austria, 6 to 10 March 2023, where PRISMAP’s activities were presented. The overall problems of pharmaceutical regulations with radiopharmaceuticals (including novel radionuclides) were discussed and a common framework was elaborated. This work resulted in a joint paper published in EJNMMI Radiopharmacy & Chemistry in January 2024 (8) with the support of PRISMAP. This is a valuable reference for users of PRISMAPs radionuclides in terms of regulatory issues. 3. Quality Data from specific radionuclides from PRISMAP`s portfolio 3.1 Terbium – 161 3.1.1 GMP considerations in the production of radiopharmaceuticals using Terbium-161 3.1.1.1 Introduction Current production of Tb-161 (9–11) is based on the irradiation of Gd-160 oxide targets in high neutron flux reactors based on the (n,γ) reaction to Gd-161 which, subsequently, decays to Tb-161. After separation from gadolinium via a cation exchange and/or extraction chromatography the n.c.a. Tb-161 is obtained in a dilute hydrochloric acid solution suitable for radiolabelling. The specific activity and radiochemical properties of Tb161 are similar to the currently widely used Lu-177. Even though Tb-161 produced within PRISMAP has not been directly used in clinical trials, examples from Tb-161 produced for nationally approved clinical trials provide guidance on how chemical grade Tb-161 may be introduced into GMP compliant radiopharmaceutical production. Deliverable D4.2 9 3.1.1.2 Example of a process for Terbium-161 radiopharmaceuticals Figure 3. Manufacturing strategy for clinical 161Tb-radiopharmaceuticals developed and approved by the Swiss national health-authority for first clinical trials in-human A process for the production of Tb-161 and subsequent manufacturing of a Tb-161 radiopharmaceutical for first in human clinical trials is shown in Figure 3. The target materials for irradiation are subject to appropriate quality control, all materials undergo a cleaning process and the target is prepared in a clean room (it was agreed with the competent authority to use an existing clean room for other radiopharmaceutical production processes in these early-phase clinical trials) under GMP conditions and stored under quarantine. The target is considered a starting material and undergoes a quality control release procedure before being sent out to irradiation. The irradiation is performed outside a GMP-quality framework under predefined conditions. The irradiated target is handled under non-GMP controlled conditions in a manual predefined process until the final chemical purification step. The solution containing the desired separated radionuclide is defined as an API starting material and the final purification is performed in a hot cell in a clean room under GMP control and then introduced into the final radiopharmaceutical manufacturing process towards final aseptic dispensing of the final IMP for use in the clinical trial. Quality control of the final product and release is performed according to GMP requirements. This process has been approved by the competent authority for clinical trials which started in 2023. The specific considerations of certain processes being outside the GMP framework cannot be generalised for radiopharmaceutical production. However, it is essential to consider less stringent requirements in the early phases of the development, which, in this case, has been accepted by the pharmaceutical authority. 3.1.2 Quality requirements for Terbium-161 – experiences from the production in the PRISMAP project The quality specification and testing methods for Tb-161 cannot be fully harmonised within PRISMAP at the current stage of development. However, Tb-161 produced by different PRISMAP partners has reached a stage of development, where main quality parameters can be defined and methods for determination be recommended. These are described further on with some “real world data” from batches of Tb-161 produced and shipped for selected projects within PRISMAP. Overall quality considerations can be related to the Deliverable D4.2 10 standards provided by the European Pharmacopoeia for Lutetium-177 in the monograph Lutetium (177Lu) solution for radiolabelling No 2798 (12), with very similar characteristics and quality considerations as compared to Tb-161. Specific considerations on the quality of Tb-161 for radiolabelling and Tb-161 radiopharmaceuticals can be found in (9–11,13). Quality considerations regarding activity measurement and calibration factors are summarised in Table 1. Table 1. Quality Control results of Tb-161 radiolabelling solution produced at PSI for PRISMAP users Date of Chemical Separation Parameter Requirement 10.11.2023 09.04.2024 28.05.2024 RCP (HPLC)* ≥99% 99% 100% 100% Identity (уspectrometry) 74.6 ± 1 keV 87.9 ± 1 keV 103.1 ± 1 keV 106.1 ± 1 keV Complies Complies Complies RNP at Expiry Date (уspectrometry) Tb-160 ≤ 0.1% Tb-161 ≥ 99.9% < 0.1% > 99.9% < 0.1% > 99.9% < 0.1% > 99.9% * AMA test: radiolabelling of DOTATOC at 100 MBq/nmol 3.1.2.1 Identity tests The identity of Tb-161 can be ensured by gamma spectrometry as the characteristic gamma emissions can be detected. The most prominent gamma photons of Tb-161 have an energy of 25.7keV (23%), 48.9 keV (17.0%), 74.6 keV (10.2%), 87.9 keV (0.18%), 103.1 keV (0.1%) and 106.1 keV (0.07%). Gamma photons with an energy of 292.4 keV (0.06%) and 550.3 keV (0.04%) are also present. High resolution gamma spectrometry can ensure presence of all peaks described for the production and release of the product, Table 1 shows compliance of batches of Tb-161 produced within PRISMAP with these specifications. However, the low abundance and close energies of several emissions makes it impractical for users in hospitals to identify all lines e.g., with an available NaI-gamma spectrometer. Therefore, users may consider to use the main gamma peaks at 48.9 keV and 74.6 keV as a main identification factor, based on risk assessment and considerations of other radionuclides used in the respective facility. A detailed description of the properties and interfering gamma emissions from other radionuclides can be found in the supplementary information of (13). 3.1.2.2 Radionuclidic purity The main radionuclidic impurity to be considered is Tb-160 with a half-life of 72.3 days, which is co-produced in the irradiation of the 160Gd2O3 target material via various routes (13). Table 1 shows the Tb-160 content at the time of expiry (4 weeks after end of bombardment) of several batches of Tb-161 produced for PRISMAP users by PSI. These data indicate that a limit of 0.1% of Tb-160 can be reached even with a shelf life of several weeks, such an impurity level will lead to an additional radiation dose of 0.35% for the patient (13). However, it is important for the user to be aware of this impurity and the specifications of certain production, as it will have a considerable effect on the waste management, especially when used clinically in hospitals. Also, late measurements e.g., in gamma counters, have to take this into account. Deliverable D4.2 11 3.1.2.3 Radiochemical purity The radiochemical purity can be determined in analogy to Lu-177 as described in the respective monograph (12). A simple ITLC test using an acidified NaCl-solution allows the separation of the Lu-177 Lutetium(III) ion from bound or hydrolysed Lu-177. Table 2 shows compliant results of batches produced for PRISMAP users at NCBJ. Table 2. TLC results of radiochemical purity testing of Tb-161 Terbium(III) produced at NCBJ for PRISMAP users NCBJ Batch Radiochemical purity [%] 01/24 99.9 02/24 99.6 03/24 99.3 04/24 99.5 01/25 99.2 3.1.2.4 Metal determination The determination of selected metals in radionuclide solutions as a release-criteria has been a matter of debate. Dedicated equipment such as ICP-MS or ICP-OES allowing the determination of metals in highly radioactive solutions is a challenge for many laboratories (Table 39. Additionally, the question arises which metals are relevant for the use of a radionuclide for radiolabelling. Toxicity of heavy metals is no concern due to the very low amounts of total metals present. The main quality concern is the radiolabelling performance of the radionuclide, which depends on the specific chelator employed, the molar radioactivities and the total amounts of radioactivity required for a particular application. In the case of Lu-177, the Pharm Eur monograph describes the determination of Lu, Cu, Fe, Pb and Zn and specifies respective limits. However, in a recent draft for the monograph on 68Ga for radiolabelling it is proposed to replace the test for metals by the Apparent Molar Activity (AMA) test (see 3.1.2.5. below). This could also be applied for Tb-161 and other radiometals. In early phase clinical trials, the establishment of the metallic impurities in the validation and release based on AMAtests can be considered acceptable. Table 3 shows results from metal determination of metals by ICP-OES in Tb-161 batches produced at NCBJ for PRISMAP users. Table 3. Metals in Tb-161 solutions determined by ICP OES of batches produced for PRISMAP users at NCBJ. NCBJ Elements 01/24 02/24 03/24 04/24 01/25 [ppm] Cu 0.111 < 0.892 1.92 < 0.586 < 3.58 Co 0.185 < 0.529 < 1.68 < 0.647 < 0.376 Fe 0.167 < 0.068 < 0.117 < 0.405 < 0.372 Ni < 0.116 < 0.839 < 2.00 < 0.376 < 0.720 Pb < 0.439 < 0.574 < 2.71 < 1.90 < 1.13 Zn 0.431 0.823 4.25 1.58 1.52 Gd 5.41 6.41 12.5 4.25 < 3.10 Tb 3.76 < 2.10 1.99 < 1.77 < 4.51 Dy 0.224 < 0.343 0.637 0.425 < 3.12 Deliverable D4.2 12 3.1.2.5 Apparent molar activity (AMA) determination A radiolabelling test with the radionuclide using a suitable chelator allows to investigate the presence of metals interfering in the radiolabelling process of the radiopharmaceutical preparation and can therefore be a substitute for the determination of individual metals as a quality test. The test can be useful for the producer of the radionuclide, but more importantly for the user. This test includes a radiolabelling step, followed by the determination of the radiolabelling yield, typically by a simple TLC-test. It can be designed as a titration of decreasing amounts of ligand to the radionuclide to see at which molar concentration the radiolabelling fails to meet a predefined limit (e.g., 95%). This requires a quite laborious test regime. Therefore, once the respective concentration has been defined in a validation, the test can be simplified to work as a limit test using a single concentration of the ligand. The user will use the same type of ligand as is used for the radiopharmaceutical in question. The producer of the radionuclide will use a standard chelator, in most cases DOTA or a DOTA derivative. Below is an example of a potential protocol of an AMA test for Tb-161 (modified from a protocol applied at NCBJ, not validated):  Test solution. Prepare a solution containing 2 nmol DOTATATE in 52 µL Ascorbic acid buffer. Add a solution of 200 MBq of Tb-161 in dilute hydrochloric acid and make up to a final volume of 500µl with ascorbic acid buffer. Heat to 95°C and react for 20 minutes.  Plate: TLC silica gel plate R; use a glass-fibre plate 1 .  Mobile phase: 1M ammonium acetate and methanol in a volume ratio of 1:1  Application: about 3 µL  Development: immediately, over a path of at least 7 cm.  Drying: in oven, 80°C  Detection: suitable detector to determine the distribution of radioactivity.  Retardation factor: [Tb-161]Tb-DOTA-TATE ≥0.9, [Tb-161]Terbium(III) ion ≤0.2  Measure the relative amount of activity of each plate.  Calculate the percentage of the radioactivity due to [Tb-161]Tb-DOTA-X of the preparation  Limit: ≥ 95% of the total radioactivity due to [Tb-161]Tb-DOTA-TATE 3.1.2.6 pH The pH can be determined using an indicator strip and should ensure the acidic character of the Tb-161 solution for radiolabelling, in which the Tb3+ ion is present. Higher pH holds the risk of generating hydrolysis products. A pH between 1 and 2, as described in the monograph Lutetium (Lu-177) solution for radiolabelling No 2798 (12), can be considered a suitable specification in view of the typical acid concentrations used in the final formulation of Tb-161 for radiolabelling. 3.1.2.7 Sterility and endotoxins There is no formal necessity from a quality aspect to have a sterile solution of the radionuclide for radiolabelling as long as the preparation process of the final radiopharmaceutical ensures that the final product is sterile. This has to be confirmed in a documented risk assessment by the user of the radionuclide. Radionuclide producers may consider providing a sterile product for wider clinical applications. In case the product is not sterile, the bioburden of the radionuclide should be established in the process validation of the radiopharmaceutical preparation. This can include determination of endotoxins. The limits of endotoxins in the Tb-161 radiolabelling solution will depend on several factors: the total volume of radiolabelling solution used, the maximum volume used for one patient dose and whether the production process of the radiopharmaceuticals includes steps with the potential of reducing the endotoxin levels. Also, the contribution to the endotoxin burden in the final product by other materials used in the synthesis process has to be taken into account. Therefore, it is not possible to give a general recommendation of Endotoxin 1 iTLC-SG (Agilent Technologies, SGI0001) is suitable Deliverable D4.2 13 values for a Tb-161 solution for radiolabelling, but again, the Lu-177 can serve as a reference which states “less than 175 IU/V, V being the maximum volume to be used for the preparation of a single patient dose, if intended for use in the manufacture of parenteral preparations without a further appropriate procedure for the removal of bacterial endotoxins.”(12) For early stages of development and early phase clinical trials the requirements for tests on endotoxins, bioburden and sterility of the Tb-161 solutions for radiolabelling as release criteria should be viewed within a risk assessment taking validation data, radiation safety aspects and the final production process of the radiopharmaceutical into account. 3.1.2.8 Labelling The vial-label of a Tb-161 solution for radiolabelling should indicate:  that the solution is not for direct administration to humans;  the maximum volume that can be used for the preparation of a single patient dose;  the concentration of hydrochloric acid;  that the solution is intended for use in the preparation of Terbium-161-labelled radiopharmaceuticals; 3.2 Copper-64 3.2.1 Quality considerations in the production of radiopharmaceuticals using Copper-64 3.2.1.1 Introduction In contrast to Tb-161, Cu-64 was provided within PRISMAP by different methods. Cu-64 from DTU and PSI originated from proton irradiation of enriched Ni-64 targets, whereas ARRONAX used deuteron irradiations of enriched Ni-64 targets. Additionally, reactor-produced Cu-64 with low specific activity was available from NCBJ. The different production pathways of Cu-64, as well as quality considerations have been summarised as an outcome of a Coordinated Research Project by the IAEA in 2022 (14). PRISMAP`s Cu-64 has not been directly used in clinical trials, but the experience within PRISMAP will contribute to standardizing the quality of Cu-64 for radiopharmaceutical preparations within the Pharm Eur, where a monograph on Cu-64 is currently being drafted. Deliverable D4.2 14 3.2.1.2 Example of a process for Cu-64 radiopharmaceuticals Figure 4. Manufacturing strategy for 64Cu-radiopharmaceuticals used for compassionate use and clinical trials developed and approved by the Danish Health Authority Figure 4 describes the overall manufacturing scheme for in-house production of a Cu-64 radiopharmaceutical for human use requiring compliance with GMP. The final radiopharmaceutical is manufactured in clean rooms according to a GMP-compliant (GMP Part I) aseptic manufacturing process with final sterile filtration and released by a qualified person based on validated QC methods. Cu-64 for this process is considered an API starting material, its production is, in many parts, under GMP control, the whole process is described in SOPs and detailed manufacturing records are kept. The target material is from qualified suppliers with defined specifications and it is released based on defined identity and purity tests. Other reagents required are also tested against predefined specifications, the target is prepared in a radiochemical laboratory in a controlled area based on predefined validated procedures, and the Ni-64 plated target becomes a starting material in the production process. Irradiation in the cyclotron is outside the GMP framework, but well controlled with defined irradiation conditions and times, and having two cyclotrons qualified for the process. The cyclotron`s maintenance and repairs are well-documented and traceable. After irradiation, the target is transferred to a controlled area and handled under strictly defined and validated procedures from dissolution, and ion exchange purification followed by batch fractionation in pharmaceutical grade glass vials, in which the solution is evaporated to dryness. These vials are the API starting material for the manufacturing of the radiopharmaceutical. QC samples of the Cu-64 solutions are taken from the batch fractionation solution and tested against predefined specifications using validated methods. The final Cu-64 vials are measured for activity and kept in quarantine until activity concentration, identity and predefined metal limits are ensured. The process up to release of Cu-64 as an API starting material follows GMP Part II (“Basic Requirements for Active Substances used as Starting Materials”), the following radiopharmaceutical preparation is according to GMP Part I (“Basic Requirements for Medicinal Products”). Deliverable D4.2 15 Overall, this setup provides a GMP-compliant process that allows the use of Cu-64 for radiopharmaceutical preparations both for routine clinical use as well as for investigational Medicinal products. Production pathways with a reduced coverage by GMP-compliant processes will be acceptable, especially if Cu-64 is used for the production of investigational radiopharmaceuticals. 3.2.2 Quality requirements for Cu-64 – experiences from the production in the PRISMAP project 3.2.2.1 Identity tests: The identity of Cu-64 can be assured by the characteristic gamma emissions with the most prominent gamma photons representing the 511 keV peak from the positron annihilation, additionally a 1345.8 keV gamma line (6%) ensuring unequivocal identification. The additional determination of the half-life, as it is required for pure positron emitters like F-18 may not be required by users of the radionuclide due to these characteristics. 3.2.2.2 Radionuclidic purity The main radionuclidic impurities for the production pathways used within PRISMAP are cobalt radioisotopes, in particular Co-56, Co-57 and Co-58. These were specified for Cu-64 from PRISMAP production to be below 0.01% at the time of production. Exact specifications depend on application and the defined shelf life of the Cu-64 for a specific production and needs to be established based on a risk assessment. 3.2.2.3 Radiochemical purity In order to evaluate the presence of the ionic forms of the radiometal isotopes and the absence of colloidal species, radio thin layer chromatography (TLC) analysis of the purified CuCl2-64 is strongly recommended. This can be done using ITLC-SG as stationary phase and 0.1M Citrate buffer pH 4-4.5, whereby the retardation factor for Cu-64 in colloidal form is <0.2 and for [Cu-64]Copper(II) ion 1.0 (14). 3.2.2.4 Metal determination & Apparent Molar Activity (AMA) determination (ILC study) Similar to Tb-161, trace metals are an important factor in relation to the radiolabelling properties for a Cu64 production. Important metals include, Fe, Ni, Zn and Cu. The exact specifications for these metals again will depend on the individual application. A radiolabelling test to determine the apparent molar activities or the concentration in which a typical chelator still provides high radiolabelling yields therefore is also recommended. To compare the quality of Cu-64 provided by different PRISMAP users an Interlaboratory Comparison (ILC) Study was initiated. One central laboratory (ARRONAX) received samples of Cu-64 produced by other members of the PRISMAP consortium (DTU, PSI, NCBJ). A standardised protocol was used to determine the activity, Apparent Molar Activity (AMA) and Molar Activity (MA). Additionally, metal determination of major metals (Co, Cu, Fe, Ni, Zn) by ICP-OES was available for some test batches. The AMA test was designed as a titration assay using decreasing concentrations of DOTA as a chelator for Cu-64. Between February 2022 and February 2024 five different batches were analysed at ARRONAX (from each producer, 2 batches from ARRONAX itself). The main results of the study are summarised in Table 4. The low molar activities found for Cu-64 from NCBJ match the reactor production pathway, all values were below 0.1 MBq/nmol. Production from other producers resulted in high molar activities of Cu-64 with values >40 MBq/nmol. For most productions the AMA was considerably lower than the MA, reflecting the influence of other metals than Copper. The lower AMA and MA for production of ARRONAX and PSI reflect the lower activities produced in the respective batches decreasing the limit of quantification. Overall, these results indicate the expected high molar activities reached for Cu-64 suitable for radiopharmaceutical preparations. Deliverable D4.2 16 Experimental details of the study can be found in Annex 6. Table 4. Summary quality data of relevant parameters for radiolabelling performance – outcome of Cu-64 ILC study Institute Production Date AMA [MBq/nmol] DOTA titration MA [MBq/nmol] ICP ESA [MBq/nmol] ICP DTU 64Ni(p,n)64Cu 14/02/2022 1060 3225* 118** PSI 64Ni(p,n)64Cu 10/10/2023 53 197 45 ARRONAX 64Ni(d,2n)64Cu 07/02/2024 78 72 (69) 48 NCBJ 63Cu(n,γ)64Cu 31/01/2024 0.012 Nd*** 0.014 * determined from DTU ICP data, Calculated from Cu 0.31nmol/GBq ** determined from DTU ICP data, Calculated from Ni+Zn+Fe+Cu 8.47nmol/GBq *** assumed that only Cu-is present n.d.: not determined 3.2.2.5 pH, Sterility and endotoxins Similar considerations as described for Tb-161 have to be taken into consideration 3.2.2.6 Labelling The label of a Cu-64 solution for radiolabelling should indicate:  that the solution is not for direct administration to humans;  the maximum volume that can be used for the preparation of a single patient dose;  the concentration of hydrochloric acid; in case it shipped in dry form this has to be indicated (including additional information on how to dissolve the preparation)  that the solution is intended for use in the preparation of Cu-64-labelled radiopharmaceuticals; 3.3 Actinium-225 3.3.1 Quality considerations in the production of radiopharmaceuticals using Actinium-225 3.3.1.1 Introduction Actinium-225 has shown great promise in a number of clinical applications (15), however no standardisation or harmonisation of production and quality parameters has been achieved so far. One reason is the variety of proposed production routes of Ac-225 (16). In 2018 the IAEA initiated a Coordinated Research Project on Ac-225 to support their member states in the development of Ac-225 radionuclide and radiopharmaceutical production. A recent Technical Document from IAEA (17) provides technical support on production and quality control of Ac-225 radiopharmaceuticals. Within PRISMAP, Ac-225 was provided by 2 production routes. The main route is Ac-225 from generator production via Thorium-229 decay. Alternatively, Ac-225 was produced by mass separation after spallation reaction of Thorium-232 using high-energy protons. Deliverable D4.2 17 3.3.1.2 Example of a process for Actinium-225 radiopharmaceuticals Figure 5. Manufacturing strategy for Ac-225 from Th-232 mass separation process (from MEDICIS) Figure 5 describes the preparation of Ac-225 from Mass separation as performed by CERN at MEDICIS for PRISMAP supply, a description is outlined below, more details can be found in (18) Target preparation: The target material suitable for isotope mass separation must adopt specific chemical and physical forms, different from those developed at cyclotron or nuclear reactor settings. For radionuclides where the targets must be irradiated at a reactor or cyclotron prior to (offline) mass separation, a processing step (e.g., chemical separation) is required to introduce the material in an isotope mass separation production unit. This requires reaching appropriate physical structures. In the case of thorium-based target materials irradiated at CERN applied to the production of mass-separated 225Ac radionuclide samples, porous materials and composites must be designed to be able to sustain extreme temperatures of more than 2000 °C. This can take the form of porous pellets of ThC2+C2, ThO2 powders, felts of ThO2 micrometric fibres. Irradiation at CERN: The exploitation of high-energy particle beams to irradiate targets requires infrastructures that are available at large accelerators and research facilities. The target irradiation step requires suitable remote transport and handling systems, diagnostics tools, exploiting thick natural target materials of several grams in dedicated irradiation stations. The CERN PS Booster, providing 1.4 GeV protons, produces a large range of reaction channels in targets (made of various materials depending on the nuclide desired). As a result, a vast number of radionuclides are produced in the thick targets that, subsequently, need to undergo a physical mass purification step. Examples include irradiating targets of 25-µm-thick foil rolls (300 g) used for terbium radioisotopes and Ba-128. When the mass separation step is performed during the target irradiation, the purification step is called ISOL (Isotope mass Separation OnLine), while if the purification occurs after irradiation directly on the target, or after a radiochemical purification step, the purification is called offline mass separation or batch(-mode) isotope mass separation. Transfer of the target in the isotope mass separation unit: The transfer of the target containing the vast number of radionuclides proceeds with operation performed in suitable shielded nuclear laboratories, with the exploitation of remote handling systems and equipped with ventilation and gas handling decay/monitoring systems, in laboratories serving the multiple research communities. The target and ion Deliverable D4.2 24 Deliverable D4.2 25 Annex 2: Letter to Members of the European Parliament regarding the revision of the EU general pharmaceuticals legislation Dear MEP The European Commission recently put forward a 'pharmaceutical package' to revise the EU pharmaceutical legislation; to foster innovation and to secure patient access to medicines. The PRISMAP-European Network for Medical Radionuclides consortium would like to highlight its impact on the future development and clinical access of radiopharmaceuticals based on innovative production of novel radionuclides. PRISMAP is a HORIZON 2020 funded project where the main goal is to provide a sustainable source of new high purity grade medical radionuclides for nuclear medicine. Involving from the onset upcoming major European infrastructures, PRISMAP provides a single-entry point for access to reliable production of medical radionuclides to researchers granted on an excellence selection basis. Currently the main source of radionuclides are research reactors, with several other technologies that use cyclotrons, linear accelerators and mass separators in use or under development. The different radionuclides and production technologies rely on highly specialised complex supply chains. In this process the current pharmaceutical regulatory framework for the scientific translational process from basic research to clinical application must to be considered. Therefore, PRISMAP supports the user community by rallying a call for EU pharmaceutical standardisation and harmonisation in the use of novel radionuclides for radiopharmaceutical development Every year, more than 9 million patients in Europe benefit from the use of radiopharmaceuticals in nuclear medicine; for unique diagnostic imaging procedures (SPECT, PET, PET/MR) and specific treatment targeting therapies (with a specific focus on cancer - more than 80% of all nuclear medicine therapies are related to cancer treatment), known as radiotheranostics, for a “see it, treat it ” patient personalised approach, holding the promise to represent a new pillar of cancer care. The radiopharmaceutical landscape has advanced significantly in the last 20 years with a diverse number of radiopharmaceuticals available on the European market and under research and development. In particular, the developments in innovative radionuclide production and availability of novel radionuclides, initiated and coordinated by the PRISMAP project, which provides the basis for access to new diagnosis and treatment options for patients in need. These advances now need to be adequately supported by a tailored regulatory framework in order to ensure Europe’s leadership position in this field. PRISMAP therefore, welcomes the European Commission's proposal for a revision, as well as the efforts and commitment of the European Parliament, to ensure that the regulatory framework for medicinal products, including radiopharmaceuticals based on novel radionuclides, is not only adapted to support the current practice, but also fosters innovation and secures patient access for the future. To support this aim, PRISMAP proposes to adapt EC`s proposal for a new directive 2023/0132 (COD) and to ameliorate it with the following aspects: 1. Definitions (article 4) should reflect today’s nuclear medicine and radiopharmacy practices. Updated definitions (substance, radiopharmaceuticals, kit, generator…) will lay the foundation of an updated regulatory framework with adjustments for unique aspects of novel Radionuclides. 2. Clarification of marketing authorisation requirements for the use of novel radionuclides 3. Clarification of the relation to BSSD and Pharma Directive. One may find that some points have already been addressed in the European parliament ENVI draft report from October 3rd 2023 (https://www.europarl.europa.eu/doceo/document/ENVI-PR-753470_EN.pdf), for more details of PRISMAP`s proposed changes please refer to the attached table. The proposed ameliorated adaptations will not only help to increase the level of compliance among EU Member States but will also support harmonisation across Europe, while also ensuring patient safety and patient access, by supporting robust radionuclide and radiopharmaceutical supply for advancements in innovation and research. Deliverable D4.2 26 Annex 3: PRISMAP`s comments to the Concept paper on the revision of the 'Guideline on Radiopharmaceuticals-Revision 1` (Extract) 1. General comments on the Concept paper on the revision of the Guideline on Radiopharmaceuticals Stakeholder name (to be repeated in all rows) General comment PRISMAP PRISMAP very much appreciates the initiative to revise the current guideline on radiopharmaceuticals, especially taking into account the tremendous developments in the field of radiopharmaceuticals and especially with theranostics and the great variety of novel radionuclides on the horizon for these applications. PRISMAP Even though PRISMAP is mainly concerned with innovation involving novel medical radionuclides, which at this stage will mainly be used in clinical trials, PRISMAP still would like to address this guideline for the following reasons: 1.) It is expected that many of the radionuclides now being addressed within the PRISMAP project will find their way into clinical development and some into marketing authorisation 2.) A lot of investment in Europe both by the public (nationally and EU) and the industry aims towards making novel production routes fit for medical use, e.g. involving mass separation or high energy particle accelerators 3.) Even though investigational use is not in its scope, the “Guideline of radiopharmaceutical” has been the only official EMA reference when it comes to quality of radiopharmaceuticals, and therefore found widespread consideration also in applications for investigational use, esp. by regulatory authorities. In the revision, either the scope of the guideline has to be more precise to specifically exclude investigational application or has to take into account that for investigational use other rules may apply (e.g. in relation to the required quality framework, validation etc.) PRISMAP In view of a recent ITF briefing meeting with EMA representatives PRISMAP has engaged in discussions on the following topics: 1.) Marketing authorisation requirements for novel radionuclides 2.) GMP requirements for production of novel radionuclides: Novel developments 3.) Radionuclidic and Radiochemical impurities: In particular the advent of radionuclides with complex decay chains (e.g. Ac-225) or where the decay product is the “active radionuclide (e.g. Pb-212 or Ba4.) A reliable and accurate determination of the amount of (radioactivity in a radiopharmaceutical to a traceable national or international metrology institute. PRISMAP Directive 2001/83, which forms the basis of the “Guideline on radiopharmaceuticals” is currently being revised, a new proposal has recently been published by the Commission. In reply to this new proposal, main stakeholders on radiopharmaceuticals including PRISMAP has proposed revision of definitions and other topics related to radiopharmaceuticals. In particular it has been suggested to revise the current definitions and especially the current legal meaning of “radionuclide precursor”, meaning that it will not be treated as a medicinal product as such. PRSMAP would like to stress that especially in the context of novel radionuclides, these should be seen as starting materials. The definition of radionuclide precursor with its requirement for marketing authorisation is a factor slowing down innovation in the field. This should also be reflected in the guideline Deliverable D4.2 27 Question 2. Specific comments on text 2.1. Introduction Line number(s) of the relevant text (e.g. 20-23) Stakeholder name (to be repeated in all rows) Comment and rationale Proposed guidance text 15-17 PRISMAP We agree with the need for revision especially with respect to novel developments of radionuclide production and radiopharmaceutical development 2.2 Problem statement Line number(s) of the relevant text (e.g. 2023) Stakeholder name (to be repeated in all rows) Comment and rationale Proposed guidance text 33 PRISMAP Even though the concept of radionuclide precursors being handled in the same way as medicinal products within Directive 2001/83, we believe that this concept should be modified and that radionuclides should be seen as starting materials in the process of radiopharmaceutical preparation, with appropriate considerations in terms of the required quality framework Delete “radionuclide precursor” 46-47 PRISMAP Potential revisions in the new directive should be considered 2.3 Discussion (on the problem statement) Line number(s) of the relevant text (e.g. 20-23) Stakeholder name (to be repeated in all rows) Comment and rationale Proposed guidance text 59 PRISMAP As outlined above radionuclides should be seen as starting materials Radionuclide precursors should be considered in module 3.2.S only 66 PRISMAP Alignment with definitions and nomenclature accepted in the scientific community should be sought (e.g., molar activity, apparent molar activity, …) Give reference to e.g.: Status of the ‘consensus nomenclature rules in radiopharmaceutical sciences’ initiative - PMC (nih.gov) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10078979/ 69 PRISMAP Novel production routes should be considered for radionuclides (e.g. inclusion of mass separation techniques) Deliverable D4.2 28 76 PRISMAP Clear indications of radionuclides as starting materials 79 PRISMAP Consider novel radionuclides with complex decay chains and their impact on description of radioanalytical procedures 87 PRISMAP Even though general thresholds are not possible due to the heterogeneous nature of radionuclides and radiopharmaceuticals, usual accepted and often applied limits should be mentioned (e.g., 95% for radiochemical purity and 99.9% for radionuclidic purity) 92 PRISMAP We very much welcome these considerations, esp. in view of novel radionuclides being developed and their challenges in appropriate metrology 2.4 Recommendation Line number(s) of the relevant text (e.g., 20-23) Stakeholder name (to be repeated in all rows) Comment and rationale Proposed guidance text 102-104 PRISMAP Consider novel developments of radionuclide production and radionuclides with complex decay chains Deliverable D4.2 29 Annex 4: Statement PRISMAP Radionuclides and GMP-Annex 3 and EMA response PRISMAP Consortium – WP4 (Harmonisation & Standardisation) PRISMAP - Background, Introduction: PRISMAP - The European medical radionuclide programme aims at providing emerging and non-conventional radionuclides for scientists across different disciplines in biomedical research. The programme’s main goal is to provide a sustainable source of high-purity novel radionuclides towards potential medical application, involving forthcoming major European infrastructures from the onset, as a single-entry point for all researchers active in this field. Figure 1: PRISMAP brings together a set of key large European, national and regional production infrastructures, chosen specifically for their expertise in the production and dispatch of non-conventional radionuclides. Historically, the development has been limited by the difficult access to radionuclides not yet commercially available. In particular, national borders often hinder the free exchange of goods. With PRISMAP, we federate a European consortium of the key intense neutron sources, isotope mass separation facilities and high-power accelerators and cyclotrons, with leading biomedical and healthcare research institutes in the active translation of emerging radionuclides towards medical diagnosis and treatment via preclinical studies (Fig 1). PRISMAP offers a single-entry point to researchers who are seeking innovative radionuclides of high purity for medical applications (Fig.2). Our aim is to enable and accelerate early-phase research on radiopharmaceuticals, targeted drugs for cancer, theranostics and personalised medicine, thus, providing novel methodologies for research and development of new efficient therapeutic drugs based on radionuclides that are not industrially available. Deliverable D4.2 30 Figure 2: PRISMAP Radionuclide portfolio and example for specification Problem Statement: Existing regulation (Directive 2001/83/EC, Definitions, Article 1) includes the definition of a radionuclide precursor as «Any other radionuclide produced for the radiolabelling of another substance prior to administration». This very wide definition potentially includes any radionuclide used in the process of radiopharmaceutical production/manufacture. From a practical perspective, this definition seems to be based on the assumption that such a radionuclide precursor (with a marketing authorisation) is combined with a (“cold”) kit (with marketing authorisation) in a simple process to yield the final radiopharmaceutical, without further purification and full testing of the final product by the production facility (usually a Nuclear Medicine facility). In such a case, the radionuclide precursor has to be handled by an external producer equivalent to a Drug Product and, therefore, GMP Part I becomes applicable. For the production of radionuclides, GMP Annex 3 defines that “Reactor/Cyclotron Production” can be outside GMP, GMP starts with the chemical synthesis of the “radioactive precursor” and “Purification steps (see Fig.3). However, this figure does not follow the API/drug product rationale that is followed in all GMP guidelines. Furthermore, it does not guide for processes that are divided over several sites. Figure 3. Extract from GMP Annex 3, describing where GMP is applicable in a process In principle, however, if the radionuclide is introduced into a manufacturing process in a facility that fulfils the required quality framework (GMP), usually within a manufacturing authorisation of this facility, the radiopharmaceutical can be considered as an API starting material and GMP part II should be applicable. This, in principle, allows one to use a radionuclide that is not prepared according to GMP (unless it is declared to be sterile). This consideration of the radionuclide as a starting material seems to be in place in many industrially manufactured radiopharmaceuticals but seems not to be fully understood in the case of small-scale preparation in hospitals and health care establishments, also within early phase developments and clinical trials. This partly seems to come from the current wording in Annex 3, which states: Deliverable D4.2 31  This annex is also applicable to radiopharmaceuticals used in clinical trials.  that it is applicable for “Radioactive Precursors for radiopharmaceutical production” (in contrast to the directive, which only defines “Radionuclide Precursors”) In a recent meeting with EMA and representatives from regulatory agencies, it became clear that GMP requirements are, in principle, not applicable for Investigational Medicinal Products (IMPs) prepared with such radionuclides, and that no GMP requirement for the radionuclide in question can be derived from the current guideline for the preparation of any radiopharmaceutical for a clinical trial. Nevertheless, even though GMP compliance for the radionuclide as starting material may not be demanded from the applicant of a clinical trial, it has become very difficult for producers of such radionuclides to provide them as starting materials without fulfilling GMP requirements. It appears that some stakeholders (including some competent authorities) do not understand that such a radionuclide can be considered as a starting material and not as a radionuclide (radioactive?) precursor. For PRISMAP, this poses particular problems. With the introduction of novel production methods (see example below) it becomes more difficult to differentiate between processes that can be defined as “Cyclotron/Reactor Production” and “Chemical synthesis” or “purification”. In particular, the introduction of mass-separation techniques requires physical processes, chemical processes, purifications etc. within facilities that cannot comply with the requirements of GMP. If GMP is required for such a producer to provide a user in hospitals or PET centres with a novel radionuclide for the preparation of a radiopharmaceutical (usually within a clinical trial), this will make certain innovative diagnostic and therapeutic approaches impossible. Practical example: The novel production methods underlying the supply chain of PRISMAP, as introduced in the first part of the document, requires a number of sequential steps that take place in multidisciplinary research centres. We provide here a concrete example with a brief introduction of the related sequential steps (Fig.4). Figure 4: Scheme of sequential steps for a radionuclide produced via mass separation Target preparation The target material suitable for isotope mass separation must adopt specific chemical and physical forms, different from those developed at cyclotron or nuclear reactor settings. For radionuclides where the targets must be irradiated at a reactor or cyclotron prior to (offline) mass separation, a processing step (e.g. chemical separation) is required to introduce the material in an isotope mass separation production unit. This requires reaching appropriate physical structures. In the case of thorium-based target materials irradiated at CERN applied to the production of massseparated 225Ac radionuclide samples, porous materials and composites must be designed to be able to sustain extreme temperatures of more than 2000 °C. Irradiation at CERN The exploitation of high-energy particle beams to irradiate targets requires infrastructures that are available at large accelerator and research facilities. The target irradiation step requires suitable remote transport and handling systems, diagnostics tools, exploiting thick natural target materials of several grams in dedicated irradiation stations. The CERN PS Booster, providing 1.4 GeV protons, produces a large range of reaction channels in targets (made of various materials depending on the nuclide desired). As a result, a vast number of radionuclides are produced in the thick targets that, subsequently, need to undergo a physical mass purification step. Examples include irradiating targets of 25-µm-thick foil rolls (300 g) used for terbium radioisotopes and Ba-128. Transfer of the target in the isotope mass separation unit The transfer of the target containing the vast number of radionuclides proceeds with operation performed in suitable shielded nuclear laboratories, with the exploitation of remote handling systems, in laboratories serving the multiple research communities. The target and ion source unit used for isotope mass separation can be transferred to the dedicated isotope separation facility, and is performed fully remotely at MEDICIS. Deliverable D4.2 32 Isotope Mass Separation Isotope mass separation is a physical radionuclide purification process. It proceeds on specialised accelerator facilities combining target unit handling, isotope release control, most often by high-temperature evaporation, isotope ionisation in a compact ion source, and mass separation in a beam line for implantation in a collector system. It separates a fraction of the radionuclides from the target that were produced during the irradiation. The online isotope mass separation proceeds while the target is irradiated. This can be performed at a few facilities such as ISOLDE at CERN, while offline isotope mass separation takes place after the irradiation and transfer of the target to the isotope mass separator. Collection The radionuclides are collected during the isotope mass separation process as ion beams by implantation into small foils at a beam energy of 1 to 100 keV. The foil material can be thin metal or salt backing on gold, salt crystals, polymer foils and frozen buffer solutions. MEDICIS collections typically proceed with gold foil backed with zinc or aluminum layers, and alternatively with aluminum foils with NaCl salt backing. This s performed in a dedicated collection chamber under vacuum with ion implantation monitoring systems. Radiochemistry Transfer of the foil to a radiochemical laboratory for radionuclide dissolution and further chemical purification, to prepare the radionuclide in an appropriate chemical form is necessary. Proposal for Annex 3 amendments:  Clarification of the term radioactive precursor vs. radionuclide precursor: It should be clearly stated that Annex 3 only fully applies to radionuclides when they fulfil the criteria of the definition in Directive 2001/83 of a radionuclide precursor, i.e., when applied for a drug product that is prepared within a marketing authorisation. It should be particularly clarified that radionuclides (but also potentially generators) that are provided as starting material should be viewed within GMP Part II (or the related document for Clinical Trial use).  Updating Annex 3 with guidance to clarify applicability of GMP part I, II or GMP for IMPs (“Detailed Commission guidelines on good manufacturing practice for investigational medicinal products for human use, pursuant to the second subparagraph of Article 63(1) of Regulation (EU) No 536/2014”) to clarify conflicting statements. In particular the statement “this annex is also applicable to radiopharmaceuticals used in clinical trials” should be removed as it is misleading and indicates that the same rules apply for radiopharmaceuticals used as IMPs as for those with MA, which is in contradiction to the Clinical Trials Regulation and related regulatory texts.  The table that differentiates non-GMP only for “reactor/cyclotron production” should be modified in that also chemical and physical processes to isolate radionuclides (e.g., for mass separation) should be exempt from GMP. Response from the GMDP IWG on the PRISMAP Proposal for Annex 3 amendment Deliverable D4.2 33 Deliverable D4.2 40 Annex 6: Report from the Interlaboratory Comparison study (ILC) of 64Cu 1. DTU Batch  CoA supplied by DTU Production laboratory: Hevesy Laboratory (Danmark) Solution: ≈ 1 mL in HCl (0.1 M) Activity/calibration time: 6,77 GBq (14 FEB 2022 16:00)  Effective specific activity determination by DOTA titration Operator: MB Site: ARRONAX Raw materials: Ammonium acetate – 99.999% Trace metal basis (Sigma – Ref: 372331 – Batch : MKBW2885V) Chelex 100 sodium form 50-100 mesh (Sigma – Ref C7901 – Batch: 031M0175V) DOTA, 6H2O (Macrocyclics – Ref:M-140 – Batch :M14010003-100127) Solution preparation (15 FEB 2022) Ammonium acetate 0.1M Ammonium acetate : 387.3 mg (ARRONAX balance reference # 162) Ammonium acetate molecular weight = 77.08 g/mol Water (from Millipore system): 50 mL Ammonium acetate concentration = 0.1 M (addition of a spatula tip of chelex resin in the final solution) DOTA 50 𝝁M DOTA: 19.7 mg (ARRONAX balance reference # 162) DOTA molecular weight = 512.5 g/mol Ammonium acetate 0.1 M: 3.845 mL (ARRONAX micropipet reference # T1763 ZCE2) DOTA concentration = 1.10-2 M 1/50th Dilution of DOTA solution: DOTA (0.01M): 100 𝜇L Ammonium acetate 0.1 M: 4.9 mL DOTA concentration = 2.10-4 M 1/200th Dilution of DOTA solution: DOTA (2.10-4M): 1 mL Ammonium acetate 0.1 M: 3 mL DOTA concentration = 50 𝜇M TLC mobile phase (H20/MeOH 1:1 + NH4CH3COO 5%) Water (from Millipore system): 23.75 mL Methanol: 23.75 mL Deliverable D4.2 41 Ammonium acetate 0.1 M: 2.5 mL DOTA Radiolabelling (15 FEB 2022 – 16h37) Vial number NH4CH3COO 0.1M (𝜇L) DOTA 50 𝜇𝑀 in NH4CH3COO 0.1M (𝜇L) Amount of DOTA (nmol) [64Cu]-CuCl2 (𝜇L) TLC ref 1 200 0 0 20 L4 2 198 2 0.1 20 L3 3 180 20 1 20 L2 4 100 100 5 20 L1 Radiolabelling condition: 90°C – 10 min. Results Vial number Amount of DOTA (nmol) TLC ref % free 64Cu % DOTA-64Cu 1 0 L4 100 0 2 0.1 L3 21.8 78.2 3 1 L2 0.1 99.9 4 5 L1 0 100 Conclusion nmol of DOTA allows 78.2 % complexation yield of 64Cu. In this condition, we could estimate the amount of competitive metal (mainly copper and zinc and partially iron) to 0.128 nmol in 20 𝜇L of 64Cu sample (i.e. 6.39 nmol/mL). Consequently, the calculate effective specific activity (ESA) is: 1.06 GBq/nmol. For comparison with ICP data provided by Hevesy Laboratory (VA = 6.77 GBq/mL @ TC): Ni: 0.41 𝜇g/GBq = 6.99 nmol/GBq = 47.29 nmol/mL  Zn: 0.03 𝜇g/GBq = 0.46 nmol/GBq = 3.11 nmol/mL  Fe: 0.04 𝜇g/GBq = 0.71 nmol/GBq = 4.85 nmol/mL  Cu: 0.02 𝜇g/GBq = 0.31 nmol/GBq = 2.13 nmol/mL Cu + Zn = 5.24 nmol/mL 2. PSI Batch  CoA supplied by PSI Chemical form: [64Cu]CuCl2 Production laboratory: PSI Solution: ≈ 0.720 mL in HCl (0.001 M) Activity/calibration time: 1,96 GBq (10 OCT 2023 13h46)  Effective specific activity determination by DOTA titration Operator: MB Site: ARRONAX Raw materials: Ammonium acetate – 99.999% Trace metal basis (Sigma – Ref: A7330-5006 – Batch : STBB9337V) Chelex 100 sodium form 50-100 mesh (Sigma – Ref C7901 – Batch: 031M0175V) DOTA (Aldrich – Ref:86734-50MG – Batch: BCCD5753) Solution preparation (06 OCT 2023) Deliverable D4.2 42 Ammonium acetate 0.1M Ammonium acetate: 387.3 mg (ARRONAX balance reference # 162) Ammonium acetate molecular weight = 77.08 g/mol Water (from Millipore system): 50 mL Ammonium acetate concentration = 0.1 M (addition of a spatula tip of chelex resin in the final solution) DOTA 50 𝝁M DOTA: 19.7 mg (ARRONAX balance reference # 162) DOTA molecular weight = 512.5 g/mol Ammonium acetate 0.1 M: 3.845 mL (ARRONAX micropipet reference # T1763 ZCE2) DOTA concentration = 1.10-2 M 1/50th Dilution of DOTA solution: DOTA (0.01M): 100 𝜇L Ammonium acetate 0.1 M: 4.9 mL DOTA concentration = 2.10-4 M 1/200th Dilution of DOTA solution: DOTA (2.10-4M): 1 mL Ammonium acetate 0.1 M: 3 mL DOTA concentration = 50 𝜇M TLC mobile phase (H20/MeOH 1:1 + NH4CH3COO 5%) / TLC Plate: Silica Water (from Millipore system): 23.75 mL Methanol: 23.75 mL Ammonium acetate 0.1 M: 2.5 mL DOTA Radiolabelling Vial number NH4CH3COO 0.1M (𝜇L) DOTA 50 𝜇𝑀 in NH4CH3COO 0.1M (𝜇L) Amount of DOTA (nmol) [64Cu]-CuCl2 (𝜇L) TLC ref 1 200 0 0 20 L4 2 198 2 0.1 20 L3 3 180 20 1 20 L2 4 100 100 5 20 L1 Radiolabelling conditions: 90°C – 10 min. Results Vial number Amount of DOTA (nmol) TLC ref % free 64Cu % DOTA-64Cu 1 0 L4 100 0 2 0.1 L3 100 0 3 1 L2 1,8 98,2 4 5 L1 0,4 99,6 Conclusion 1.0 nmol of DOTA allows 98.2 % complexation yield of 64Cu Deliverable D4.2 43 In this condition, we could estimate the amount of competitive metal (mainly copper and zinc and partially iron) to: 1.02 nmol in 20𝜇L of 64Cu sample (i.e. 50.92 nmol/mL). Consequently, the calculate concentration of copper competitive metal is 50.92 nmol/mL. The activity concentration @TC is 2.72 GBq/mL Consequently, the calculate effective specific activity (ESA) is comprised between: 0.053 GBq/nmol @TC.  Specific activity determination by ICP-OES Analysis Site: ARRONAX Raw materials Ultra-pure nitric acid 67-70% (SCP Science) Milli Q water (18.2MΩ.cm) Results The volume activity was determined using our HPGe detector. An aliquot of the sample was diluted to obtain a dead time lower as 10% in our geometry counting conditions. The calibration time is defined as 10 OCT 2023 01:46 PM to be in accordance with the delivered activity. A vol = 2680 ± 180 MBq/mL The Cu-64 batch was diluted by 50 to perform ICP-OES analysis. The results are presented in the table below: Element Concentration (ppm) Co <0.06 Cu 0.87±0.01 Fe 0.040±0.001 Ni <0.03 Zn 2.98±0.04 Considering these concentrations and the volume activity of the Cu-64 batch, we calculated the specific activity of 64Cu (197±14 MBq/nmol) and the effective specific activity (45±3 MBq/nmol). Conclusion The results obtained with ICP-OES analysis are in good agreement with the results obtained from DOTA titration (53 MBq/nmol). We can thus be confident concerning the results. 3. Polatom Batch  CoA supplied by Polatom Chemical form: [64Cu]-CuCl2 from natCu(n,γ)64Cu production route Production laboratory: Polatom Solution: ≈ 4,998 mL in HCl Activity/calibration time: 2 GBq ± 10% (31 JAN 2024 12h00) Radioactive concentration : 400.190 MBq/mL Specific activity: 234.40 MBq/mg Cu at CT? Radionuclidic purity: >99.5% Deliverable D4.2 44  Effective specific activity determination by DOTA titration Site: ARRONAX Raw materials Ammonium acetate – 99.999% Trace metal basis (Sigma – Ref: A7330-5006 – Batch : STBB9337V) Chelex 100 sodium form 50-100 mesh (Sigma – Ref C7901 – Batch: 031M0175V) DOTA (Aldrich – Ref:86734-50MG – Batch :BCCD5753) Solution preparation sodium acetate 0.1M Sodium acetate: 683.6 mg (ARRONAX balance reference # 162) sodium acetate molecular weight = 136.08 g/mol Water (from Millipore system): 50 mL Ammonium acetate concentration = 0.1 M (addition of a spatula tip of chelex resin in the final solution) DOTA 8900 𝝁M DOTA: 13.68 mg (ARRONAX balance reference # 162) DOTA molecular weight = 512.5 g/mol Sodium acetate 0.1 M : 3 mL DOTA concentration = 8.9 mM TLC mobile phase (H20/MeOH 1:1 + NH4CH3COO 5%) / TLC Plate: Silica Water (from Millipore system): 20 mL Methanol: 20 mL Ammonium acetate: 2.0244 g DOTA Radiolabelling Essai Sodium acetate 0.1M (µL) V DOTA (µL) DOTA (nmol) Cu-64 (µL) n metal (nmol) % Cu-DOTA 1 100 0 0.00 20 537.30 0.4 2 100 10 8.90 20 537.30 1.6 3 100 20 17.80 20 537.30 3.3 4 100 30 26.69 20 537.30 4.3 5 100 45 40.04 20 537.30 5.9 6 100 60 53.39 20 537.30 8.2 7 100 90 80.08 20 537.30 12.8 8 100 120 106.77 20 537.30 15 Deliverable D4.2 45 Figure 1: radiolabelling of 64Cu with DOTA Conclusion From figure 1, we can determine the quantity of metals in the final solution. And we can deduce the apparent specific activity of the Cu-64 batch. A spe apparent = 0.012±0.002 MBq/nmol Considering that only copper is significantly present in the final solution we deduce : A spe = 185±30 MBq/mg  Specific activity determination by ICP-OES Analysis Site: ARRONAX Raw materials: Ultra-pure nitric acid 67-70% (SCP Science) Miili Q water (18.2MΩ.cm) Results The volume activity was determined using our HPGe detector. An aliquot of the sample was counted in our geometry counting conditions. The calibration time is defined as 31 JAN 2024 at 12:00 AM to be in accordance with the delivered activity. A vol = 392 ± 25 MBq/mL No radionuclide impurities were determined. The Co-57/Cu-64 and Co-58/Cu-64 ratios are respectively under 1.9E-08 and 4.3E-09. The Cu-64 batch was diluted by 20000 to perform ICP-OES analysis. The results are presented in the table below: Deliverable D4.2 46 Elements Concentration (ppm) Co <10 Cu 1740±92 Fe <10 Ni <10 Zn 60±10 Considering these concentrations and the volume activity of the Cu-64 batch, we calculated the specific activity of Cu-64 : 225±19 MBq/mg or 0.014±0.001 MBq/nmol Conclusion The results obtained with ICP-OES analysis are in good agreement with the results supplied by Polatom (234.4 mBq/mg). We can thus be confident concerning the results. 4. ARRONAX Batch (Cu64_240207)  CoA supplied by ARRONAX Chemical form: [64Cu]CuCl2 from 64Ni(d,2n)64Cu production route Production laboratory: GIP ARRONAX Radioactive concentration : 2550±120 MBq/mL at CT (07 FEV 2024 08:00) Specific activity: 72±6 MBq/nmol Cu at CT Apparent specific activity: 48.4±3.1 MBq/nmol at CT Radionuclidic purity: >99.97%  Specific activity determination by ICP-OES Analysis Site: ARRONAX Raw materials: Ultra pure nitric acid 67-70% (SCP Science) Miili Q water (18.2MΩ.cm) Results The volume activity was determined using our HPGe detector. An aliquot of the sample was counted in our geometry counting conditions. The calibration time is defined as 07 FEV 2024 at 08:00 AM. The dilution factor used to perform analysis is equal to 500. A vol = 2550 ± 120 MBq/mL No radionuclide impurities were determined. The Co-57/Cu-64 and Co-58/Cu-64 ratios are respectively under 2.3E-04 and 4.4E-05. The Cu-64 batch was diluted by 500 to perform ICP-OES analysis. The results are presented in the table below: Elements Concentration (ppm) Co <0.39 Cu 2.25±0.14 Fe <0.18 Ni <0.30 Zn 1.127±0.038 Deliverable D4.2 47 Considering these concentrations and the volume activity of the Cu-64 batch, we calculated the specific activity of Cu-64: 1133± 78 GBq/mg or 72±5 MBq/nmol The apparent specific activity is equal to 755±52 GBq/mg or 48±3 MBq/nmol  Effective specific activity determination by DOTA titration Site: ARRONAX Raw material Ammonium acetate – 99.999% Trace metal basis (Sigma – Ref: A7330-5006 – Batch : STBB9337V) Chelex 100 sodium form 50-100 mesh (Sigma – Ref C7901 – Batch: 031M0175V) DOTA (Aldrich – Ref:86734-50MG – Batch :BCCD5753) Solution preparation sodium acetate 0.1M Sodium acetate: 683.6 mg (ARRONAX balance reference # 162) sodium acetate molecular weight = 136.08 g/mol Water (from Millipore system): 50 mL Ammonium acetate concentration = 0.1 M (addition of a spatula tip of chelex resin in the final solution) DOTA 8900 𝝁M DOTA: 13.68 mg (ARRONAX balance reference # 162) DOTA molecular weight = 512.5 g/mol Sodium acetate 0.1 M : 3 mL DOTA concentration = 8.9 mM DOTA 25.06 𝝁M 0.025 mL fo DOTA 8900 µM solution + 8.85mL of sodium acetate solution TLC mobile phase (H20/MeOH 1:1 + NH4CH3COO 5%) / TLC Plate: Silica Water (from Millipore system): 20 mL Methanol: 20mL Ammonium acetate: 2.0188 g DOTA Radiolabeling Essai Sodium acetate 0.1M (µL) V DOTA (µL) DOTA (nmol) Cu-64 (µL) n metal (nmol) % Cu-DOTA 1 100 0 0.00 20 0.31 0.4 2 100 10 0.25 20 0.31 38.6 3 100 20 0.50 20 0.31 76.7 4 100 30 0.75 20 0.31 98.2 5 100 45 1.13 20 0.31 99 6 100 60 1.50 20 0.31 98.6 7 100 90 2.26 20 0.31 98.2 8 100 120 3.01 20 0.31 98 Deliverable D4.2 48 Figure 2: radiolabelling of 64Cu with DOTA (ARRONAX batch) Conclusion From figure 2, we can determine the quantity of metals in the final solution. And we can deduce the apparent specific activity of the 64Cu batch. A spe apparent = 78±4 MBq/nmol Considering that only copper is significantly present in the final solution we deduce : A spe = 1230±58 GBq/mg As regard of DOTA titration results which seems to conclude that only copper is present in the final solution, a new ICP-OES analysis was performed with weaker dilution to minimize the uncertainties. The results are presented below: Elements Concentration (ppm) Co <0.39 Cu 2.36±0.14 Fe <0.12 Ni <0.09 Zn <0.09 These results confirm the DOTA titration results. No significant metal impurities are present in the final solution. The specific activities from these new results can be calculated: A spe = 69±5 MBq/nmol Considering that only copper is significantly present in the final solution we deduce: A spe = 1082±74 GBq/mg Conclusion The important dilution factor used for ICP-analysis (with regard to radiosafety considerations) can be a source of uncertainties for some metals determination (Zn for example).