Development of an automated radiosynthesis for [68Ga]Ga-NODAGA[40Lys]-Exendin-4 in sodium acetate buffer

Development of an automated radiosynthesis for [68Ga]Ga-NODAGA[40Lys]-Exendin-4 in sodium acetate buffer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Development of an automated radiosynthesis for [68Ga]Ga-NODAGA[40Lys]-Exendin-4 in sodium acetate buffer Maïlys RAGOT, Pauline REYNAUD-ORHON, Alban REVY, Marie BATISSE-LIGNIER, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8944286/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Background [ 68 Ga]Ga-NODAGA[40Lys]-Exendin-4 ( 68 Ga-Ex-4) is a new PET radiopharmaceutical targeting GLP-1-R that showed great specificity and sensitivity in the topographic diagnosis of benign insulinoma. We herein report the development of a new method for 68 Ga-Ex-4 radiolabeling in sodium acetate buffer, automated on the miniAllinOne synthesizer. Results Key parameters were reaction volume, due to low amount of peptide, and pH. At a reaction volume of 2.65 and pH 2.9, the reaction yielded 80% of 68 Ga-Ex-4. The use of polysorbate and cooling of the solution allows to decrease the adsorption on the reaction vial (from 29 to 2% of total activity remaining on the vial). 68 Ga-Ex-4 is purified on a HLB cartridge. Final product was obtained within 40 minutes, with a mean radiochemical purity of 97% and a production yield of 67%. Conclusions We have successfully developed a new radiolabeling method for [ 68 Ga]Ga-NODAGA-[40Lys]Exendin-4, automated on a miniAllinOne synthesizer and using sodium acetate buffer, suitable for clinical application. Exendin-4 gallium-68 radiolabeling radiopharmaceutical automation insulinoma PET Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Insulinoma is a pancreatic neuroendocrine tumor, most often benign but associated with a significant risk of hypoglycemia due to unregulated insulin secretion, which can be life-threatening. Precise topographical diagnosis and tumor extension is required for surgery, which remains the first-line treatment. However, it is made difficult by the small size of these tumors (90% are less than 2 cm and 30% less than 1 cm). While there has been a lot of improvement these past years, especially with the development of new PET tracers, the lack of sensitivity and specificity of current imaging modalities remains challenging (1). Exendin-4 positron emission tomography (PET) imaging has shown great promises for localization of benign insulinoma, being both highly specific and sensitive (2–4). GLP-1 (glucagon like peptide 1) receptors, targeted by Exendin-4, are expressed by more than 90% of insulinomas, with an extremely high receptor density (5). When available, Exendin-4 PET imaging is recommended as the first-line modality for topographic localization of benign insulinomas, following biochemical confirmation and initial conventional imaging with CT or MRI (6,7). In France, [68Ga]Ga-NODAGA[40Lys]-Exendin-4 (68Ga-Ex-4) is authorized under compassionate use and requires in situ radiolabeling, using 10 µg of commercially available precursor (NODAGA[40Lys]-Exendin-4) and gallium-68 chloride. Gallium-68 ( 68 Ga) is a widely used positron-emitting radionuclide for PET imaging because of its favorable physical and chemical properties, including a short half-life of 68 minutes and on-site availability from a 68 Ge/ 68 Ga generator. Automated synthesis has become the clinical standard for the production of 68 Ga radiopharmaceuticals, offering improved radioprotection and reproducibility compared with manual methods. Given the growing clinical interest in 68 Ga-Ex-4, the establishment of a robust and efficient automated radiolabeling process is essential. Radiolabeling of NODAGA-Exendin-4 with gallium-68 at routine clinical scale has previously been described using HEPES buffer, on a Scintomics synthesizer (8,9). In both cases, the gallium eluate is purified on a strong cations exchange (SCX) cartridge, before being transferred into the reaction vial containing the buffered peptide. Then, radiolabeling takes place between 95 and 100°C for 10 to 15 min. 68 Ga-Ex-4 is purified on a hydrophilic-lipophilic balance (HLB) cartridge. Before purification, Boss et al. let the vial cool and add ethylenediaminetetraacetic acid (EDTA)/polysorbate 80, supposedly to prevent adsorption, an issue that was not described by the other team. Exendin-4 is also a peptide known to be susceptible to oxidation, mainly because of its methionine in position 14 (10): antioxidant can be added, such as ascorbic acid (8), or a modified peptide has been synthetized, replacing the methionine residue by a norleucine (9). However, only the unmodified peptide is authorized in France under compassionate use. The ideal buffer for gallium-68 radiolabeling must maintain an appropriate pH (usually between 3 and 5) with the lowest possible reaction volume, while having a low complexation constant with gallium-68 (8). HEPES (4-(2-hydroxyethyl)-1-piperazine ethane sulphonic acid) (pKa = 3.0) and sodium acetate (pKa = 4.75) provide relevant pH and function as stabilizing agents (11,12). However, HEPES is considered a chemical impurity in the European Pharmacopoeia (Ph. Eur.), and is only authorized for use in injectable radiopharmaceuticals in humans up to a limit of 200 µg per dose, due to limited toxicological data (13). A colorimetric method is described in several monographs, comparing the respective deposits of the test solution and a reference solution on a silica gel, but this method is described as unreliable in the literature (14–16) and requires precious time. Sodium acetate is fully approved for human use, with no additional quality control required. We herein report the development of a new method for the radiolabeling of [ 68 Ga]Ga-NODAGA-[40Lys]Exendin-4 in sodium acetate buffer, automated on the miniAllinOne (miniAiO) synthesizer. By addressing key parameters such as reaction conditions (reaction volume, pH), transfer and purification processes, this work seeks to improve radiolabeling efficiency and radiochemical purity of the final product, ensuring the method’s suitability for routine clinical use. Results The first synthesis yielded only 1% of [ 68 Ga]Ga-NODAGA-Exendin-4, with most of the activity found as free gallium species (95.8%). Two uncharacterised radiolabelled impurities were observed at 10.9 and 28.7 mn, representing 1.5 and 1.1% of the activity. Radiosynthesis conditions are detailed on Fig. 1 . Based on the data in the literature and the stability of the peptide, the heating was set to maintain the temperature in the reaction vial between 95°C and 100°C for 15 min. Adjustments of the programming were made to achieve a rapid temperature rise and maintain a stable temperature thereafter (Supp. Figure 1 ). The TRASIS “68Ga labelling with prepurification” kit (2.2 mL of SCX eluent and 3 mL of sodium acetate buffer) and sequence are designed to obtain a reaction pH of 4, the pH value measured in the reaction medium was 4.2. The reaction vial was recovered at the end of production. Despite rinsing the cassette with NaCl 0.9%, a white deposit was observed on the walls of the vial. A new synthesis was performed in the same conditions, with an antioxidant added to the peptide prior to synthesis: 50 µL of ascorbic acid (100 mg/mL). Radiochemical purity (RCP) at the end of the radiolabeling step was 2.7%. After adding a surfactant (polysorbate) to the reaction medium, RCP increased to 7.3%. Based on these results, ascorbic acid and polysorbate were systematically used hereinafter. One of the most critical parameters was the composition of the reaction medium, particularly volume (as it is directly linked to the peptide concentration) and pH during the radiolabeling step. The initial reaction medium was composed of the reconstituted peptide in water for injection (wfi) (100 µL), ascorbic acid (50 µL), acetate buffer (3 mL) and the prepurification eluate (2.2 mL). The buffer and the prepurification eluate represented 97% of the total volume, and also determined the pH of the solution, as the prepurification eluate contains HCl 0.1M. Therefore, pH and reaction volume (V r ) are evaluated together. Increase of pH was obtained by adding NaOH (120 mg/mL, 0.15 mL) to the reaction medium after the prepurification step: at pH 4.45 and V r 5.50 mL, RCP was 3.7%. A decrease of both pH and reaction volume was obtained by reducing the volume of acetate buffer (1 mL instead of 3 mL): at pH 1.34 and V r 3.35 mL, RCP was 54%. To obtain a pH between 2 and 3 without increasing the reaction volume, the prepurification eluate volume was reduced. As the volume of prepurification eluate directly affects the prepurification efficacy, three volumes were tested: 0.7, 1.5 and 2.2 mL, resulting in a purification yield of respectively 46, 76 and 89%. A volume of 1.5 mL was chosen, as the loss of activity on the cartridge was considered too great at the lowest volume. With 1.5 mL of SCX eluent and 1 mL of buffer, i.e. pH 2.9 and V r 2.65, RCP was 83% (Fig. 2 ). Five syntheses were performed under these conditions, resulting in RCP from 78 to 84%. The final stage of the synthesis is the purification of the solution on a HLB cartridge, to ensure less than 3% of free gallium species in the final product. This step also allows to formulate the final product in excipients adapted to intravenous injection (NaCl 0.9%, with an adequate pH between 4 and 8). The HLB cartridge is prepared beforehand by passing 1 mL of ethanol (EtOH) and 10 mL of wfi. The cartridge retains 68 Ga-Ex-4, while the main impurities (free gallium-68 and 68 Ga-EDTA) are eliminated in the waste vial. Desorption of 68 Ga-Ex-4 is achieved by washing with 1.6 mL of EtOH/wfi. For this stage, the main parameter to adjust is the composition of the eluent (EtOH/wfi ratio and volume), ideally using the cartridge supplied with the cassette. Different ratios of EtOH/wfi have been tested: increasing the percentage of EtOH is associated with an increase of purification efficacy, allowing for a better desorption of 68 Ga-Ex-4 (Table 1 ). However, the Eur. Ph. sets a 10% limit for EtOH level in the final product, for radiopharmaceuticals. A dilution with NaCl 0.9% is done to ensure an EtOH level below 10%. Consequently, the volume of the final product is directly linked to the amount of EtOH in the eluent. A ratio of 60/40 EtOH/wfi (v/v), corresponding to 0.96 mL of EtOH and 0.64 mL wfi, is selected to obtain a sufficient volume activity and limit the injection volume for the patient: 9 mL of NaCl 0.9% is added to the purification eluate, for a total volume of 10.6 mL and 9% level of EtOH. In terms of efficacy, less than 1% of free gallium-68 and [ 68 Ga]Ga-EDTA is found in the final product after purification, for the three EtOH/wfi ratios tested. Sterility of the final product is ensured by the solution passing through a 0.22 µm sterilizing filter, into a glass sterile vial, with a mean activity of 35 MBq remaining on the filter. Table 1 Purification yield according to EtOH/wfi ratio. EtOH/wfi ratio (% v/v) 80/20 70/30 60/40 Purification yield, decay corrected 84% 82% 75% Ethanol volume (mL) 1.28 1.12 0.96 Minimal final volume required to ensure a level of ethanol below 10% (mL) 12.8 11.2 9.6 Two solutions were explored to limit the adsorption of 68 Ga-Ex-4 observed on the reaction vial: cooling the solution and adding a surfactant, polysorbate 80 (Tween® 80). As active cooling is not available on our synthesizer, decrease of the reaction medium temperature was achieved by either turning the oven off or manually moving the vial to a lead container. Since the Tween-EDTA solution is stored at room temperature and of a comparable volume to the reaction volume (respectively 2 and 2.65 mL), a fast decrease in the reaction medium temperature is observed when it is added. However, if the vial is left in the oven, despite turning it off, temperature rises again with a final temperature of 77°C after 5 minutes. Manually moving the vial to a lead container allows for the temperature to keep decreasing with a final temperature of 55°C. When Tween-EDTA is not added, the temperature only decreases to 69°C (Fig. 3 ). When the vial is moved, adding Tween-EDTA decreases the mean residual activity in the reaction vial from 29 to 2% (Table 2 ). Consequently, it was decided to both add Tween-EDTA and manually move the vial to a lead container. Table 2 Residual activity in the reaction vial and activity in the final product vial, with and without adding Tween-EDTA. Residual activity in the reaction vial (MBq) (% of total activity) Without adding Tween-EDTA Adding Tween-EDTA 132 (30%) 218 (28%) 11 (2%) 9 (2%) 10 (2%) 11 (2%) Activity in the final product (MBq) (% of total activity) 70 (16%) 137 (18%) 286 (62%) 331 (70%) 344 (73%) 354 (73%) Total activity (MBq) 447 773 461 476 471 487 Residual activities at end of synthesis (39 min after the start of elution), in the final conditions (Fig. 4 ), are shown in Fig. 5 . Activity is mainly lost during the two purification stages, on the cartridge and in the waste vial. Almost 7% of the total activity remains on the sterilizing filter. 67% of the total activity is found in the final product. Discussion This novel method enables the radiolabeling of Ex-4 in sodium acetate buffer in under one hour, achieving a high radiochemical purity (free and insoluble gallium species < 1%). The final activity is suitable for clinical applications, with a decay-corrected yield of 67%. Given the low peptide amount (10 µg), a low reaction volume proved critical, increasing the peptide concentration and therefore the reaction yield. This was achieved by pre-purifying the eluate and reducing the volume used to eluate the SCX cartridge, which subsequently decreased the amount of buffer needed to neutralize the hydrochloric acid. Alternatively, eluate volume could be reduced via fractional elution or by utilizing a Galliad® generator (IRE), which requires only 1.1 mL of HCl. Under final conditions, the reaction yield reached 81%. However, the presence of residual free gallium suggests that further optimization is possible by refining the pH, heating parameters, or further volume reduction. While increasing the initial peptide amount would likely improve yield, Ex-4 dosage must remain minimal to avoid pharmacological side effects, such as nausea and hypoglycemia (17). A purification step is mandatory to remove residual free gallium and formulate the product for injection. By adding a surfactant and implementing a cooling step (manual transfer to a lead container), activity loss in the reaction vial was reduced from 30% to less than 3%. Final purification was conducted using an HLB cartridge, which is highly effective at removing free and insoluble gallium-68 species (18). Although 7% of the activity is currently lost during sterile filtration, this step is non-negotiable for intravenous administration. Loss of activity might be mitigated by wetting the filter prior to the filtration or evaluating alternative filter materials. Literature regarding the automated radiolabeling of Exendin-4, especially NODAGA(40Lys)-Exendin-4, remains sparse. Boss et al. (8) described in 2020 an automated method using HEPES buffer and a Scintomics system, but did not report synthesis yields, as their focus was on dosimetry. Migliari et al. (9) reported an automated HEPES-based method for a modified peptide [Nle14-Lys40-(NODAGA)NH2]Exendin-4 (Nle14-Ex-4), achieving a decay-corrected yield of only 23.5% and an RCP of 97.7%. Manual synthesis of Nle14-Ex-4 has been described using ammonium acetate (Kaeppeli et al. (19)) and sodium acetate (Kirsi et al. (20)) buffers. However, they involved very low activities (14 MBq) or high peptide amount (> 20 µg) in sub-milliliter volumes. These conditions are not representative of, or compatible with, routine clinical production. Unlike existing methods that rely on HEPES buffer – which requires additional quality control – our protocol utilizes sodium acetate, a buffer fully approved for human use. This method represents the first automated radiolabeling process for Ex-4 using sodium acetate that is directly transferable to clinical radiopharmacy practice. Conclusions We successfully developed an automated radiosynthesis for [ 68 Ga]Ga-NODAGA(40Lys)-Exendin-4 using a sodium acetate buffer, with high radiochemical purity and a good radiochemical yield, allowing for activities compatible with clinical use. Materials and methods Materials Gallium-68 is obtained as [ 68 Ga]gallium chloride ([ 68 Ga]GaCl 3 ) from a TiO 2 based 68 Ge/ 68 Ga generator, GalliaPharm®, produced by Eckert&Ziegler (Berlin, Germany). The precursor NODAGA-Exendin(40Lys)-4 was obtained from PiChem (Grambach, Austria). Radiolabeling is automated on a miniAiO® synthesizer (Trasis, Belgium), using a disposable cassette. All chemical reagents were of Eur. Ph. quality and used without further purification unless otherwise specified: HCl 0.1M (Eckert&Ziegler, Germany), L-ascorbic acid (Merck, Germany), SCX eluent and acetate buffer (Trasis, Belgium), Tween-EDTA (ABX, Germany), Ethanol EMPROVE® (Merck, Germany). All HPLC solvents are from Merck (Germany): trifluoroacetic acid (TFA), water with 0.1% v:v TFA and acetonitrile (ACN). Radiochemical synthesis optimization This method was adapted from the TRASIS sequence and cassette for 68 Ga synthesis with prepurification. Based on the data in the literature and the stability of the peptide, the heating was set to maintain the temperature in the reaction vial between 95°C and 100°C for 15 min. The following parameters were optimized to increase final yield: composition of the reaction medium (volume, pH, composition), cooling and use of a surfactant to improve the transfer of 68 Ga-Ex-4 at the end of the reaction and composition of the eluate for the purification step and final formulation. The radiochemical synthesis was automated on a MiniAllinOne® (miniAIO) synthesizer (Trasis). 10 µg of the peptide were reconstituted with 100 µL of water for injection, sodium acetate buffer (several volumes tested, between 0.95 and 3 mL) and 50 µL of ascorbic acid (100 mg/mL), and placed in the reaction vial. The 68 Ge/ 68 Ga generator was eluted with 5 mL of HCl 0.1 M. The [ 68 Ga]GaCl 3 obtained was then purified on a cation exchange (SCX) cartridge and eluted with 0.7, 1.5 or 2.2 mL of SCX eluent (NaCl 5M, HCl 0.1 M) into the reaction vial. Reaction took place at 100°C for 15 min. Afterwards, 2 mL of Tween 80 0.15%/EDTA 50 mM were added to the mixture, and the vial was left to cool down for 5 min, either in the turned off oven or in a lead container. The solution was then transferred to a HLB cartridge, preconditioned with 1 mL EtOH and 10 mL wfi. The cartridge was rinsed with NaCl 0.9%. 68 Ga-Ex-4 was eluted with 1.6 mL of EtOH/wfi (three ratios tested: 60/40, 70/30 and 80/20). The final product was diluted with NaCl 0.9% to obtain an EtOH level below 10%, through a 0.22 µm sterilizing filter. Radiochemical purity (RCP) was determined by a validated radio-HPLC analytical method, described below. It was monitored either at the end of radiolabeling, in the reaction vial, or at the end of synthesis, in the final product. pH was initially determined by indicator paper, and then by pH-meter after radioactive decay. At the end of the synthesis, activity was measured on a MEDI 405 dose calibrator, at different points: on the two cartridges (pre-purification and purification), in the reaction vial, in the waste vial, on the sterilization filter and in the final product vial. Radiochemical purity testing by radio-HPLC Radio-HPLC was performed on a SIL-20AHT HPLC (Shimadzu) equipped with a UV SPD-20A detector (Shimadzu) and a Flow-Ram radiometric detector (LabLogic). An Alltima C18 column, 5µm, 250x4.6 mm (HiChrom®) was eluted by gradient elution (1 mL/min) of water/trifluoroacetic acid 0.1% (solvent A) and acetonitrile/trifluoroacetic acid 0.1% (solvent B): 0–5 mn, 3% B; 5–15 mn, 3 to 100% B; 15–25 mn, 100% B; 25–35 mn, 3% B. The reference standard [ 69,71 Ga]NODAGA(40Lys)-Exendin-4 was obtained from PiChem (Grambach, Austria) to determine retention time. RCP in the final product has a 90% compliance threshold, with less than 3% of free gallium species. Abbreviations 68 Ga-Ex-4 [ 68 Ga]Ga-NODAGA(40Lys)-Exendin-4 EtOH ethanol Ex-4 NODAGA(40Lys)-Exendin-4 HCl hydrochloric acid HLB hydrophilic-lipophilic cartridge HPLC high performance liquid chromatography miniAiO miniAllinOne Nle14-Ex-4 [Nle14-Lys40-(NODAGA)NH2]Exendin-4 PET positron emission tomography RCP radiochemical purity RCY radiochemical yield wfi water for injection Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and material The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding No funding was received to assist with the preparation of this manuscript. Authors' contributions MR and MT contributed to experimental design. Radiosynthesis and quality control were performed by MR and MT. The first draft of the manuscript was written by MR. All authors have revised the manuscript, and read and approved its final version. Acknowledgements Not applicable References Imperiale A, Boursier C, Sahakian N, Ouvrard E, Chevalier E, Sebag F, et al. Value of 68 Ga-DOTATOC and Carbidopa-Assisted 18 F-DOPA PET/CT for Insulinoma Localization. J Nucl Med. 2022 Mar;63(3):384–8. doi:10.2967/jnumed.121.262401 Sidrak MMA, De Feo MS, Corica F, Gorica J, Conte M, Filippi L, et al. Role of Exendin-4 Functional Imaging in Diagnosis of Insulinoma: A Systematic Review. Life. 2023 Apr 11;13(4):989. doi:10.3390/life13040989 Qi-chang W, Lan W, Bin J. Diagnostic Performance of Exendin-4 PET/CT in Localizing Insulinomas: A Systematic Review and Meta-analysis. Acad Radiol. 2025 Dec;32(12):7526–36. doi:10.1016/j.acra.2025.09.020 Boss M, Eriksson O, Mikkola K, Eek A, Brom M, Buitinga M, et al. Improved Localization of Insulinomas Using 68 Ga-NODAGA-Exendin-4 PET/CT. J Nucl Med. 2024 Oct 17;jnumed.124.268158. doi:10.2967/jnumed.124.268158 Reubi JC, Waser B. Concomitant expression of several peptide receptors in neuroendocrine tumours: molecular basis for in vivo multireceptor tumour targeting. Eur J Nucl Med Mol Imaging. 2003 May 1;30(5):781–93. doi:10.1007/s00259-003-1184-3 Hofland J, Falconi M, Christ E, Castaño JP, Faggiano A, Lamarca A, et al. European Neuroendocrine Tumor Society 2023 guidance paper for functioning pancreatic neuroendocrine tumour syndromes. J Neuroendocrinol. 2023 Aug;35(8):e13318. doi:10.1111/jne.13318 Antwi K, Nicolas G, Wild D, Christ E. Molecular imaging for neuroendocrine tumours: This article was corrected and republished online on April 3, 2019. Please see Erratum (Swiss Med Wkly. 2019;149:w20076). Swiss Med Wkly. 2019 Mar 10;149(0910):w20017. doi:10.4414/smw.2019.20017 Boss M, Buitinga M, Jansen TJP, Brom M, Visser EP, Gotthardt M. PET-Based Human Dosimetry of 68Ga-NODAGA-Exendin-4, a Tracer for β-Cell Imaging. J Nucl Med. 2020 Jan 1;61(1):112–6. doi:10.2967/jnumed.119.228627 Migliari S, Sammartano A, Scarlattei M, Baldari G, Janota B, Bonadonna RC, et al. Feasibility of a Scale-down Production of [68Ga]Ga-NODAGA-Exendin-4 in a Hospital Based Radiopharmacy. Curr Radiopharm. 2021;15(1):63–75. Janota B, Karczmarczyk U, Laszuk E, Garnuszek P, Mikołajczak R. Oxidation of methionine — is it limiting the diagnostic properties of 99mTc-labeled Exendin-4, a Glucagon-Like Peptide-1 receptor agonist? Nucl Med Rev. 2016 Jul 29;19(2):104–10. doi:10.5603/NMR.2016.0021 Bauwens M, Chekol R, Vanbilloen H, Bormans G, Verbruggen A. Optimal buffer choice of the radiosynthesis of 68Ga–Dotatoc for clinical application. Nucl Med Commun. 2010 Aug;31(8):753–8. doi:10.1097/MNM.0b013e32833acb99 Velikyan I. 68Ga-Based Radiopharmaceuticals: Production and Application Relationship. Molecules. 2015 Jul 16;20(7):12913–43. doi:10.3390/molecules200712913 Gallium (68Ga) DOTANOC, monograph 3051. Ph. Eur. Suppl. 11.5. Strasbourg, France: Council of Europe; 2025. Pfaff S, Nehring T, Pichler V, Cardinale J, Mitterhauser M, Hacker M, et al. Development and evaluation of a rapid analysis for HEPES determination in 68Ga-radiotracers. EJNMMI Res. 2018 Dec;8(1):95. doi:10.1186/s13550-018-0449-6 Sasson R, Vaknin D, Bross A, Lavie E. Determination of HEPES in 68Ga-labeled peptide solutions. J Radioanal Nucl Chem. 2010 Mar;283(3):753–6. doi:10.1007/s10967-010-0449-0 Antunes IF, Franssen GM, Zijlma R, Laverman P, Boersma HH, Elsinga PH. New sensitive method for HEPES quantification in 68Ga-radiopharmaceuticals. EJNMMI Radiopharm Chem. 2020 May 14;5(1):12. doi:10.1186/s41181-020-00093-x Jansen TomJP, Van Lith SanneAM, Boss M, Brom M, Joosten L, Béhé M, et al. Exendin‐4 analogs in insulinoma theranostics. J Label Compd Radiopharm. 2019 Aug;62(10):656–72. doi:10.1002/jlcr.3750 Brom M, Franssen G, Joosten L, Gotthardt M, Boerman O. The effect of purification of Ga-68-labeled exendin on in vivo distribution. EJNMMI Res. 2016 Aug 12;6. doi:10.1186/s13550-016-0221-8 Kaeppeli SAM, Schibli R, Mindt TL, Behe M. Comparison of desferrioxamine and NODAGA for the gallium-68 labeling of exendin-4. EJNMMI Radiopharm Chem. 2019 May 16;4(1):9. doi:10.1186/s41181-019-0060-9 Kirsi M, Cheng-Bin Y, Veronica F, Tamiko I, Viki-Veikko E, Johan R, et al. 64Cu- and 68Ga-Labelled [Nle14,Lys40(Ahx-NODAGA)NH2]-Exendin-4 for Pancreatic Beta Cell Imaging in Rats. Mol Imaging Biol. 2014 Apr 1;16(2):255–63. doi:10.1007/s11307-013-0691-2 Supplementary Files SupplementaryFigure1.tif Supplementary Figure 1. Temperature in the reaction vial, from the start of the synthesis to the end of the radiolabeling step (n=6). Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 19 Mar, 2026 Reviewers agreed at journal 09 Mar, 2026 Reviewers invited by journal 09 Mar, 2026 Editor assigned by journal 09 Mar, 2026 First submitted to journal 05 Mar, 2026 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8944286","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":602929843,"identity":"46756866-feff-440e-b0f0-754ed2c2777d","order_by":0,"name":"Maïlys RAGOT","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+klEQVRIiWNgGAWjYFACxsYDcPYHIGZjJ6ylAaaFsXEGSAszEfbAtTTzgChCWnTbDzcc5vnDkM/fv/j4Y5tf2+T5mBkYP3zMwa3F7Exiw2HeNgbLGTeeJTbn9t02bGNmYJacuQ2PlgMgLQ0MBgw3zhg25/bcZgRqYWPmxafl/EOwwwzkQVose27bE9ZyA2gLDxuDgcH5HsNmhh+3E4nQ8rDh4Nw2CQPDG2yJM3sbbie3MTM24/fL+fSHD978sTGQO3/4wIcff27bzm9vPvjhIx4tIMDEwyDBwCCRAIyZNhCfsQG/epCSHyCS/wCQ+ENQ8SgYBaNgFIxAAAC5GFiBEj3OzAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-9100-3965","institution":"CLCC Jean Perrin: Centre Jean Perrin","correspondingAuthor":true,"prefix":"","firstName":"Maïlys","middleName":"","lastName":"RAGOT","suffix":""},{"id":602929855,"identity":"84614e07-54d8-42d5-bf14-94516a256f36","order_by":1,"name":"Pauline REYNAUD-ORHON","email":"","orcid":"","institution":"CLCC Jean Perrin: Centre Jean Perrin","correspondingAuthor":false,"prefix":"","firstName":"Pauline","middleName":"","lastName":"REYNAUD-ORHON","suffix":""},{"id":602929859,"identity":"d5617c9b-4922-4104-a2ba-139216e05581","order_by":2,"name":"Alban REVY","email":"","orcid":"","institution":"CLCC Jean Perrin: Centre Jean Perrin","correspondingAuthor":false,"prefix":"","firstName":"Alban","middleName":"","lastName":"REVY","suffix":""},{"id":602929860,"identity":"87a0ccb6-96da-409a-a016-4f96f69b9134","order_by":3,"name":"Marie BATISSE-LIGNIER","email":"","orcid":"","institution":"CLCC Jean Perrin: Centre Jean Perrin","correspondingAuthor":false,"prefix":"","firstName":"Marie","middleName":"","lastName":"BATISSE-LIGNIER","suffix":""},{"id":602929862,"identity":"17ba0e99-46aa-48f8-9dbb-3058d18952f5","order_by":4,"name":"Valentin BRUSSEAU","email":"","orcid":"","institution":"CLCC Jean Perrin: Centre Jean Perrin","correspondingAuthor":false,"prefix":"","firstName":"Valentin","middleName":"","lastName":"BRUSSEAU","suffix":""},{"id":602929863,"identity":"40fd743d-32e1-46cd-9f8a-4b6b280f07d6","order_by":5,"name":"Antony KELLY","email":"","orcid":"","institution":"CLCC Jean Perrin: Centre Jean Perrin","correspondingAuthor":false,"prefix":"","firstName":"Antony","middleName":"","lastName":"KELLY","suffix":""},{"id":602929865,"identity":"c83fd20f-c317-41e4-8137-d41720c8c63c","order_by":6,"name":"Hosameldin OTMAN","email":"","orcid":"","institution":"CLCC Jean Perrin: Centre Jean Perrin","correspondingAuthor":false,"prefix":"","firstName":"Hosameldin","middleName":"","lastName":"OTMAN","suffix":""},{"id":602929867,"identity":"736ebbeb-c6aa-4ba6-8a80-ca6091a16f46","order_by":7,"name":"Florent CACHIN","email":"","orcid":"","institution":"CLCC Jean Perrin: Centre Jean Perrin","correspondingAuthor":false,"prefix":"","firstName":"Florent","middleName":"","lastName":"CACHIN","suffix":""},{"id":602929868,"identity":"ea12a982-9e54-4e92-86d1-ca80cebffd7e","order_by":8,"name":"Marion CHANCHOU","email":"","orcid":"","institution":"CLCC Jean Perrin: Centre Jean Perrin","correspondingAuthor":false,"prefix":"","firstName":"Marion","middleName":"","lastName":"CHANCHOU","suffix":""},{"id":602929869,"identity":"1829edcb-9938-4cdf-82e6-4b87ad0378b6","order_by":9,"name":"Sophie LEVESQUE","email":"","orcid":"https://orcid.org/0000-0003-3046-1804","institution":"CLCC Jean Perrin: Centre Jean Perrin","correspondingAuthor":false,"prefix":"","firstName":"Sophie","middleName":"","lastName":"LEVESQUE","suffix":""},{"id":602929870,"identity":"c7649f73-5dd8-4659-861d-c23345a2b91f","order_by":10,"name":"Marion TEMPIER","email":"","orcid":"","institution":"CLCC Jean Perrin: Centre Jean Perrin","correspondingAuthor":false,"prefix":"","firstName":"Marion","middleName":"","lastName":"TEMPIER","suffix":""}],"badges":[],"createdAt":"2026-02-23 07:45:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8944286/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8944286/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104781521,"identity":"1b3d0977-5ea9-48a2-a8fb-e0f653b490dc","added_by":"auto","created_at":"2026-03-17 07:55:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":966661,"visible":true,"origin":"","legend":"\u003cp\u003eFirst synthesis (a) Process flow-chart (b) Photography of the reaction vial (c) Radiochromatogram\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8944286/v1/7c1f04ced8a7e07984b23ec8.png"},{"id":104781585,"identity":"d5b3a9a5-ee39-4a1e-874a-07bd5c8d85b1","added_by":"auto","created_at":"2026-03-17 07:55:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":371110,"visible":true,"origin":"","legend":"\u003cp\u003eFinal radiolabeling conditions (a) Process flow-chart (b) Radiochromatogram\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8944286/v1/0a9cb5306eeac057308c09f6.png"},{"id":104572086,"identity":"06d9f77b-e2b8-4f19-9c43-656a9f6e01fd","added_by":"auto","created_at":"2026-03-13 12:59:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1098594,"visible":true,"origin":"","legend":"\u003cp\u003eTemperature in the reaction medium during the cooling step, in 4 conditions.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8944286/v1/df94c58e87eb356ca0ba03cf.png"},{"id":104572091,"identity":"0a8a73fa-d005-4c6a-bf58-f11183b73dec","added_by":"auto","created_at":"2026-03-13 12:59:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":500435,"visible":true,"origin":"","legend":"\u003cp\u003eFinal synthesis conditions (a) Process flow-chart (b) Radiochromatogram\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8944286/v1/80ad17e14f34743ec9cbd32e.png"},{"id":104572089,"identity":"f09ea825-b804-4d10-8f8c-2dbb06fa9dd9","added_by":"auto","created_at":"2026-03-13 12:59:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":499347,"visible":true,"origin":"","legend":"\u003cp\u003eMean residual activities at end of synthesis (EoS) (n=4).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8944286/v1/b72eabb3c7dc2c87a475dd38.png"},{"id":104808565,"identity":"a552de91-db8e-4286-b886-6f3263f1b366","added_by":"auto","created_at":"2026-03-17 12:38:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3968408,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8944286/v1/8a02dc8c-c849-475a-902f-ad38c9497fa3.pdf"},{"id":104781129,"identity":"d5ce53da-1ee2-40d0-93f7-a80034d06072","added_by":"auto","created_at":"2026-03-17 07:54:53","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":250256,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure 1. Temperature in the reaction vial, from the start of the synthesis to the end of the radiolabeling step (n=6).\u003c/p\u003e","description":"","filename":"SupplementaryFigure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-8944286/v1/7e926857944ff377729b29f5.tif"}],"financialInterests":"","formattedTitle":"Development of an automated radiosynthesis for [68Ga]Ga-NODAGA[40Lys]-Exendin-4 in sodium acetate buffer","fulltext":[{"header":"Background","content":"\u003cp\u003eInsulinoma is a pancreatic neuroendocrine tumor, most often benign but associated with a significant risk of hypoglycemia due to unregulated insulin secretion, which can be life-threatening. Precise topographical diagnosis and tumor extension is required for surgery, which remains the first-line treatment. However, it is made difficult by the small size of these tumors (90% are less than 2 cm and 30% less than 1 cm). While there has been a lot of improvement these past years, especially with the development of new PET tracers, the lack of sensitivity and specificity of current imaging modalities remains challenging (1). Exendin-4 positron emission tomography (PET) imaging has shown great promises for localization of benign insulinoma, being both highly specific and sensitive (2\u0026ndash;4). GLP-1 (glucagon like peptide 1) receptors, targeted by Exendin-4, are expressed by more than 90% of insulinomas, with an extremely high receptor density (5). When available, Exendin-4 PET imaging is recommended as the first-line modality for topographic localization of benign insulinomas, following biochemical confirmation and initial conventional imaging with CT or MRI (6,7). In France, [68Ga]Ga-NODAGA[40Lys]-Exendin-4 (68Ga-Ex-4) is authorized under compassionate use and requires in situ radiolabeling, using 10 \u0026micro;g of commercially available precursor (NODAGA[40Lys]-Exendin-4) and gallium-68 chloride.\u003c/p\u003e \u003cp\u003eGallium-68 (\u003csup\u003e68\u003c/sup\u003eGa) is a widely used positron-emitting radionuclide for PET imaging because of its favorable physical and chemical properties, including a short half-life of 68 minutes and on-site availability from a \u003csup\u003e68\u003c/sup\u003eGe/\u003csup\u003e68\u003c/sup\u003eGa generator. Automated synthesis has become the clinical standard for the production of \u003csup\u003e68\u003c/sup\u003eGa radiopharmaceuticals, offering improved radioprotection and reproducibility compared with manual methods. Given the growing clinical interest in \u003csup\u003e68\u003c/sup\u003eGa-Ex-4, the establishment of a robust and efficient automated radiolabeling process is essential.\u003c/p\u003e \u003cp\u003eRadiolabeling of NODAGA-Exendin-4 with gallium-68 at routine clinical scale has previously been described using HEPES buffer, on a Scintomics synthesizer (8,9). In both cases, the gallium eluate is purified on a strong cations exchange (SCX) cartridge, before being transferred into the reaction vial containing the buffered peptide. Then, radiolabeling takes place between 95 and 100\u0026deg;C for 10 to 15 min. \u003csup\u003e68\u003c/sup\u003eGa-Ex-4 is purified on a hydrophilic-lipophilic balance (HLB) cartridge. Before purification, Boss \u003cem\u003eet al.\u003c/em\u003e let the vial cool and add ethylenediaminetetraacetic acid (EDTA)/polysorbate 80, supposedly to prevent adsorption, an issue that was not described by the other team. Exendin-4 is also a peptide known to be susceptible to oxidation, mainly because of its methionine in position 14 (10): antioxidant can be added, such as ascorbic acid (8), or a modified peptide has been synthetized, replacing the methionine residue by a norleucine (9). However, only the unmodified peptide is authorized in France under compassionate use.\u003c/p\u003e \u003cp\u003eThe ideal buffer for gallium-68 radiolabeling must maintain an appropriate pH (usually between 3 and 5) with the lowest possible reaction volume, while having a low complexation constant with gallium-68 (8). HEPES (4-(2-hydroxyethyl)-1-piperazine ethane sulphonic acid) (pKa\u0026thinsp;=\u0026thinsp;3.0) and sodium acetate (pKa\u0026thinsp;=\u0026thinsp;4.75) provide relevant pH and function as stabilizing agents (11,12). However, HEPES is considered a chemical impurity in the European Pharmacopoeia (Ph. Eur.), and is only authorized for use in injectable radiopharmaceuticals in humans up to a limit of 200 \u0026micro;g per dose, due to limited toxicological data (13). A colorimetric method is described in several monographs, comparing the respective deposits of the test solution and a reference solution on a silica gel, but this method is described as unreliable in the literature (14\u0026ndash;16) and requires precious time. Sodium acetate is fully approved for human use, with no additional quality control required.\u003c/p\u003e \u003cp\u003eWe herein report the development of a new method for the radiolabeling of [\u003csup\u003e68\u003c/sup\u003eGa]Ga-NODAGA-[40Lys]Exendin-4 in sodium acetate buffer, automated on the miniAllinOne (miniAiO) synthesizer. By addressing key parameters such as reaction conditions (reaction volume, pH), transfer and purification processes, this work seeks to improve radiolabeling efficiency and radiochemical purity of the final product, ensuring the method\u0026rsquo;s suitability for routine clinical use.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe first synthesis yielded only 1% of [\u003csup\u003e68\u003c/sup\u003eGa]Ga-NODAGA-Exendin-4, with most of the activity found as free gallium species (95.8%). Two uncharacterised radiolabelled impurities were observed at 10.9 and 28.7 mn, representing 1.5 and 1.1% of the activity. Radiosynthesis conditions are detailed on Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Based on the data in the literature and the stability of the peptide, the heating was set to maintain the temperature in the reaction vial between 95\u0026deg;C and 100\u0026deg;C for 15 min. Adjustments of the programming were made to achieve a rapid temperature rise and maintain a stable temperature thereafter (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The TRASIS \u0026ldquo;68Ga labelling with prepurification\u0026rdquo; kit (2.2 mL of SCX eluent and 3 mL of sodium acetate buffer) and sequence are designed to obtain a reaction pH of 4, the pH value measured in the reaction medium was 4.2. The reaction vial was recovered at the end of production. Despite rinsing the cassette with NaCl 0.9%, a white deposit was observed on the walls of the vial.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA new synthesis was performed in the same conditions, with an antioxidant added to the peptide prior to synthesis: 50 \u0026micro;L of ascorbic acid (100 mg/mL). Radiochemical purity (RCP) at the end of the radiolabeling step was 2.7%. After adding a surfactant (polysorbate) to the reaction medium, RCP increased to 7.3%. Based on these results, ascorbic acid and polysorbate were systematically used hereinafter.\u003c/p\u003e \u003cp\u003eOne of the most critical parameters was the composition of the reaction medium, particularly volume (as it is directly linked to the peptide concentration) and pH during the radiolabeling step. The initial reaction medium was composed of the reconstituted peptide in water for injection (wfi) (100 \u0026micro;L), ascorbic acid (50 \u0026micro;L), acetate buffer (3 mL) and the prepurification eluate (2.2 mL). The buffer and the prepurification eluate represented 97% of the total volume, and also determined the pH of the solution, as the prepurification eluate contains HCl 0.1M. Therefore, pH and reaction volume (V\u003csub\u003er\u003c/sub\u003e) are evaluated together. Increase of pH was obtained by adding NaOH (120 mg/mL, 0.15 mL) to the reaction medium after the prepurification step: at pH 4.45 and V\u003csub\u003er\u003c/sub\u003e 5.50 mL, RCP was 3.7%. A decrease of both pH and reaction volume was obtained by reducing the volume of acetate buffer (1 mL instead of 3 mL): at pH 1.34 and V\u003csub\u003er\u003c/sub\u003e 3.35 mL, RCP was 54%. To obtain a pH between 2 and 3 without increasing the reaction volume, the prepurification eluate volume was reduced. As the volume of prepurification eluate directly affects the prepurification efficacy, three volumes were tested: 0.7, 1.5 and 2.2 mL, resulting in a purification yield of respectively 46, 76 and 89%. A volume of 1.5 mL was chosen, as the loss of activity on the cartridge was considered too great at the lowest volume. With 1.5 mL of SCX eluent and 1 mL of buffer, i.e. pH 2.9 and V\u003csub\u003er\u003c/sub\u003e 2.65, RCP was 83% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Five syntheses were performed under these conditions, resulting in RCP from 78 to 84%.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe final stage of the synthesis is the purification of the solution on a HLB cartridge, to ensure less than 3% of free gallium species in the final product. This step also allows to formulate the final product in excipients adapted to intravenous injection (NaCl 0.9%, with an adequate pH between 4 and 8). The HLB cartridge is prepared beforehand by passing 1 mL of ethanol (EtOH) and 10 mL of wfi. The cartridge retains \u003csup\u003e68\u003c/sup\u003eGa-Ex-4, while the main impurities (free gallium-68 and \u003csup\u003e68\u003c/sup\u003eGa-EDTA) are eliminated in the waste vial. Desorption of \u003csup\u003e68\u003c/sup\u003eGa-Ex-4 is achieved by washing with 1.6 mL of EtOH/wfi. For this stage, the main parameter to adjust is the composition of the eluent (EtOH/wfi ratio and volume), ideally using the cartridge supplied with the cassette. Different ratios of EtOH/wfi have been tested: increasing the percentage of EtOH is associated with an increase of purification efficacy, allowing for a better desorption of \u003csup\u003e68\u003c/sup\u003eGa-Ex-4 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, the Eur. Ph. sets a 10% limit for EtOH level in the final product, for radiopharmaceuticals. A dilution with NaCl 0.9% is done to ensure an EtOH level below 10%. Consequently, the volume of the final product is directly linked to the amount of EtOH in the eluent. A ratio of 60/40 EtOH/wfi (v/v), corresponding to 0.96 mL of EtOH and 0.64 mL wfi, is selected to obtain a sufficient volume activity and limit the injection volume for the patient: 9 mL of NaCl 0.9% is added to the purification eluate, for a total volume of 10.6 mL and 9% level of EtOH. In terms of efficacy, less than 1% of free gallium-68 and [\u003csup\u003e68\u003c/sup\u003eGa]Ga-EDTA is found in the final product after purification, for the three EtOH/wfi ratios tested. Sterility of the final product is ensured by the solution passing through a 0.22 \u0026micro;m sterilizing filter, into a glass sterile vial, with a mean activity of 35 MBq remaining on the filter.\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\u003ePurification yield according to EtOH/wfi ratio.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEtOH/wfi ratio (% v/v)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e80/20\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70/30\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e60/40\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePurification yield, decay corrected\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e84%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e82%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e75%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEthanol volume (mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMinimal final volume required to ensure a level of ethanol below 10% (mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.6\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\u003eTwo solutions were explored to limit the adsorption of \u003csup\u003e68\u003c/sup\u003eGa-Ex-4 observed on the reaction vial: cooling the solution and adding a surfactant, polysorbate 80 (Tween\u0026reg; 80). As active cooling is not available on our synthesizer, decrease of the reaction medium temperature was achieved by either turning the oven off or manually moving the vial to a lead container. Since the Tween-EDTA solution is stored at room temperature and of a comparable volume to the reaction volume (respectively 2 and 2.65 mL), a fast decrease in the reaction medium temperature is observed when it is added. However, if the vial is left in the oven, despite turning it off, temperature rises again with a final temperature of 77\u0026deg;C after 5 minutes. Manually moving the vial to a lead container allows for the temperature to keep decreasing with a final temperature of 55\u0026deg;C. When Tween-EDTA is not added, the temperature only decreases to 69\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). When the vial is moved, adding Tween-EDTA decreases the mean residual activity in the reaction vial from 29 to 2% (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Consequently, it was decided to both add Tween-EDTA and manually move the vial to a lead container.\u003c/p\u003e \u003cp\u003e \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\u003eResidual activity in the reaction vial and activity in the final product vial, with and without adding Tween-EDTA.\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=\"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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eResidual activity in the reaction vial (MBq) (% of total activity)\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e\u003cem\u003eWithout adding\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eTween-EDTA\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c7\" namest=\"c4\"\u003e \u003cp\u003e\u003cem\u003eAdding Tween-EDTA\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e132 (30%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e218 (28%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003cp\u003e(2%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9\u003c/p\u003e \u003cp\u003e(2%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10\u003c/p\u003e \u003cp\u003e(2%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e11\u003c/p\u003e \u003cp\u003e(2%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eActivity in the final product (MBq)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(% of total activity)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e70 (16%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e137 (18%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e286 (62%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e331 (70%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e344 (73%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e354 (73%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal activity (MBq)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e447\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e773\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e461\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e476\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e471\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e487\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\u003eResidual activities at end of synthesis (39 min after the start of elution), in the final conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Activity is mainly lost during the two purification stages, on the cartridge and in the waste vial. Almost 7% of the total activity remains on the sterilizing filter. 67% of the total activity is found in the final product.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis novel method enables the radiolabeling of Ex-4 in sodium acetate buffer in under one hour, achieving a high radiochemical purity (free and insoluble gallium species\u0026thinsp;\u0026lt;\u0026thinsp;1%). The final activity is suitable for clinical applications, with a decay-corrected yield of 67%. Given the low peptide amount (10 \u0026micro;g), a low reaction volume proved critical, increasing the peptide concentration and therefore the reaction yield. This was achieved by pre-purifying the eluate and reducing the volume used to eluate the SCX cartridge, which subsequently decreased the amount of buffer needed to neutralize the hydrochloric acid. Alternatively, eluate volume could be reduced via fractional elution or by utilizing a Galliad\u0026reg; generator (IRE), which requires only 1.1 mL of HCl. Under final conditions, the reaction yield reached 81%. However, the presence of residual free gallium suggests that further optimization is possible by refining the pH, heating parameters, or further volume reduction. While increasing the initial peptide amount would likely improve yield, Ex-4 dosage must remain minimal to avoid pharmacological side effects, such as nausea and hypoglycemia (17). A purification step is mandatory to remove residual free gallium and formulate the product for injection. By adding a surfactant and implementing a cooling step (manual transfer to a lead container), activity loss in the reaction vial was reduced from 30% to less than 3%. Final purification was conducted using an HLB cartridge, which is highly effective at removing free and insoluble gallium-68 species (18). Although 7% of the activity is currently lost during sterile filtration, this step is non-negotiable for intravenous administration. Loss of activity might be mitigated by wetting the filter prior to the filtration or evaluating alternative filter materials.\u003c/p\u003e \u003cp\u003eLiterature regarding the automated radiolabeling of Exendin-4, especially NODAGA(40Lys)-Exendin-4, remains sparse. Boss \u003cem\u003eet al.\u003c/em\u003e (8) described in 2020 an automated method using HEPES buffer and a Scintomics system, but did not report synthesis yields, as their focus was on dosimetry. Migliari \u003cem\u003eet al.\u003c/em\u003e (9) reported an automated HEPES-based method for a modified peptide [Nle14-Lys40-(NODAGA)NH2]Exendin-4 (Nle14-Ex-4), achieving a decay-corrected yield of only 23.5% and an RCP of 97.7%. Manual synthesis of Nle14-Ex-4 has been described using ammonium acetate (Kaeppeli \u003cem\u003eet al.\u003c/em\u003e (19)) and sodium acetate (Kirsi \u003cem\u003eet al.\u003c/em\u003e (20)) buffers. However, they involved very low activities (14 MBq) or high peptide amount (\u0026gt;\u0026thinsp;20 \u0026micro;g) in sub-milliliter volumes. These conditions are not representative of, or compatible with, routine clinical production.\u003c/p\u003e \u003cp\u003eUnlike existing methods that rely on HEPES buffer \u0026ndash; which requires additional quality control \u0026ndash; our protocol utilizes sodium acetate, a buffer fully approved for human use. This method represents the first automated radiolabeling process for Ex-4 using sodium acetate that is directly transferable to clinical radiopharmacy practice.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWe successfully developed an automated radiosynthesis for [\u003csup\u003e68\u003c/sup\u003eGa]Ga-NODAGA(40Lys)-Exendin-4 using a sodium acetate buffer, with high radiochemical purity and a good radiochemical yield, allowing for activities compatible with clinical use.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eGallium-68 is obtained as [\u003csup\u003e68\u003c/sup\u003eGa]gallium chloride ([\u003csup\u003e68\u003c/sup\u003eGa]GaCl\u003csub\u003e3\u003c/sub\u003e) from a TiO\u003csub\u003e2\u003c/sub\u003e based \u003csup\u003e68\u003c/sup\u003eGe/\u003csup\u003e68\u003c/sup\u003eGa generator, GalliaPharm\u0026reg;, produced by Eckert\u0026amp;Ziegler (Berlin, Germany). The precursor NODAGA-Exendin(40Lys)-4 was obtained from PiChem (Grambach, Austria). Radiolabeling is automated on a miniAiO\u0026reg; synthesizer (Trasis, Belgium), using a disposable cassette. All chemical reagents were of Eur. Ph. quality and used without further purification unless otherwise specified: HCl 0.1M (Eckert\u0026amp;Ziegler, Germany), L-ascorbic acid (Merck, Germany), SCX eluent and acetate buffer (Trasis, Belgium), Tween-EDTA (ABX, Germany), Ethanol EMPROVE\u0026reg; (Merck, Germany). All HPLC solvents are from Merck (Germany): trifluoroacetic acid (TFA), water with 0.1% v:v TFA and acetonitrile (ACN).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRadiochemical synthesis optimization\u003c/h3\u003e\n\u003cp\u003eThis method was adapted from the TRASIS sequence and cassette for \u003csup\u003e68\u003c/sup\u003eGa synthesis with prepurification. Based on the data in the literature and the stability of the peptide, the heating was set to maintain the temperature in the reaction vial between 95\u0026deg;C and 100\u0026deg;C for 15 min. The following parameters were optimized to increase final yield: composition of the reaction medium (volume, pH, composition), cooling and use of a surfactant to improve the transfer of \u003csup\u003e68\u003c/sup\u003eGa-Ex-4 at the end of the reaction and composition of the eluate for the purification step and final formulation.\u003c/p\u003e \u003cp\u003eThe radiochemical synthesis was automated on a MiniAllinOne\u0026reg; (miniAIO) synthesizer (Trasis). 10 \u0026micro;g of the peptide were reconstituted with 100 \u0026micro;L of water for injection, sodium acetate buffer (several volumes tested, between 0.95 and 3 mL) and 50 \u0026micro;L of ascorbic acid (100 mg/mL), and placed in the reaction vial. The \u003csup\u003e68\u003c/sup\u003eGe/\u003csup\u003e68\u003c/sup\u003eGa generator was eluted with 5 mL of HCl 0.1 M. The [\u003csup\u003e68\u003c/sup\u003eGa]GaCl\u003csub\u003e3\u003c/sub\u003e obtained was then purified on a cation exchange (SCX) cartridge and eluted with 0.7, 1.5 or 2.2 mL of SCX eluent (NaCl 5M, HCl 0.1 M) into the reaction vial. Reaction took place at 100\u0026deg;C for 15 min. Afterwards, 2 mL of Tween 80 0.15%/EDTA 50 mM were added to the mixture, and the vial was left to cool down for 5 min, either in the turned off oven or in a lead container. The solution was then transferred to a HLB cartridge, preconditioned with 1 mL EtOH and 10 mL wfi. The cartridge was rinsed with NaCl 0.9%. \u003csup\u003e68\u003c/sup\u003eGa-Ex-4 was eluted with 1.6 mL of EtOH/wfi (three ratios tested: 60/40, 70/30 and 80/20). The final product was diluted with NaCl 0.9% to obtain an EtOH level below 10%, through a 0.22 \u0026micro;m sterilizing filter.\u003c/p\u003e \u003cp\u003eRadiochemical purity (RCP) was determined by a validated radio-HPLC analytical method, described below. It was monitored either at the end of radiolabeling, in the reaction vial, or at the end of synthesis, in the final product. pH was initially determined by indicator paper, and then by pH-meter after radioactive decay. At the end of the synthesis, activity was measured on a MEDI 405 dose calibrator, at different points: on the two cartridges (pre-purification and purification), in the reaction vial, in the waste vial, on the sterilization filter and in the final product vial.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRadiochemical purity testing by radio-HPLC\u003c/h2\u003e \u003cp\u003eRadio-HPLC was performed on a SIL-20AHT HPLC (Shimadzu) equipped with a UV SPD-20A detector (Shimadzu) and a Flow-Ram radiometric detector (LabLogic). An Alltima C18 column, 5\u0026micro;m, 250x4.6 mm (HiChrom\u0026reg;) was eluted by gradient elution (1 mL/min) of water/trifluoroacetic acid 0.1% (solvent A) and acetonitrile/trifluoroacetic acid 0.1% (solvent B): 0\u0026ndash;5 mn, 3% B; 5\u0026ndash;15 mn, 3 to 100% B; 15\u0026ndash;25 mn, 100% B; 25\u0026ndash;35 mn, 3% B. The reference standard [\u003csup\u003e69,71\u003c/sup\u003eGa]NODAGA(40Lys)-Exendin-4 was obtained from PiChem (Grambach, Austria) to determine retention time. RCP in the final product has a 90% compliance threshold, with less than 3% of free gallium species.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003csup\u003e68\u003c/sup\u003eGa-Ex-4\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e[\u003csup\u003e68\u003c/sup\u003eGa]Ga-NODAGA(40Lys)-Exendin-4\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEtOH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eethanol\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEx-4\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNODAGA(40Lys)-Exendin-4\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHCl\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehydrochloric acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHLB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehydrophilic-lipophilic cartridge\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHPLC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehigh performance liquid chromatography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eminiAiO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eminiAllinOne\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNle14-Ex-4\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e[Nle14-Lys40-(NODAGA)NH2]Exendin-4\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePET\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epositron emission tomography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRCP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eradiochemical purity\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRCY\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eradiochemical yield\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ewfi\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ewater for injection\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003ch2\u003eAvailability of data and material\u003c/h2\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eNo funding was received to assist with the preparation of this manuscript.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eAuthors\u0026apos; contributions\u003c/h2\u003e\n\u003cp\u003eMR and MT contributed to experimental design. Radiosynthesis and quality control were performed by MR and MT. The first draft of the manuscript was written by MR. All authors have revised the manuscript, and read and approved its final version.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eImperiale A, Boursier C, Sahakian N, Ouvrard E, Chevalier E, Sebag F, et al. Value of\u003csup\u003e68\u003c/sup\u003e Ga-DOTATOC and Carbidopa-Assisted\u003csup\u003e18\u003c/sup\u003e F-DOPA PET/CT for Insulinoma Localization. J Nucl Med. 2022 Mar;63(3):384\u0026ndash;8. doi:10.2967/jnumed.121.262401\u003c/li\u003e\n\u003cli\u003eSidrak MMA, De Feo MS, Corica F, Gorica J, Conte M, Filippi L, et al. Role of Exendin-4 Functional Imaging in Diagnosis of Insulinoma: A Systematic Review. Life. 2023 Apr 11;13(4):989. doi:10.3390/life13040989\u003c/li\u003e\n\u003cli\u003eQi-chang W, Lan W, Bin J. Diagnostic Performance of Exendin-4 PET/CT in Localizing Insulinomas: A Systematic Review and Meta-analysis. Acad Radiol. 2025 Dec;32(12):7526\u0026ndash;36. doi:10.1016/j.acra.2025.09.020\u003c/li\u003e\n\u003cli\u003eBoss M, Eriksson O, Mikkola K, Eek A, Brom M, Buitinga M, et al. Improved Localization of Insulinomas Using\u003csup\u003e68\u003c/sup\u003e Ga-NODAGA-Exendin-4 PET/CT. J Nucl Med. 2024 Oct 17;jnumed.124.268158. doi:10.2967/jnumed.124.268158\u003c/li\u003e\n\u003cli\u003eReubi JC, Waser B. Concomitant expression of several peptide receptors in neuroendocrine tumours: molecular basis for in vivo multireceptor tumour targeting. Eur J Nucl Med Mol Imaging. 2003 May 1;30(5):781\u0026ndash;93. doi:10.1007/s00259-003-1184-3\u003c/li\u003e\n\u003cli\u003eHofland J, Falconi M, Christ E, Casta\u0026ntilde;o JP, Faggiano A, Lamarca A, et al. European Neuroendocrine Tumor Society 2023 guidance paper for functioning pancreatic neuroendocrine tumour syndromes. J Neuroendocrinol. 2023 Aug;35(8):e13318. doi:10.1111/jne.13318\u003c/li\u003e\n\u003cli\u003eAntwi K, Nicolas G, Wild D, Christ E. Molecular imaging for neuroendocrine tumours: This article was corrected and republished online on April 3, 2019. Please see Erratum (Swiss Med Wkly. 2019;149:w20076). Swiss Med Wkly. 2019 Mar 10;149(0910):w20017. doi:10.4414/smw.2019.20017\u003c/li\u003e\n\u003cli\u003eBoss M, Buitinga M, Jansen TJP, Brom M, Visser EP, Gotthardt M. PET-Based Human Dosimetry of 68Ga-NODAGA-Exendin-4, a Tracer for \u0026beta;-Cell Imaging. J Nucl Med. 2020 Jan 1;61(1):112\u0026ndash;6. doi:10.2967/jnumed.119.228627\u003c/li\u003e\n\u003cli\u003eMigliari S, Sammartano A, Scarlattei M, Baldari G, Janota B, Bonadonna RC, et al. Feasibility of a Scale-down Production of [68Ga]Ga-NODAGA-Exendin-4 in a Hospital Based Radiopharmacy. Curr Radiopharm. 2021;15(1):63\u0026ndash;75.\u003c/li\u003e\n\u003cli\u003eJanota B, Karczmarczyk U, Laszuk E, Garnuszek P, Mikołajczak R. Oxidation of methionine \u0026mdash; is it limiting the diagnostic properties of 99mTc-labeled Exendin-4, a Glucagon-Like Peptide-1 receptor agonist? Nucl Med Rev. 2016 Jul 29;19(2):104\u0026ndash;10. doi:10.5603/NMR.2016.0021\u003c/li\u003e\n\u003cli\u003eBauwens M, Chekol R, Vanbilloen H, Bormans G, Verbruggen A. Optimal buffer choice of the radiosynthesis of 68Ga\u0026ndash;Dotatoc for clinical application. Nucl Med Commun. 2010 Aug;31(8):753\u0026ndash;8. doi:10.1097/MNM.0b013e32833acb99\u003c/li\u003e\n\u003cli\u003eVelikyan I. 68Ga-Based Radiopharmaceuticals: Production and Application Relationship. Molecules. 2015 Jul 16;20(7):12913\u0026ndash;43. doi:10.3390/molecules200712913\u003c/li\u003e\n\u003cli\u003eGallium (68Ga) DOTANOC, monograph 3051. Ph. Eur. Suppl. 11.5. Strasbourg, France: Council of Europe; 2025.\u003c/li\u003e\n\u003cli\u003ePfaff S, Nehring T, Pichler V, Cardinale J, Mitterhauser M, Hacker M, et al. Development and evaluation of a rapid analysis for HEPES determination in 68Ga-radiotracers. EJNMMI Res. 2018 Dec;8(1):95. doi:10.1186/s13550-018-0449-6\u003c/li\u003e\n\u003cli\u003eSasson R, Vaknin D, Bross A, Lavie E. Determination of HEPES in 68Ga-labeled peptide solutions. J Radioanal Nucl Chem. 2010 Mar;283(3):753\u0026ndash;6. doi:10.1007/s10967-010-0449-0\u003c/li\u003e\n\u003cli\u003eAntunes IF, Franssen GM, Zijlma R, Laverman P, Boersma HH, Elsinga PH. New sensitive method for HEPES quantification in 68Ga-radiopharmaceuticals. EJNMMI Radiopharm Chem. 2020 May 14;5(1):12. doi:10.1186/s41181-020-00093-x\u003c/li\u003e\n\u003cli\u003eJansen TomJP, Van Lith SanneAM, Boss M, Brom M, Joosten L, B\u0026eacute;h\u0026eacute; M, et al. Exendin‐4 analogs in insulinoma theranostics. J Label Compd Radiopharm. 2019 Aug;62(10):656\u0026ndash;72. doi:10.1002/jlcr.3750\u003c/li\u003e\n\u003cli\u003eBrom M, Franssen G, Joosten L, Gotthardt M, Boerman O. The effect of purification of Ga-68-labeled exendin on in vivo distribution. EJNMMI Res. 2016 Aug 12;6. doi:10.1186/s13550-016-0221-8\u003c/li\u003e\n\u003cli\u003eKaeppeli SAM, Schibli R, Mindt TL, Behe M. Comparison of desferrioxamine and NODAGA for the gallium-68 labeling of exendin-4. EJNMMI Radiopharm Chem. 2019 May 16;4(1):9. doi:10.1186/s41181-019-0060-9\u003c/li\u003e\n\u003cli\u003eKirsi M, Cheng-Bin Y, Veronica F, Tamiko I, Viki-Veikko E, Johan R, et al. 64Cu- and 68Ga-Labelled [Nle14,Lys40(Ahx-NODAGA)NH2]-Exendin-4 for Pancreatic Beta Cell Imaging in Rats. Mol Imaging Biol. 2014 Apr 1;16(2):255\u0026ndash;63. doi:10.1007/s11307-013-0691-2\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":false,"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":"Exendin-4, gallium-68, radiolabeling, radiopharmaceutical, automation, insulinoma, PET","lastPublishedDoi":"10.21203/rs.3.rs-8944286/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8944286/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003e[\u003csup\u003e68\u003c/sup\u003eGa]Ga-NODAGA[40Lys]-Exendin-4 (\u003csup\u003e68\u003c/sup\u003eGa-Ex-4) is a new PET radiopharmaceutical targeting GLP-1-R that showed great specificity and sensitivity in the topographic diagnosis of benign insulinoma. We herein report the development of a new method for \u003csup\u003e68\u003c/sup\u003eGa-Ex-4 radiolabeling in sodium acetate buffer, automated on the miniAllinOne synthesizer.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eKey parameters were reaction volume, due to low amount of peptide, and pH. At a reaction volume of 2.65 and pH 2.9, the reaction yielded 80% of \u003csup\u003e68\u003c/sup\u003eGa-Ex-4. The use of polysorbate and cooling of the solution allows to decrease the adsorption on the reaction vial (from 29 to 2% of total activity remaining on the vial). \u003csup\u003e68\u003c/sup\u003eGa-Ex-4 is purified on a HLB cartridge. Final product was obtained within 40 minutes, with a mean radiochemical purity of 97% and a production yield of 67%.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eWe have successfully developed a new radiolabeling method for [\u003csup\u003e68\u003c/sup\u003eGa]Ga-NODAGA-[40Lys]Exendin-4, automated on a miniAllinOne synthesizer and using sodium acetate buffer, suitable for clinical application.\u003c/p\u003e","manuscriptTitle":"Development of an automated radiosynthesis for [68Ga]Ga-NODAGA[40Lys]-Exendin-4 in sodium acetate buffer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-13 12:59:07","doi":"10.21203/rs.3.rs-8944286/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2026-03-19T10:02:43+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2026-03-09T08:29:02+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-09T07:43:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-09T07:11:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"EJNMMI Radiopharmacy and Chemistry","date":"2026-03-06T04:35:32+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":"e7742908-2434-4d0a-8ddc-ae1a789d39c4","owner":[],"postedDate":"March 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-04T10:48:43+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-13 12:59:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8944286","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8944286","identity":"rs-8944286","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}