Development and optimization of [68Ga]Ga-DOTA-EMP-100 for non-invasive PET imaging and targeted radioligand therapy of c-MET overactivation in cancer

Development and optimization of [68Ga]Ga-DOTA-EMP-100 for non-invasive PET imaging and targeted radioligand therapy of c-MET overactivation in cancer | 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 and optimization of [68Ga]Ga-DOTA-EMP-100 for non-invasive PET imaging and targeted radioligand therapy of c-MET overactivation in cancer Silvia Migliari, Anna Gagliardi, Alessandra Guercio, Maura Scarlattei, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7127095/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background. Overactivation of the HGF/c-MET pathway is implicated in various cancers, making its inhibition a promising therapeutic strategy. While several MET-targeting agents are currently approved or in advanced clinical development, patient selection often relies on invasive tissue-based assays. The development of a specific c-MET radioligand for PET imaging and radioligand therapy represents a non-invasive alternative, enabling real-time monitoring of target expression and offering a pathway to personalized treatment. Results. An optimized formulation of [⁶⁸Ga]Ga-DOTA-EMP-100, using 40 µg of precursor, provided the best outcome in terms of radiochemical performance. Process validation across three independent productions confirmed a consistent radiochemical yield of 64.5%, high radiochemical purity (100%), and a molar activity of 53.41 GBq/µmol. Conclusions. [⁶⁸Ga]Ga-DOTA-EMP-100 was successfully synthesized with high purity and reproducibility, supporting its potential for multi-dose application in clinical PET imaging and targeted radioligand therapy. [68Ga]Ga-radiopharmaceuticals [68Ga]Ga-DOTA-EMP-100 HGF/c-MET pathway PET imaging Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The cellular mesenchymal-epithelial transcription factor (c-MET) is a transmembrane receptor tyrosine kinase, encoded by the MET gene in humans. It is primarily involved in various cellular processes, including growth, differentiation, motility, and survival, particularly in response to its ligand hepatocyte growth factor (HGF), also known as scatter factor [ 1 ]. The binding of HGF and c-MET triggers several downstream signaling pathways such as phosphoinositide 3-kinase/threonine-protein kinase (PI3K/AKT) pathway, wingless-related integration site (Wnt) pathway, and other tumor-related functions [ 2 – 5 ]. The HGF/c-MET intracellular signalling pathway promotes cellular growth, invasion, and migration, which are important in normal development as well as in cancer progression [ 2 – 4 ] and it is significantly overactivated in several solid cancers. [ 6 ]. An increasing number of studies have confirmed that inhibition of HGF/c-MET signalling is an effective therapeutic strategy for suppression of multiple human cancers, such as non-small cell lung cancer (NSCLC), hepatocellular carcinoma (HCC), gastric cancer, colorectal cancer, ovarian cancer, bladder cancer, head and neck cancer and cervical cancer [2; 7–12] High c-MET levels are associated with poorer overall and progression-free survival [ 13 ]. MET amplification, overexpression, and super-activation have been implicated in chemotherapy resistance and consequently, c-MET inhibition to overcome resistance has been explored as a single agent or in combination with chemotherapy [ 14 ]. However, most clinical trials testing c-MET inhibitors in cancer have yielded inconsistent results and the lack of reliable biomarkers to identify responsive patients pose challenges [ 15 – 16 ]. Currently, patient eligibility for targeted MET therapy is determined by tissue-based assays, as immunostaining, FISH and NGS which have many limitations, including sampling bias due to the intratumoral and intertumoral heterogeneity, temporal and spatial heterogeneity, in addition to sampling difficulties on inaccessible sites or multisampling, such as the brain. Molecular imaging using positron emission tomography (PET) by enabling in vivo visualisation and quantification of cellular and subcellular mechanisms using targeted radioligands, may overcome limits of tissue based assays. PET detects radioligands in tissues with high sensitivity, at the picomolar level, providing a noninvasive, real-time map of target expression throughout the body. Finally, PET signals can be assessed quantitatively using standardised uptake value (SUV) metrics or derived quantification methods. A number of PET probes for imaging c-MET have been reported, and these are based on the HGF ligand, antibodies, peptides, and small molecules [ 17 ]. Several radiopharmaceuticals (RPs) targeting the MET pathway, including antibodies, peptides, and small molecules, have been radiolabeled for cancer detection ([64Cu]Cu-NOTA-rh-HGF, [89Zr]Zr-onartuzumab, [18F]F-AH113804, [11C]C-SU11274) [ 18 – 22 ], but these are not yet used in routine clinical practice. Current targeted therapies for the MET pathway primarily include tyrosine kinase inhibitors such as crizotinib, capmatinib, and tepotinib [ 23 ], along with the MET-specific monoclonal antibody onartuzumab [ 24 – 25 ] and the antibody-drug conjugate ABBV-399 [ 26 ]. The development of a specific c-MET radioligand for PET imaging could nonetheless pave the way for radioligand therapies, similar to how PSMA ligands are used in prostate cancer [ 27 ] or somatostatin ligands in neuroendocrine tumors [ 28 ]. Peptide probes based on the structure of EMI-137, a clinical stage optical imaging agent, appear most promising [ 29 ]. One of the radiopharmaceuticals in this group is EMP-100, a water-soluble 26-amino acid cyclic oligopeptide. It binds with nanomolar affinity (3.0 ± 0.5 nM) to the human c-MET receptor, as determined by fluorescence polarization [ 30 ]. By conjugating EMP-100 to a DOTA chelator, [68Ga]Ga-DOTA-EMP-100 is developed as a PET ligand, building upon the same c-MET binding peptide used in EMI-137 [ 29 ]. The peptide binding moiety was initially identified using phage display technology, selecting for binding to the extracellular domain of c-Met in the presence of its endogenous ligand, hepatocyte growth factor (HGF). This results in the peptide binding to a distinct site on the c-Met receptor, separate from HGF, ensuring high specificity for human c-Met across various conjugates. Crucially, the peptide does not compete with the native ligand nor interfere with the HGF/c-Met signaling pathway. Similarly, radiolabeling EMP-100 with Ga-68 does not impact receptor activation, proliferation, or phosphorylation within the HGF/c-Met pathway and shows no significant off-target binding across a panel of 70 therapeutically relevant receptors (yet unpublished data) [ 29 ]. [68Ga]Ga-DOTA-EMP-100 has already been administered in forty-two clinical cases without any observed adverse effects, delivering promising imaging results in metastatic renal cell carcinoma (mRCC), non-small cell lung cancer (NSCLC), and hepatocellular carcinoma [ 29 ]. The goal of the present research is to develop an automated radiosynthesis method for standardized production of the radioligand clinical batches. Automation improves consistency, quality, and operator safety in producing radiopharmaceuticals, facilitating clinical translation. Finally, we will validate the radiosynthesis and quality control methods to produce [68Ga]Ga-DOTA-EMP-100 using the GMP-grade precursor, proving that any used procedure, process, equipment, material, activity or system leads to the expected results. Results Labeling and quality control results for different DOTA-EMP-100 loads and the validated synthesis The fully automated production of [68Ga]Ga-DOTA-EMP-100 was conducted by a scale down method from 50 µg to 20 µg different precursor amounts of DOTA-EMP-100 (50-40-30-20 µg) of peptide precursor for radiolabelling with gallium-68. Immediately after each synthesis the overall QCs of the final product were performed in order to evaluate the best setup conditions and to determine the best precursor amount from which to start to obtain [68Ga]Ga-DOTA-EMP-100, guaranteeing the highest and optimal paramount parameters as well as to optimize the entire production process. As shown in Table 1 the precursor amount of 40 µg allows the best radiochemical purity (100%), high radiochemical yield (64.93%) (n.d.c.) as well as good molar activity (53.075 GBq/µmol). Consequently, once the automated synthesis process is optimized, the production procedures will be validated using 40 µg of peptide precursor, in compliance with regulatory standards to ensure the robustness of the gallium-68 labelling methods for DOTA-EMP-100. Some of the quality control (QC) parameters tested were based on the European Pharmacopoeia (11.0/0125) (Table 2 ). The radiochemical purity (RCP% = 100% - colloids - ions) was assessed by checking for the presence of free gallium (using Radio-UV-HPLC) and gallium colloids (using Radio-TLC). Using Radio-UV-HPLC, free gallium-68 was identified at Rt = 1.433 min (Fig. 1 a), while gallium-68 bound to DOTA-EMP-100 was detected at Rt = 7.620 min (Fig. 1 b), with a purity of 100%. Table 1 Summary data of [68Ga]Ga-DOTA-EMP-100 quality controls (20–50 µg, n = 3) Peptide (DOTA-EMP-100) PM = 3709.7 g/mol 50 µg (500 µl, 0.0135 µmol) 40 µg (400 µl, 0.0108µmol) 30 µg (300 µl, 0.008 µmol) 20 µg (200 µl, 0.005 µmol) Radiochemical purity (Radio-UV-HPLC) 99.31% 100,00% 99,73% 99,52% Radiochemical purity (Radio-TLC) 100% 100% 100% 100% pH 7 7 7 7 Radiochemical yield (n.d.c) 68,17% 64.93% 64,56% 54,75% Volume 10 10 10 10 Colour Colourless Colourless Colourless Colourless Molar activity 37.42 GBq/µmol 53.08 GBq/µmol 64,63 GBq/µmol 83,80 GBq/µmol Table 2 Summary data of three consecutive validation batches of [68Ga]Ga-DOTA-EMP-100 (40 µg) Test Batch 1 Batch 2 Batch 3 Acceptance criteria Radiochemical purity (Radio-UV-HPLC) 99.31% 100,00% 99,73% > 95% Radiochemical purity (Radio-TLC) 100% 100% 100% > 95% pH 7 7 7 4–8.5 Radiochemical yield (n.d.c) 64,37% 64.58% 64,56% > 40% Radioactivity concentration 75.6-52.48 75.6-52.77 75.6–52.70 > 50 MBq Radioactivity 756–524.82 756–527.67 756–527.03 > 150 MBq Volume 10 10 10 2–10 mL Colour Colourless Colourless Colourless Colourless Molar activity 53.26 GBq/µmol 53.52 GBq/µmol 53.46 GBq/µmol 1–60 GBq/µmol Radionuclidic purity 100% 100% 100% > 99.9% Ge-68 breakthrough 0.00000036% 0.00000033% 0.00000035% < 0.001% EtOH amount 3.73% 3.68% 3.45% < 10% (V/V) (< 2.5 g) HEPES content 9.45 µg/mL 9.45 µg/mL 9.45 µg/mL Less than 200 µg/V of HEPES in test solution Endotoxins < 17.5 IU/mL < 17.5 IU/mL < 17.5 IU/mL 95% The reference solution of DOTA-EMP-100 exhibited a slightly different retention time (Rt = 7.428 min) compared to [68Ga]Ga-DOTA-EMP-100, which had a retention time of Rt = 7.620 min, as shown in Fig. 1 c. The difference in retention times is attributed to the use of different detectors (Radio and UV–VIS), as well as to a variation in the charge of the DOTA chelator following the incorporation of gallium-68. This change in charge affects the interaction with the column, leading to a slight alteration in the hydrophobicity of the entire molecule. The absence of the 68Ga-chelate in the standard solution leaves three free carboxylic acid groups, increasing the hydrophilicity of the standard and consequently modifying its retention time. With Radio-TLC no [68Ga]Ga-colloids could be detected at Rf = 0.2 and the radiopharmaceutical product was detected at Rf = 0.8 (Fig. 2 ). HPLC performed on the final radiopharmaceutical solution showed that the residual content of HEPES in the final preparation was lower in the HEPES test solution (12.5 µg/mL) (Fig. 3 ). Additionally, the product was tested for endotoxins, and the concentration was found to be below 17.5 EU/mL in all samples. Sterility testing was conducted on all samples, and they resulted sterile. The stability of [68Ga]Ga-DOTA-EMP-100 in buffer solution at room temperature was evaluated for up to 4 hours using Radio-UV-HPLC, RadioTLC, and pH measurements. As shown in Fig. 4 , [68Ga]Ga-DOTA-EMP-100 remains stable under the test conditions. No additional radioactive by-products or free gallium-68 were detected during this period, and the RCP% stayed 100% over time. The RCP% was also assessed and confirmed by Radio-TLC (Fig. 5 ), while the pH value remained stable at 7 throughout the 4-hour period. Discussion The c-MET protein, a mesenchymal-epithelial transcription factor, is a transmembrane receptor tyrosine kinase encoded by the human MET gene. It is involved in various cellular functions, including growth, differentiation, motility, making it an active target for drug discovery and development. We hereby describe the development and validation of an automated synthesis method and QC system to label c-MET ligand (DOTA-EMP-100) with gallium-68. The process of developing and designing a new radiopharmaceutical typically involves the establishment and setup of the radiosynthesis, along with the implementation of quality assessment methods for the final product. These assessments include evaluating release specifications such as RCP%, specific activity (As or Am), radionuclidic purity, chemical purity, radiochemical yield (RCY%), pH, sterility, and stability. A critical parameter in this process is the specific activity or molar activity of the final product, which refers to the ratio of the labeling isotope (in Bq) to the amount of peptide (in grams) or the unit mole of the compound (mol). For optimal synthesis setup, high molar activity (Am) is crucial, as it affects the peptide's receptor affinity and available receptor sites, limiting the peptide dose. A suboptimal Am can lead to receptor saturation or side effects. However, Am should not be excessively high, as sufficient peptide mass is needed to ensure a good radiochemical yield and reliable biodistribution for imaging. For these reasons, five different amounts of DOTA-EMP-100 (50-40-30-20 µg) were evaluated for the production of [68Ga]Ga-DOTA-EMP-100 considering the affinity and specificity of the c-Met peptide EMP-100 Kd 3.0 ± 0.5 nM [ 30 ]. The results summarized in Table 1 demonstrate that the amount of 40 µg of DOTA-EMP-100 allows the best radiochemical purity (100%), high radiochemical yield (64.93%) (n.d.c.) as well as good molar activity (53.075 GBq/µmol). We noticed an increasing radiochemical yield corresponding to increasing ligand amount up to 50 µg, but a lower RCP% for all the amount peptide precursor except for 40 µg (Table 1 ), allowing to determine the best amount of peptide for the production of [68Ga]Ga-DOTA-EMP-100. The higher molar activity (83,80 GBq/µmol) was observed for 20 µg of peptide precursor, but the lower radiochemical yield (54,75%) and radiochemical purity (99,52%) lead us to validate the radiosynthesis of [68Ga]Ga-DOTA-EMP-100 starting from 40 µg of DOTA-EMP-100. Standardization and harmonization of radiopharmaceutical production are essential for ensuring that radiopharmaceutical research can be reliably tested and transferred across laboratories. After optimizing the method, the process and final product must be validated, as required by regulations, to confirm the robustness of the gallium-68 labeling method for EMP-100, ensuring its clinical applicability and compliance with established standard operating procedures. As seen in Table 2 , the three consecutive syntheses and QC results show mean values of RCP% of 100%, confirmed by both Radio-TLC and Radio-UV-HPLC, radiochemical yield of 64.50% and a molar activity (Am) of 53.41 GBq/µmol. Moreover, we achieved an endotoxin-free, sterile and stable solution of [68Ga]Ga-DOTA-EMP-100 maintaining a radiochemical purity of 100% over a period of 4 h. All quality control parameters tested were in accordance with the standards set by the European Pharmacopoeia (Ph.Eur.), confirming the high reproducibility of the [68Ga]Ga-DOTA-EMP-100 production method. This ensures that it is not only safe but also suitable for daily production and transfer to other radiopharmaceutical laboratories. Based on these results, [68Ga]Ga-DOTA-EMP-100 holds significant potential for evaluating therapeutic strategies involving tyrosine kinase inhibitors by assessing c-MET expression at tumor sites prior to systemic treatments. Additionally, preclinical evaluation of this novel radiotracer could be instrumental in detecting altered c-MET expression, aiding in the identification of associated pathologies and advancing its potential clinical applications, as demonstrated by its use in metastatic renal cell carcinoma (mRCC) [ 29 ]. With the incorporation of a DOTA-chelator for EMP-100, a theranostic approach is now a feasible option. This opens up promising opportunities, such as the use of cMET-PET to assess uptake intensity before potential therapy with 177Lu-DOTA-labeled ligands targeting c-MET, a strategy currently under investigation. Conclusions The synthesis of [68Ga]Ga-DOTA-EMP-100 was successfully carried out through a fully automated process using the GRP Scintomics module. All quality control parameters, including radiochemical purity, pH, endotoxins, and sterility, were in compliance with the European Pharmacopoeia standards. Additionally, the product solution demonstrated stability for at least 3 hours after production, as confirmed by Radio-UV-HPLC. As a result, [68Ga]Ga-DOTA-EMP-100 can be consistently and efficiently produced for routine clinical applications. Materials and methods Materials The precursor to [68Ga]Ga-DOTA-EMP-100, namely EMP-100 was purchased from Edinburgh Molecular Imaging Ltd. An aqueous stock solution of 1 mg/mL was prepared and kept at -20°C. All chemicals used for the radiolabelling reaction, i.e. saline (NaCl), ethanol, 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid (HEPES) buffer solution, PBS buffer solution and water were of the highest available purity grade and commercially obtained as a single disposable kit (SC-01, ABX Radeberg, Germany). All chemicals used to perform quality controls, i.e. trifluoroacetic acid (TFA), water and acetonitrile used for Radio-UV-HPLC, as well as ammonium acetate and methanol, were metal-free and purchased from Sigma Aldrich (Saint Louis, Missouri, USA). All the medicinal products used in this study are commercially available and authorized for clinical use. An automated synthesis module (Scintomics GRP® module, Germany) equipped with a disposable single-use cassette (SC-01, ABX) and a pharmaceutical grade, GMP certified and compliant with European Pharmacopoeia 68Ge/68Ga generator (1850 MBq, GalliaPharm® Eckert & Ziegler, Berlin, Germany) were used and both placed in a GMP grade A hot cell (NMC Ga-68, Tema Sinergie) to assess aseptic production. The amount of detected metal impurities/68Ge breakthrough as provided by the manufacturer was less than the defined limit in the European Pharmacopeia monograph [ 31 ]. Activity counting was determined using a borehole counter (CRC® 25-PET, Capintec). Radio-UV-HPLC was performed using a Dionex Ultimate 3000 HPLC system (Thermo Fisher Scientific) equipped with a BioBasic-18 column 5µm 300Å (250 mm × 4.6 mm) and coupled with UV and a γ-detector (Berthold Technologies, Milan, Italy). The Radio-TLC scanner used was Cyclone® Plus Storage Phosphor system (Perkin Elmer). The test for endotoxins was performed with Nexgen PTS (Charles River) Radiosynthesis The synthesis template was identical to the already established synthesis template for [68Ga]Ga-PSMA [ 32 ] as well as [68Ga]Ga-DOTA-ECL1i [ 33 ]. The elution of 68Ge/68Ga generator (GalliaPharma®) was carried out using the GRP module 3 V automated synthesis system (Scintomics GRP® module). The generator was eluted with 0.1 M HCl 24 h before labelling to remove the accumulated stable Zn-68 from Ga-68 decay and the elute [68Ga]GaCl 3 , obtained from the generator elution, was pre-concentrated on a strong cation exchange (SCX) cartridge, which separates the ions based on their net total surface area change. [68Ga]GaCl 3 was recovered from the SCX by the eluent 5 M NaCl. The eluate was transferred into the reaction vial, previously loaded with DOTA-EMP-100 ( 20–30–40–50 µg in 1.5 M 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid (HEPES buffer solution) at pH = 4–4.5. The mixture was incubated at 95°C for 10 min. After the completion of the labeling reaction the crude product was cooled down and trapped onto Sep Pak C18 RP cartridge, washed with water for injection Ph. Eur., and eluted with 2 mL of Ethanol/Water 1/1. The final product was diluted with phosphate buffered saline (PBS) and sterilized through a 0.2 µm filter (millex GV) into a sterile 25 mL capped glass vial and diluted with PBS for the final formulation. The entire radiopharmaceutical production takes 35 min. Quality control and process validation To ensure that the final injectable radiopharmaceutical product fulfils regulatory requirements relating to contaminants, suitable production and quality control are crucial [ 34 ]. After synthesis, the radiopharmaceutical product was evaluated determining the following parameters: total product activity, gallium-68 ion identity via half-life time and gamma spectroscopy, chemical and radiochemical purity by Radio-UV-HPLC and Radio-TLC, pH, radionuclide purity for 68Ge-breakthrough and sterility/endotoxin assay (sterility test and LAL test). The radiochemical purity and the stability of [68Ga]Ga-EMP-100 at room temperature was evaluated by Radio-TLC and Radio-UV-HPLC for 4 h. For Radio-TLC, ITLC-SG (8-cm length, 1 cm thick) (Agilent Technologies) was used as stationary phase and ammonium acetate/methanol (1/1) as mobile phase. The software OptiQuantTM was used to analyse the chromatograms. The percentages of each fraction were determined relative to the total activity of the chromatogram. For Radio-UV-HPLC a standardized method was performed. Flow rate of the mobile phase was set at 0.6 mL/min, and the mobile phases used were A) 0.1% TFA in water and B) 0.1% TFA in acetonitrile, following a phase gradient: 0-1.7 min 0% B, 1.7-9 min 70% Band 9–12 min 3% B. The column temperature was kept at 25°C and the samples were also monitored with an UV detector at 220 nm to detect chemical impurities in the final product. The software system Chromeleon 7 was used to assemble the information. Reference solutions of [68Ga]GaCl 3 , DOTA-EMP-100 and the final radiopharmaceutical [68Ga]Ga-DOTA-EMP-100 were assessed using the same analytical conditions. The chemical purity of [68Ga]Ga-DOTA-EMP-100 concerning the residual HEPES content was assessed according to Ph. Eur. Monograph (Gallium 2482), following our validated HPLC method [ 35 ]. For HPLC a Waters Xbridge® column C18 (150 mm×4.6 mm, 3.5 µm), as stationary phase was used, connected to an UV detector set to a wavelength of 195 nm and a γ-detector (Berthold Technologies, Milan, Italy) and ammonium formate 20 mM pH 9.5, as mobile phase, at an isocratic flow of 0.7 mL/min. The sterility tests were performed as described in the European Pharmacopoeia (EMA/CHMP/ICH/645592/2008) and LAL test with Nexgen PTS (Charles River). To validate the entire process of radiopharmaceutical production and quality control, three batches of [68Ga]Ga-DOTA-EMP-100 were produced in three different days under the same conditions set for typical routine preparations. Every batch was fully characterized from the analytical point of view, with the aim to verify that the product met the acceptance criteria for all the established quality parameters. Abbreviations As specific activity CT Computed Tomography GMP Good Manufacturing Practice GRP Good Radiopharmaceutical Practices EANM European Association of Nuclear Medicine Eur. Ph. European Pharmacopeia HEPES 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid HPLC High Pressure Liquid Chromatography NBP-MN Norme di Buona Preparazione in Nuclear Medicine TLC Thin Layer Chromatography TFA trifluoroacetic acid PET Positron Emission Tomography QC quality control RCY radiochemical yield RPC radiochemical purity Declarations Acknowledgements Not applicable Availability of data and materials Not applicable. Authors’ contributions SM, AG, AG, MS, GB, AG, CP and LV have contributed to the organization of the content for this manuscript. SM, AG, AG, CP and AG collected relevant information and prepared the draft. SM, AG, AG, MS, GB, AG and CP drafted and LV revised the manuscript. Funding This study did not receive any financial support. Competing interests The authors declare that they have no competing interests. Consent for publication Not applicable Ethics approval and consent to participate Not applicable. References Fu J, Su X, Li Z, Deng L, Liu X, Feng X, Peng J. HGF/c-MET pathway in cancer: from molecular characterization to clinical evidence. Oncogene. 2021;40(28):4625–51. 10.1038/s41388-021-01863-w . Epub 2021 Jun 18. PMID: 34145400. Konstorum A, Lowengrub JS. Activation of the HGF/c-Met axis in the tumor microenvironment: a multispecies model. J Theor Biol. 2018;439:86–99. 2. Boromand N, Hasanzadeh M, ShahidSales S, Farazestanian M, Gharib M, Fiuji H, Behboodi N, Ghobadi N, Hassanian SM, Ferns GA, et al. Clinical and prognostic value of the C-Met/HGF signaling pathway in cervical cancer. 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In vivo positron emission tomography (PET) imaging of mesenchymal – epithelial transition (MET) receptor. J Med Chem. 2010;53(1):139–46. Remon J, Hendriks LEL, Mountzios G, García-Campelo R, Saw SPL, Uprety D, et al. MET alterations in NSCLC—current perspectives and future challenges. J Thorac Oncol. 2023;18(4):419–35. Spigel DR, Edelman MJ, O’Byrne K, Paz-Ares L, Mocci S, Phan S, et al. Results from the phase III randomized trial of onartuzumab plus erlotinib versus erlotinib in previously treated stage IIIB or IV non-small-cell lung cancer: METLung. JCO. 2017;35(4):412–20. Comoglio PM, Trusolino L, Boccaccio C. Known and novel roles of the MET oncogene in cancer: a coherent approach to targeted therapy. Nat Rev Cancer. 2018;18:341–58. Wang J, Anderson MG, Oleksijew A, Vaidya KS, Boghaert ER, Tucker L, et al. ABBV-399, a c-Met antibody-drug conjugate that targets both MET-amplified and c-Met-overexpressing tumors, irrespective of MET pathway dependence. Clin Cancer Res. 2017;23(4):992–1000. Sartor O, de Bono J, Chi KN, Fizazi K, Herrmann K, Rahbar K, et al. Lutetium-177–PSMA-617 for metastatic castration-resistant prostate cancer. N Engl J Med. 2021;385(12):1091–103. Strosberg J, El-Haddad G, Wolin E, Hendifar A, Yao J, Chasen B, et al. Phase 3 trial of 177Lu-dotatate for midgut neuroendocrine tumors. N Engl J Med. 2017;376(2):125–35. Mittlmeier LM, Todica A, Gildehaus FJ, Unterrainer M, Beyer L, Brendel M, Albert NL, Ledderose ST, Vettermann FJ, Schott M, Rodler S, Marcon J, Ilhan H, Cyran CC, Stief CG, Staehler M, Bartenstein P. 68Ga-EMP-100 PET/CT-a novel ligand for visualizing c-MET expression in metastatic renal cell carcinoma-first in-human biodistribution and imaging results. Eur J Nucl Med Mol Imaging. 2022;49(5):1711–20. 10.1007/s00259-021-05596-6 . Epub 2021 Oct 28. PMID: 34708249; PMCID: PMC8940803. Rusu T, Delion M, Pirot C, Blin A, Rodenas A, Talbot JN, Veran N, Portal C, Montravers F, Cadranel J, Prignon A. Fully automated radiolabeling of [68Ga]Ga-EMP100 targeting c-MET for PET-CT clinical imaging. EJNMMI Radiopharm Chem. 2023;8(1):30. 10.1186/s41181-023-00213-3 . PMID: 37843660; PMCID: PMC10579204. Gallium Chloride. (68Ga) solution for labeling (Monograph 2464) In: European Pharmacopoeia European Directorate for the Quality of Medicines < za Migliari S, Sammartano A, Scarlattei M, Serreli G, Ghetti C, Cidda C, Baldari G, Ortenzia O, Ruffini L. Development and Validation of a High-Pressure Liquid Chromatography Method for the Determination of Chemical Purity and Radiochemical Purity of a [. ACS Omega. 2017;2(10):7120–6. 10.1021/acsomega.7b00677 . Epub 2017 Oct 25. PMID: 29520394; PMCID: PMC5837251. Migliari S, Scarlattei M, Baldari G, Ruffini L. Scale down and optimized automated production of [68Ga]68Ga-DOTA-ECL1i PET tracer targeting CCR2 expression. EJNMMI Radiopharm Chem. 2023;8(1):3. 10.1186/s41181-023-00188-1 . PMID: 36729317; PMCID: PMC9895323. European Pharmacopoeia Commission. Council of Europe. European pharmacopoeia. 8th ed. Strasbourg: European Directorate for the Quality of Medicines & Health Care; 2013. Migliari S, Scarlattei M, Baldari G, Silva C, Ruffini L. A specific HPLC method to determine residual HEPES in [68Ga]Ga radiopharmaceuticals: development and validation. Molecules. 2022;27(14):4477. https://doi.org/10.3390/molec ules27144477 . (PMID: 35889351 PMCID: PMC9323806. Cite Share Download PDF Status: Posted Version 1 posted 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7127095","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":486159191,"identity":"a7ba2528-ae70-4290-ac8f-4156ee658465","order_by":0,"name":"Silvia Migliari","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1klEQVRIiWNgGAWjYFACxgaGBwYMDGzsDRA+G1FaEkBaeA5AtRChh4EhAURIJDAQZ438tMPNLxIK7PL5JB8/fMC44x4Dn3wDfi0GtxPbLBIMki3bpNOMDRjPFBN2mIF0YptBggGzAZt0gpkEY1sCYS3ys8Fa6g3YJI9/I04Lw+3E5gcJBocN2CR4iLQF5BdgIB83YOPJKTZIPJPAw8aWQMhh6Y8/fPhTbSDffnzjg487EuTkmw8QchkDmwScmdjAwENQPRAwf4AzgSlhFIyCUTAKRgEGAAC4czkIKxoOGwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-2082-773X","institution":"University Hospital of Parma: Azienda Ospedaliero-Universitaria di Parma","correspondingAuthor":true,"prefix":"","firstName":"Silvia","middleName":"","lastName":"Migliari","suffix":""},{"id":486159192,"identity":"b9bce867-1424-43d5-a6d9-9e79aaae8153","order_by":1,"name":"Anna Gagliardi","email":"","orcid":"","institution":"University Hospital of Parma: Azienda Ospedaliero-Universitaria di Parma","correspondingAuthor":false,"prefix":"","firstName":"Anna","middleName":"","lastName":"Gagliardi","suffix":""},{"id":486159193,"identity":"77d6b422-ac73-4732-9759-20ef254e875c","order_by":2,"name":"Alessandra Guercio","email":"","orcid":"","institution":"University Hospital of Parma: Azienda Ospedaliero-Universitaria di Parma","correspondingAuthor":false,"prefix":"","firstName":"Alessandra","middleName":"","lastName":"Guercio","suffix":""},{"id":486159194,"identity":"490d67ad-a408-412d-a612-7b958add84f6","order_by":3,"name":"Maura Scarlattei","email":"","orcid":"","institution":"University Hospital of Parma: Azienda Ospedaliero-Universitaria di Parma","correspondingAuthor":false,"prefix":"","firstName":"Maura","middleName":"","lastName":"Scarlattei","suffix":""},{"id":486159195,"identity":"912ef97c-ed74-4fcf-8468-9511211850ac","order_by":4,"name":"Giorgio Baldari","email":"","orcid":"","institution":"University Hospital of Parma: Azienda Ospedaliero-Universitaria di Parma","correspondingAuthor":false,"prefix":"","firstName":"Giorgio","middleName":"","lastName":"Baldari","suffix":""},{"id":486159196,"identity":"48aaefd6-8945-4ff0-a7cf-fe8702fbe10c","order_by":5,"name":"Alex Gibson","email":"","orcid":"","institution":"Edinburgh Molecular Imaging Ltd","correspondingAuthor":false,"prefix":"","firstName":"Alex","middleName":"","lastName":"Gibson","suffix":""},{"id":486159197,"identity":"1d7ace99-2c0e-456d-bb26-7781ff8bc7cc","order_by":6,"name":"Christophe Portal","email":"","orcid":"","institution":"Edinburgh Molecular Imaging Ltd","correspondingAuthor":false,"prefix":"","firstName":"Christophe","middleName":"","lastName":"Portal","suffix":""},{"id":486159198,"identity":"0c3ba2f2-6427-4cff-9452-6aaf827d964d","order_by":7,"name":"Livia Ruffini","email":"","orcid":"","institution":"University Hospital of Parma: Azienda Ospedaliero-Universitaria di Parma","correspondingAuthor":false,"prefix":"","firstName":"Livia","middleName":"","lastName":"Ruffini","suffix":""}],"badges":[],"createdAt":"2025-07-15 06:41:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7127095/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7127095/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87360630,"identity":"99915ae4-53f2-4cce-92e5-7bceb541f578","added_by":"auto","created_at":"2025-07-23 05:48:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":33788,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Radio-UV-HPLC chromatogram of the eluate [68Ga]GaCl\u003csub\u003e3\u003c/sub\u003e; (b) Radio-UV-HPLC chromatogram of [68Ga]Ga-DOTA-EMP-100; (c) Radio-UV-HPLC chromatogram of DOTA-EMP-100.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7127095/v1/3e52a8b1b9e4b47adf31aaf5.png"},{"id":87363303,"identity":"b2d0e35e-4c4e-4124-bc00-b7a1255807cc","added_by":"auto","created_at":"2025-07-23 06:04:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":189902,"visible":true,"origin":"","legend":"\u003cp\u003eRadio-TLC chromatogram of [68Ga]Ga-DOTA-EMP-100.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7127095/v1/00ca611f1b4c88937bbfc0f0.png"},{"id":87360631,"identity":"18e7cf83-0315-413b-8175-5ef7888356c7","added_by":"auto","created_at":"2025-07-23 05:48:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":39640,"visible":true,"origin":"","legend":"\u003cp\u003eHPLC chromatogram of HEPES content in [68Ga]Ga-DOTA-EMP-100.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7127095/v1/8a3f986751533afae9270138.png"},{"id":87360632,"identity":"e8889516-08d3-4a6d-a78e-ed0af132e40b","added_by":"auto","created_at":"2025-07-23 05:48:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":128895,"visible":true,"origin":"","legend":"\u003cp\u003eStability of [68Ga]Ga-DOTA-EMP-100 tested with Radio-UV-HPLC (blue dash: 0 h, pink dash: 1 h, red dash: 2 h, green dash: 3 h and black dash: 4h)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7127095/v1/8f10b59957bc53d656e8816b.png"},{"id":87364123,"identity":"d4ed053a-3a3e-40cb-8e19-e74bb34055d8","added_by":"auto","created_at":"2025-07-23 06:12:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":401801,"visible":true,"origin":"","legend":"\u003cp\u003eStability of [68Ga]Ga-DOTA-EMP-100 tested with Radio-TLC (pink dash: 0 h, red dash: 1 h, green dash: 2 h, blue dash: 3 h and yellow dash: 4h)\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7127095/v1/f507835abe3cf737cef414a1.png"},{"id":88251409,"identity":"35843de5-5bbf-450f-a91f-5d4b11e61a2a","added_by":"auto","created_at":"2025-08-04 13:37:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1455542,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7127095/v1/46b75baf-8c81-4ae7-b05b-017e65d14b06.pdf"}],"financialInterests":"","formattedTitle":"Development and optimization of [68Ga]Ga-DOTA-EMP-100 for non-invasive PET imaging and targeted radioligand therapy of c-MET overactivation in cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe cellular mesenchymal-epithelial transcription factor (c-MET) is a transmembrane receptor tyrosine kinase, encoded by the MET gene in humans. It is primarily involved in various cellular processes, including growth, differentiation, motility, and survival, particularly in response to its ligand hepatocyte growth factor (HGF), also known as scatter factor [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe binding of HGF and c-MET triggers several downstream signaling pathways such as phosphoinositide 3-kinase/threonine-protein kinase (PI3K/AKT) pathway, wingless-related integration site (Wnt) pathway, and other tumor-related functions [\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The HGF/c-MET intracellular signalling pathway promotes cellular growth, invasion, and migration, which are important in normal development as well as in cancer progression [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] and it is significantly overactivated in several solid cancers. [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAn increasing number of studies have confirmed that inhibition of HGF/c-MET signalling is an effective therapeutic strategy for suppression of multiple human cancers, such as non-small cell lung cancer (NSCLC), hepatocellular carcinoma (HCC), gastric cancer, colorectal cancer, ovarian cancer, bladder cancer, head and neck cancer and cervical cancer [2; 7\u0026ndash;12]\u003c/p\u003e\u003cp\u003eHigh c-MET levels are associated with poorer overall and progression-free survival [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. \u003cem\u003eMET\u003c/em\u003e amplification, overexpression, and super-activation have been implicated in chemotherapy resistance and consequently, c-MET inhibition to overcome resistance has been explored as a single agent or in combination with chemotherapy [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, most clinical trials testing c-MET inhibitors in cancer have yielded inconsistent results and the lack of reliable biomarkers to identify responsive patients pose challenges [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Currently, patient eligibility for targeted MET therapy is determined by tissue-based assays, as immunostaining, FISH and NGS which have many limitations, including sampling bias due to the intratumoral and intertumoral heterogeneity, temporal and spatial heterogeneity, in addition to sampling difficulties on inaccessible sites or multisampling, such as the brain.\u003c/p\u003e\u003cp\u003eMolecular imaging using positron emission tomography (PET) by enabling \u003cem\u003ein vivo\u003c/em\u003e visualisation and quantification of cellular and subcellular mechanisms using targeted radioligands, may overcome limits of tissue based assays. PET detects radioligands in tissues with high sensitivity, at the picomolar level, providing a noninvasive, real-time map of target expression throughout the body. Finally, PET signals can be assessed quantitatively using standardised uptake value (SUV) metrics or derived quantification methods. A number of PET probes for imaging c-MET have been reported, and these are based on the HGF ligand, antibodies, peptides, and small molecules [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSeveral radiopharmaceuticals (RPs) targeting the MET pathway, including antibodies, peptides, and small molecules, have been radiolabeled for cancer detection ([64Cu]Cu-NOTA-rh-HGF, [89Zr]Zr-onartuzumab, [18F]F-AH113804, [11C]C-SU11274) [\u003cspan additionalcitationids=\"CR19 CR20 CR21\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], but these are not yet used in routine clinical practice. Current targeted therapies for the MET pathway primarily include tyrosine kinase inhibitors such as crizotinib, capmatinib, and tepotinib [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], along with the MET-specific monoclonal antibody onartuzumab [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and the antibody-drug conjugate ABBV-399 [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The development of a specific c-MET radioligand for PET imaging could nonetheless pave the way for radioligand therapies, similar to how PSMA ligands are used in prostate cancer [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] or somatostatin ligands in neuroendocrine tumors [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Peptide probes based on the structure of EMI-137, a clinical stage optical imaging agent, appear most promising [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. One of the radiopharmaceuticals in this group is EMP-100, a water-soluble 26-amino acid cyclic oligopeptide. It binds with nanomolar affinity (3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 nM) to the human c-MET receptor, as determined by fluorescence polarization [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. By conjugating EMP-100 to a DOTA chelator, [68Ga]Ga-DOTA-EMP-100 is developed as a PET ligand, building upon the same c-MET binding peptide used in EMI-137 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The peptide binding moiety was initially identified using phage display technology, selecting for binding to the extracellular domain of c-Met in the presence of its endogenous ligand, hepatocyte growth factor (HGF). This results in the peptide binding to a distinct site on the c-Met receptor, separate from HGF, ensuring high specificity for human c-Met across various conjugates. Crucially, the peptide does not compete with the native ligand nor interfere with the HGF/c-Met signaling pathway. Similarly, radiolabeling EMP-100 with Ga-68 does not impact receptor activation, proliferation, or phosphorylation within the HGF/c-Met pathway and shows no significant off-target binding across a panel of 70 therapeutically relevant receptors (yet unpublished data) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. [68Ga]Ga-DOTA-EMP-100 has already been administered in forty-two clinical cases without any observed adverse effects, delivering promising imaging results in metastatic renal cell carcinoma (mRCC), non-small cell lung cancer (NSCLC), and hepatocellular carcinoma [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe goal of the present research is to develop an automated radiosynthesis method for standardized production of the radioligand clinical batches. Automation improves consistency, quality, and operator safety in producing radiopharmaceuticals, facilitating clinical translation. Finally, we will validate the radiosynthesis and quality control methods to produce [68Ga]Ga-DOTA-EMP-100 using the GMP-grade precursor, proving that any used procedure, process, equipment, material, activity or system leads to the expected results.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eLabeling and quality control results for different DOTA-EMP-100 loads and the validated synthesis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe fully automated production of [68Ga]Ga-DOTA-EMP-100 was conducted by a scale down method from 50 \u0026micro;g to 20 \u0026micro;g different precursor amounts of DOTA-EMP-100 (50-40-30-20 \u0026micro;g) of peptide precursor for radiolabelling with gallium-68. Immediately after each synthesis the overall QCs of the final product were performed in order to evaluate the best setup conditions and to determine the best precursor amount from which to start to obtain [68Ga]Ga-DOTA-EMP-100, guaranteeing the highest and optimal paramount parameters as well as to optimize the entire production process.\u003c/p\u003e\u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e the precursor amount of 40 \u0026micro;g allows the best radiochemical purity (100%), high radiochemical yield (64.93%) (n.d.c.) as well as good molar activity (53.075 GBq/\u0026micro;mol).\u003c/p\u003e\u003cp\u003eConsequently, once the automated synthesis process is optimized, the production procedures will be validated using 40 \u0026micro;g of peptide precursor, in compliance with regulatory standards to ensure the robustness of the gallium-68 labelling methods for DOTA-EMP-100. Some of the quality control (QC) parameters tested were based on the European Pharmacopoeia (11.0/0125) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe radiochemical purity (RCP% = 100% - colloids - ions) was assessed by checking for the presence of free gallium (using Radio-UV-HPLC) and gallium colloids (using Radio-TLC). Using Radio-UV-HPLC, free gallium-68 was identified at Rt\u0026thinsp;=\u0026thinsp;1.433 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), while gallium-68 bound to DOTA-EMP-100 was detected at Rt\u0026thinsp;=\u0026thinsp;7.620 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), with a purity of 100%.\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\u003eSummary data of [68Ga]Ga-DOTA-EMP-100 quality controls (20\u0026ndash;50 \u0026micro;g, n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePeptide (DOTA-EMP-100)\u003c/p\u003e\u003cp\u003ePM\u0026thinsp;=\u0026thinsp;3709.7 g/mol\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e50 \u0026micro;g (500 \u0026micro;l, 0.0135 \u0026micro;mol)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e40 \u0026micro;g (400 \u0026micro;l, 0.0108\u0026micro;mol)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e30 \u0026micro;g (300 \u0026micro;l, 0.008 \u0026micro;mol)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e20 \u0026micro;g (200 \u0026micro;l, 0.005 \u0026micro;mol)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRadiochemical purity (Radio-UV-HPLC)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e99.31%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e100,00%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e99,73%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e99,52%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRadiochemical purity (Radio-TLC)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e100%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e100%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e100%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003epH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRadiochemical yield (n.d.c)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e68,17%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e64.93%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e64,56%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e54,75%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVolume\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eColour\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eColourless\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eColourless\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eColourless\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eColourless\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMolar activity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e37.42 GBq/\u0026micro;mol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e53.08 GBq/\u0026micro;mol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e64,63 GBq/\u0026micro;mol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e83,80 GBq/\u0026micro;mol\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\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\u003eSummary data of three consecutive validation batches of [68Ga]Ga-DOTA-EMP-100 (40 \u0026micro;g)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTest\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBatch 1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBatch 2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eBatch 3\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAcceptance criteria\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRadiochemical purity (Radio-UV-HPLC)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e99.31%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e100,00%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e99,73%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;95%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRadiochemical purity (Radio-TLC)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e100%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e100%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;95%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003epH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4\u0026ndash;8.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRadiochemical yield (n.d.c)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e64,37%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e64.58%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e64,56%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;40%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRadioactivity concentration\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e75.6-52.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e75.6-52.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e75.6\u0026ndash;52.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;50 MBq\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRadioactivity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e756\u0026ndash;524.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e756\u0026ndash;527.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e756\u0026ndash;527.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;150 MBq\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVolume\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2\u0026ndash;10 mL\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eColour\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eColourless\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eColourless\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eColourless\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eColourless\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMolar activity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e53.26 GBq/\u0026micro;mol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e53.52 GBq/\u0026micro;mol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e53.46 GBq/\u0026micro;mol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1\u0026ndash;60 GBq/\u0026micro;mol\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRadionuclidic purity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e100%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e100%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;99.9%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGe-68 breakthrough\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.00000036%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00000033%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.00000035%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEtOH amount\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.73%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.68%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.45%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;10% (V/V) (\u0026lt;\u0026thinsp;2.5 g)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHEPES content\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9.45 \u0026micro;g/mL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9.45 \u0026micro;g/mL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9.45 \u0026micro;g/mL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLess than 200 \u0026micro;g/V of HEPES in test solution\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEndotoxins\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;17.5 IU/mL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;17.5 IU/mL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;17.5 IU/mL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;17.5 IU/mL\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSterility test\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSterile\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSterile\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSterile\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSterile\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStability over 4 h (RCP%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e100%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e100%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;95%\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\u003e\u003c/p\u003e\u003cp\u003eThe reference solution of DOTA-EMP-100 exhibited a slightly different retention time (Rt\u0026thinsp;=\u0026thinsp;7.428 min) compared to [68Ga]Ga-DOTA-EMP-100, which had a retention time of Rt\u0026thinsp;=\u0026thinsp;7.620 min, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec. The difference in retention times is attributed to the use of different detectors (Radio and UV\u0026ndash;VIS), as well as to a variation in the charge of the DOTA chelator following the incorporation of gallium-68. This change in charge affects the interaction with the column, leading to a slight alteration in the hydrophobicity of the entire molecule. The absence of the 68Ga-chelate in the standard solution leaves three free carboxylic acid groups, increasing the hydrophilicity of the standard and consequently modifying its retention time.\u003c/p\u003e\u003cp\u003eWith Radio-TLC no [68Ga]Ga-colloids could be detected at Rf\u0026thinsp;=\u0026thinsp;0.2 and the radiopharmaceutical product was detected at Rf\u0026thinsp;=\u0026thinsp;0.8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eHPLC performed on the final radiopharmaceutical solution showed that the residual content of HEPES in the final preparation was lower in the HEPES test solution (12.5 \u0026micro;g/mL) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAdditionally, the product was tested for endotoxins, and the concentration was found to be below 17.5 EU/mL in all samples. Sterility testing was conducted on all samples, and they resulted sterile.\u003c/p\u003e\u003cp\u003eThe stability of [68Ga]Ga-DOTA-EMP-100 in buffer solution at room temperature was evaluated for up to 4 hours using Radio-UV-HPLC, RadioTLC, and pH measurements. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, [68Ga]Ga-DOTA-EMP-100 remains stable under the test conditions. No additional radioactive by-products or free gallium-68 were detected during this period, and the RCP% stayed 100% over time.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe RCP% was also assessed and confirmed by Radio-TLC (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), while the pH value remained stable at 7 throughout the 4-hour period.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe c-MET protein, a mesenchymal-epithelial transcription factor, is a transmembrane receptor tyrosine kinase encoded by the human MET gene. It is involved in various cellular functions, including growth, differentiation, motility, making it an active target for drug discovery and development.\u003c/p\u003e\u003cp\u003eWe hereby describe the development and validation of an automated synthesis method and QC system to label c-MET ligand (DOTA-EMP-100) with gallium-68. The process of developing and designing a new radiopharmaceutical typically involves the establishment and setup of the radiosynthesis, along with the implementation of quality assessment methods for the final product. These assessments include evaluating release specifications such as RCP%, specific activity (As or Am), radionuclidic purity, chemical purity, radiochemical yield (RCY%), pH, sterility, and stability. A critical parameter in this process is the specific activity or molar activity of the final product, which refers to the ratio of the labeling isotope (in Bq) to the amount of peptide (in grams) or the unit mole of the compound (mol). For optimal synthesis setup, high molar activity (Am) is crucial, as it affects the peptide's receptor affinity and available receptor sites, limiting the peptide dose. A suboptimal Am can lead to receptor saturation or side effects. However, Am should not be excessively high, as sufficient peptide mass is needed to ensure a good radiochemical yield and reliable biodistribution for imaging. For these reasons, five different amounts of DOTA-EMP-100 (50-40-30-20 \u0026micro;g) were evaluated for the production of [68Ga]Ga-DOTA-EMP-100 considering the affinity and specificity of the c-Met peptide EMP-100 Kd 3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 nM [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The results summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e demonstrate that the amount of 40 \u0026micro;g of DOTA-EMP-100 allows the best radiochemical purity (100%), high radiochemical yield (64.93%) (n.d.c.) as well as good molar activity (53.075 GBq/\u0026micro;mol). We noticed an increasing radiochemical yield corresponding to increasing ligand amount up to 50 \u0026micro;g, but a lower RCP% for all the amount peptide precursor except for 40 \u0026micro;g (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), allowing to determine the best amount of peptide for the production of [68Ga]Ga-DOTA-EMP-100. The higher molar activity (83,80 GBq/\u0026micro;mol) was observed for 20 \u0026micro;g of peptide precursor, but the lower radiochemical yield (54,75%) and radiochemical purity (99,52%) lead us to validate the radiosynthesis of [68Ga]Ga-DOTA-EMP-100 starting from 40 \u0026micro;g of DOTA-EMP-100.\u003c/p\u003e\u003cp\u003eStandardization and harmonization of radiopharmaceutical production are essential for ensuring that radiopharmaceutical research can be reliably tested and transferred across laboratories. After optimizing the method, the process and final product must be validated, as required by regulations, to confirm the robustness of the gallium-68 labeling method for EMP-100, ensuring its clinical applicability and compliance with established standard operating procedures. As seen in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the three consecutive syntheses and QC results show mean values of RCP% of 100%, confirmed by both Radio-TLC and Radio-UV-HPLC, radiochemical yield of 64.50% and a molar activity (Am) of 53.41 GBq/\u0026micro;mol. Moreover, we achieved an endotoxin-free, sterile and stable solution of [68Ga]Ga-DOTA-EMP-100 maintaining a radiochemical purity of 100% over a period of 4 h.\u003c/p\u003e\u003cp\u003eAll quality control parameters tested were in accordance with the standards set by the European Pharmacopoeia (Ph.Eur.), confirming the high reproducibility of the [68Ga]Ga-DOTA-EMP-100 production method. This ensures that it is not only safe but also suitable for daily production and transfer to other radiopharmaceutical laboratories. Based on these results, [68Ga]Ga-DOTA-EMP-100 holds significant potential for evaluating therapeutic strategies involving tyrosine kinase inhibitors by assessing c-MET expression at tumor sites prior to systemic treatments. Additionally, preclinical evaluation of this novel radiotracer could be instrumental in detecting altered c-MET expression, aiding in the identification of associated pathologies and advancing its potential clinical applications, as demonstrated by its use in metastatic renal cell carcinoma (mRCC) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. With the incorporation of a DOTA-chelator for EMP-100, a theranostic approach is now a feasible option. This opens up promising opportunities, such as the use of cMET-PET to assess uptake intensity before potential therapy with 177Lu-DOTA-labeled ligands targeting c-MET, a strategy currently under investigation.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe synthesis of [68Ga]Ga-DOTA-EMP-100 was successfully carried out through a fully automated process using the GRP Scintomics module. All quality control parameters, including radiochemical purity, pH, endotoxins, and sterility, were in compliance with the European Pharmacopoeia standards. Additionally, the product solution demonstrated stability for at least 3 hours after production, as confirmed by Radio-UV-HPLC. As a result, [68Ga]Ga-DOTA-EMP-100 can be consistently and efficiently produced for routine clinical applications.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cb\u003eMaterials\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe precursor to [68Ga]Ga-DOTA-EMP-100, namely EMP-100 was purchased from Edinburgh Molecular Imaging Ltd. An aqueous stock solution of 1 mg/mL was prepared and kept at -20\u0026deg;C.\u003c/p\u003e\u003cp\u003eAll chemicals used for the radiolabelling reaction, i.e. saline (NaCl), ethanol, 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid (HEPES) buffer solution, PBS buffer solution and water were of the highest available purity grade and commercially obtained as a single disposable kit (SC-01, ABX Radeberg, Germany).\u003c/p\u003e\u003cp\u003eAll chemicals used to perform quality controls, i.e. trifluoroacetic acid (TFA), water and acetonitrile used for Radio-UV-HPLC, as well as ammonium acetate and methanol, were metal-free and purchased from Sigma Aldrich (Saint Louis, Missouri, USA).\u003c/p\u003e\u003cp\u003eAll the medicinal products used in this study are commercially available and authorized for clinical use.\u003c/p\u003e\u003cp\u003eAn automated synthesis module (Scintomics GRP\u0026reg; module, Germany) equipped with a disposable single-use cassette (SC-01, ABX) and a pharmaceutical grade, GMP certified and compliant with European Pharmacopoeia 68Ge/68Ga generator (1850 MBq, GalliaPharm\u0026reg; Eckert \u0026amp; Ziegler, Berlin, Germany) were used and both placed in a GMP grade A hot cell (NMC Ga-68, Tema Sinergie) to assess aseptic production.\u003c/p\u003e\u003cp\u003eThe amount of detected metal impurities/68Ge breakthrough as provided by the manufacturer was less than the defined limit in the European Pharmacopeia monograph [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eActivity counting was determined using a borehole counter (CRC\u0026reg; 25-PET, Capintec). Radio-UV-HPLC was performed using a Dionex Ultimate 3000 HPLC system (Thermo Fisher Scientific) equipped with a BioBasic-18 column 5\u0026micro;m 300\u0026Aring; (250 mm \u0026times; 4.6 mm) and coupled with UV and a γ-detector (Berthold Technologies, Milan, Italy). The Radio-TLC scanner used was Cyclone\u0026reg; Plus Storage Phosphor system (Perkin Elmer). The test for endotoxins was performed with Nexgen PTS (Charles River)\u003c/p\u003e\u003cp\u003e\u003cb\u003eRadiosynthesis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe synthesis template was identical to the already established synthesis template for [68Ga]Ga-PSMA [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] as well as [68Ga]Ga-DOTA-ECL1i [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe elution of 68Ge/68Ga generator (GalliaPharma\u0026reg;) was carried out using the GRP module 3 V automated synthesis system (Scintomics GRP\u0026reg; module). The generator was eluted with 0.1 M HCl 24 h before labelling to remove the accumulated stable Zn-68 from Ga-68 decay and the elute [68Ga]GaCl\u003csub\u003e3\u003c/sub\u003e, obtained from the generator elution, was pre-concentrated on a strong cation exchange (SCX) cartridge, which separates the ions based on their net total surface area change. [68Ga]GaCl\u003csub\u003e3\u003c/sub\u003e was recovered from the SCX by the eluent 5 M NaCl.\u003c/p\u003e\u003cp\u003eThe eluate was transferred into the reaction vial, previously loaded with DOTA-EMP-100 ( 20\u0026ndash;30\u0026ndash;40\u0026ndash;50 \u0026micro;g in 1.5 M 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid (HEPES buffer solution) at pH\u0026thinsp;=\u0026thinsp;4\u0026ndash;4.5. The mixture was incubated at 95\u0026deg;C for 10 min. After the completion of the labeling reaction the crude product was cooled down and trapped onto Sep Pak C18 RP cartridge, washed with water for injection Ph. Eur., and eluted with 2 mL of Ethanol/Water 1/1. The final product was diluted with phosphate buffered saline (PBS) and sterilized through a 0.2 \u0026micro;m filter (millex GV) into a sterile 25 mL capped glass vial and diluted with PBS for the final formulation. The entire radiopharmaceutical production takes 35 min.\u003c/p\u003e\u003cp\u003e\u003cb\u003eQuality control and process validation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo ensure that the final injectable radiopharmaceutical product fulfils regulatory requirements relating to contaminants, suitable production and quality control are crucial [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAfter synthesis, the radiopharmaceutical product was evaluated determining the following parameters: total product activity, gallium-68 ion identity via half-life time and gamma spectroscopy, chemical and radiochemical purity by Radio-UV-HPLC and Radio-TLC, pH, radionuclide purity for 68Ge-breakthrough and sterility/endotoxin assay (sterility test and LAL test). The radiochemical purity and the stability of [68Ga]Ga-EMP-100 at room temperature was evaluated by Radio-TLC and Radio-UV-HPLC for 4 h.\u003c/p\u003e\u003cp\u003eFor Radio-TLC, ITLC-SG (8-cm length, 1 cm thick) (Agilent Technologies) was used as stationary phase and ammonium acetate/methanol (1/1) as mobile phase. The software OptiQuantTM was used to analyse the chromatograms. The percentages of each fraction were determined relative to the total activity of the chromatogram.\u003c/p\u003e\u003cp\u003eFor Radio-UV-HPLC a standardized method was performed. Flow rate of the mobile phase was set at 0.6 mL/min, and the mobile phases used were A) 0.1% TFA in water and B) 0.1% TFA in acetonitrile, following a phase gradient: 0-1.7 min 0% B, 1.7-9 min 70% Band 9\u0026ndash;12 min 3% B. The column temperature was kept at 25\u0026deg;C and the samples were also monitored with an UV detector at 220 nm to detect chemical impurities in the final product. The software system Chromeleon 7 was used to assemble the information.\u003c/p\u003e\u003cp\u003eReference solutions of [68Ga]GaCl\u003csub\u003e3\u003c/sub\u003e, DOTA-EMP-100 and the final radiopharmaceutical [68Ga]Ga-DOTA-EMP-100 were assessed using the same analytical conditions.\u003c/p\u003e\u003cp\u003eThe chemical purity of [68Ga]Ga-DOTA-EMP-100 concerning the residual HEPES content was assessed according to Ph. Eur. Monograph (Gallium 2482), following our validated HPLC method [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. For HPLC a Waters Xbridge\u0026reg; column C18 (150 mm\u0026times;4.6 mm, 3.5 \u0026micro;m), as stationary phase was used, connected to an UV detector set to a wavelength of 195 nm and a γ-detector (Berthold Technologies, Milan, Italy) and ammonium formate 20 mM pH 9.5, as mobile phase, at an isocratic flow of 0.7 mL/min.\u003c/p\u003e\u003cp\u003eThe sterility tests were performed as described in the European Pharmacopoeia (EMA/CHMP/ICH/645592/2008) and LAL test with Nexgen PTS (Charles River).\u003c/p\u003e\u003cp\u003eTo validate the entire process of radiopharmaceutical production and quality control, three batches of [68Ga]Ga-DOTA-EMP-100 were produced in three different days under the same conditions set for typical routine preparations. Every batch was fully characterized from the analytical point of view, with the aim to verify that the product met the acceptance criteria for all the established quality parameters.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003especific activity\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eComputed Tomography\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGMP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGood Manufacturing Practice\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGRP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGood Radiopharmaceutical Practices\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eEANM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eEuropean Association of Nuclear Medicine\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eEur. Ph.\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eEuropean Pharmacopeia\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHEPES\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid\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 Pressure Liquid Chromatography\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNBP-MN\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eNorme di Buona Preparazione in Nuclear Medicine\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTLC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eThin Layer Chromatography\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTFA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003etrifluoroacetic acid\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\"\u003eQC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003equality control\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\"\u003eRPC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eradiochemical purity\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eAuthors\u0026rsquo; contributions\u003c/p\u003e\n\u003cp\u003eSM, AG, AG, MS, GB, AG, CP and LV have contributed to the organization of the content for this manuscript. SM, AG, AG, CP and AG collected relevant information and prepared the draft. SM, AG, AG, MS, GB, AG and CP drafted and LV revised the manuscript.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis study did not receive any financial support.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFu J, Su X, Li Z, Deng L, Liu X, Feng X, Peng J. HGF/c-MET pathway in cancer: from molecular characterization to clinical evidence. 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(PMID: 35889351 PMCID: PMC9323806.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"[68Ga]Ga-radiopharmaceuticals, [68Ga]Ga-DOTA-EMP-100, HGF/c-MET pathway, PET imaging","lastPublishedDoi":"10.21203/rs.3.rs-7127095/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7127095/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground.\u003c/h2\u003e\u003cp\u003eOveractivation of the HGF/c-MET pathway is implicated in various cancers, making its inhibition a promising therapeutic strategy. While several MET-targeting agents are currently approved or in advanced clinical development, patient selection often relies on invasive tissue-based assays. The development of a specific c-MET radioligand for PET imaging and radioligand therapy represents a non-invasive alternative, enabling real-time monitoring of target expression and offering a pathway to personalized treatment.\u003c/p\u003e\u003ch2\u003eResults.\u003c/h2\u003e\u003cp\u003eAn optimized formulation of [⁶⁸Ga]Ga-DOTA-EMP-100, using 40 \u0026micro;g of precursor, provided the best outcome in terms of radiochemical performance. Process validation across three independent productions confirmed a consistent radiochemical yield of 64.5%, high radiochemical purity (100%), and a molar activity of 53.41 GBq/\u0026micro;mol.\u003c/p\u003e\u003ch2\u003eConclusions.\u003c/h2\u003e\u003cp\u003e[⁶⁸Ga]Ga-DOTA-EMP-100 was successfully synthesized with high purity and reproducibility, supporting its potential for multi-dose application in clinical PET imaging and targeted radioligand therapy.\u003c/p\u003e","manuscriptTitle":"Development and optimization of [68Ga]Ga-DOTA-EMP-100 for non-invasive PET imaging and targeted radioligand therapy of c-MET overactivation in cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-23 05:48:12","doi":"10.21203/rs.3.rs-7127095/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"870b1e1b-8b2c-4d51-96ef-d864c000081c","owner":[],"postedDate":"July 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-04T13:29:22+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-23 05:48:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7127095","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7127095","identity":"rs-7127095","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}